CHAPTER 10 CYTOGENETICS AND GENE REARRANGEMENT

CHAPTER 10 CYTOGENETICS AND GENE REARRANGEMENT
Williams Hematology

CHAPTER 10 CYTOGENETICS AND GENE REARRANGEMENT

MICHELLE M. LE BEAU

Genetic Consequences of Chromosomal Rearrangements
Methods
Chromosome Terminology
Specific Disorders

Chronic Myelogenous Leukemia

Other Myeloproliferative Disorders

Myelodysplastic Syndromes

Acute Myeloid Leukemia De Novo

Acute Myeloid Leukemia and Myelodysplastic Syndromes Associated with Prior Cytotoxic Treatment (T-MDS and T-AML)

Malignant Lymphoproliferative Disorders

T Cell Acute Lymphoblastic Leukemia

Chronic Lymphocytic Leukemia

Lymphoma
Chapter References

Cytogenetic analysis provides pathologists and clinicians with a powerful tool for the diagnosis and classification of hematologic malignant diseases. The detection of an acquired, somatic mutation establishes the diagnosis of a neoplastic disorder and rules out a reactive hyperplasia or morphological changes due to toxic injury or vitamin deficiency. Specific cytogenetic abnormalities identify homogeneous subsets of various malignant diseases and enable clinicians to predict their clinical course and their likelihood of responding to particular treatments. In many cases, the prognostic information derived from cytogenetic analysis is independent of that provided by other clinical features. Patients with favorable prognostic features benefit from standard therapies with well-known spectra of toxicities, whereas those with less favorable clinical or cytogenetic characteristics may be better treated with more intensive or investigational therapies. The disappearance of a chromosomal abnormality present at diagnosis is an important indicator of complete remission following treatment, and its reappearance invariably heralds relapse of the disease. Pretreatment cytogenetic analysis can be useful in choosing among postremission therapies that differ widely in cost, acute and chronic morbidity, and effectiveness.

Acronyms and abbreviations that appear in this chapter include: ALCL, anaplastic large cell lymphoma; ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; AML-M2, AML with maturation; AMMoL-M4Eo, acute myelomonocytic leukemia with abnormal eosinophils; APL, acute promyelocytic leukemia; BL, Burkitt lymphoma; CD30, cluster of differentiation; CLL, chronic lymphocytic leukemia; CML, chronic myelogenous leukemia; CMMoL, chronic myelomonocytic leukemia; CNS, central nervous system; CTCL, cutaneous T-cell lymphoma; DLCL, diffuse large cell lymphoma; FAB, French-American-British; FISH, fluorescence in situ hybridization; FITC, fluorescein isothiocyanate; LPL, lymphoplasmacytoid lymphoma; MALT, mucosa-associated lymphoid tumor; MCL, mantle cell lymphoma; MDS, myelodysplastic syndrome; EFS, event-free survival; PML, promyelocytic leukemia; RA, refractory anemia; RAEB, RA with excess blasts; RAEB-T, RAEB in transformation; RARS, RA with ringed sideroblasts; RT-PCR, reverse transcriptase polymerase chain reaction; SIg, surface immunoglobulin; SLL, small lymphocytic lymphoma; WBC, white blood cell.

The malignant cells in many patients who have leukemia or lymphoma have acquired clonal chromosomal abnormalities. A number of specific cytogenetic abnormalities have been recognized that are very closely, and sometimes uniquely, associated with morphologically and clinically distinct subsets of leukemia or lymphoma.1,2,3 and 4 The detection of one of these recurring abnormalities can be helpful in establishing the correct diagnosis, influencing selection of therapy, and providing prognostic information. The appearance of new abnormalities in the karyotype of a patient under observation often signals a change in the pace of the disease, usually to a more aggressive disorder. Further detailed information regarding recurring chromosomal rearrangements is contained in reviews.1,2,3,4 and 5
GENETIC CONSEQUENCES OF CHROMOSOMAL REARRANGEMENTS
The genes that are located at the breakpoints of a number of the recurring chromosomal translocations have been identified (Table 10-1). Alterations in the expression of the genes or in the properties of the encoded proteins resulting from the rearrangement play an integral role in the process of malignant transformation.5,6 The altered genes fall into several functional classes, including those that encode for tyrosine or serine protein kinases, cell surface receptors, and growth factors (see Table 10-1). However, the largest class are those that encode transcriptional regulating factors; these proteins are involved in the initiation of gene transcription, often functioning in a tissue-specific fashion to regulate growth and differentiation.

TABLE 10-1 EXAMPLES OF TRANSFORMING GENES FOUND AT TRANSLOCATION JUNCTIONS: FUNCTIONAL CLASSIFICATION

There are two general mechanisms by which chromosomal translocations result in altered gene function. The first is deregulation of gene expression. This mechanism is characteristic of the translocations in lymphoid neoplasms that involve the immunoglobulin genes in B lineage tumors and the T cell–receptor genes in T lineage tumors. These rearrangements result in inappropriate expression of an oncogene with no alteration in its protein structure. The second mechanism is the expression of a novel fusion protein, resulting from the juxtaposition of coding sequences from two genes that are normally located on different chromosomes. Such chimeric proteins are “tumor-specific” in that the fusion gene does not exist in nonmalignant cells; thus, the detection of such a fusion gene or its protein product can be important in diagnosis, or in the detection of residual disease or early relapse. Moreover, they may also be appropriate targets for tumor-specific therapies. An example is the chimeric BCR/ABL protein resulting from the t(9;22) in chronic myelogenous leukemia. All of the translocations cloned to date in the myeloid leukemias result in a fusion protein.
Chromosomal translocations result in the activation of genes in a dominant fashion. A number of human tumors are believed to result from homozygous, recessive mutations.7 These mutations lead to the absence of a functional protein product, suggesting that these genes function as “suppressor” genes whose normal role is to limit cellular proliferation. The hallmark of tumor suppressor genes is the loss of genetic material in malignant cells, resulting from chromosomal loss or deletion, as well as by other genetic mechanisms.
METHODS
Cytogenetic analysis of malignant diseases is based upon the study of the tumor cells themselves. In leukemia, the specimen is usually obtained by marrow aspiration and is either processed immediately (direct preparation) or cultured for 24–72 hours. When a marrow aspirate cannot be obtained, a marrow biopsy (bone core specimen) or a blood sample, for patients who have circulating immature myeloid or lymphoid cells, can often be studied successfully. An involved lymph node or tumor mass may be sampled. The use of amethopterin to synchronize dividing cells in culture, combined with brief exposures to mitotic inhibitors such as colchicine or to DNA binding agents (ethidium bromide) are used by some laboratories to obtain elongated chromosomes that have an increased number of bands.
For specimen collection, 1 to 5 ml of marrow are aspirated aseptically into a syringe coated with preservative-free sodium heparin (preservatives in heparin can suppress cell growth) and transferred to a sterile 15-ml centrifuge tube containing 5 ml of culture medium (RPMI 1640, 100 units sodium heparin). If a marrow aspirate cannot be obtained, a bone marrow biopsy may be taken and placed into the collection tube. For blood specimens, 10 ml are drawn aseptically by venipuncture into a syringe coated with preservative-free heparin. Specimens should be maintained at room temperature and transported in culture medium. To avoid loss of cell viability, it is critical that the specimen be transported to the cytogenetics laboratory without delay. Overnight shipment of specimens frequently results in loss of cell viability, and most laboratories experience a high proportion (25–50%) of inadequate analyses using such specimens. For specimens handled optimally, 95 to 98 percent of all cases should be adequate for cytogenetic analysis. Those cases that are inadequate generally represent samples from patients with hypocellular bone marrows. Overall, approximately 75 percent of bone core biopsies will yield adequate numbers of metaphase cells for complete analysis.
To prepare metaphase cells, the sample is exposed sequentially to mitotic inhibitors to accumulate cells in mitosis, hypotonic KCl (0.075 M) to swell the cells, and fixative (absolute methanol:glacial acetic acid, 3:1). Slides are prepared by dropping the cell suspension onto precleaned glass microscope slides, and the slides are air dried. The most popular chromosomal banding techniques is trypsin-Giemsa banding. Using this technique, a consistent chromosome banding pattern is induced by exposing cells to a dilute trypsin solution (0.1–0.25 percent), followed by staining in phosphate-buffered Giemsa stain.
Cytogenetic analysis of human tumors is often technically difficult, due to the presence of multiple abnormalities and multiple cell lines, and requires highly skilled personnel. These factors have led investigators to seek alternative methods for identifying chromosomal abnormalities, such as Southern blot analysis of DNA, RT-PCR analysis of RNA from tumor cells, or FISH.8
The technique of FISH is based upon the same principle as Southern blot analysis, namely, the ability of single-stranded DNA to anneal to complementary DNA. In the case of FISH, the target DNA is the nuclear DNA of interphase cells, or the DNA of metaphase chromosomes that are affixed to a glass microscope slide (FISH can also be accomplished with bone marrow or blood films or with fixed and sectioned tissue). The test probe is labeled with biotin- or digoxigenin-labeled nucleotides and detected with fluorescein isothiocyanate (FITC)- or CY3-conjugated avidin or with rhodamine-labeled anti-digoxigenin antibodies. Probes that are directly labeled with fluorochrome are also available for hybridization, thereby simplifying the technique by eliminating the probe detection steps. With the development of dual- and triple-pass filters, most laboratories now have the capacity to hybridize and detect 2 to 3 probes simultaneously.
Several types of probes can be used to detect chromosomal abnormalities by FISH. Hybridization of centromere-specific probes has been used to detect monosomy, trisomy, and other aneuploidies in both leukemias and solid tumors (Plate XXIV-A). Chromosome-specific libraries, which paint the chromosomes, are particularly useful in identifying marker chromosomes (rearranged chromosomes of unidentified origin), or structural rearrangements such as translocations (Plate XXIV-B). Translocations and deletions can also be identified in interphase or metaphase cells by using genomic probes that are derived from the breakpoints of recurring translocations or within the deleted segment (Plate XXIV-C). The newest innovation in FISH technology is spectral karyotyping, or multiplex FISH.9 Using this approach 24 differentially labeled painting probes representing each chromosome are cohybridized; Fourier spectroscopy is used to distinguish each spectrally overlapping probe and imaging software assigns a unique color to each chromosome (Plate XXIV-D). Often referred to as “color karyotyping,” this method is applicable to the identification of numerical abnormalities as well as many structural abnormalities.
FISH techniques have a number of applications (Table 10-2). In some cases, FISH analysis provides more sensitivity, in that cytogenetic abnormalities have been identified by FISH in samples that appeared to be normal by morphological and conventional cytogenetic analyses. FISH is most powerful when the analysis is targeted towards those abnormalities that are known to be associated with a particular tumor or disease. An example of how FISH could be used in a clinical setting is as follows: Cytogenetic analysis could be performed at the time of diagnosis to identify the chromosomal abnormalities in an individual patient’s malignant cells. Thereafter, FISH with the appropriate probes could be used to detect residual disease or early relapse and to assess the efficacy of therapeutic regimens. For example, the use of FISH to detect the t(9;22) in CML patients following transplantation or interferon therapy and for the sex chromosome composition of cells in the recipient after a sex-mismatched transplant has become frequent.

TABLE 10-2 APPLICATIONS AND ADVANTAGES OF FISH

CHROMOSOME TERMINOLOGY
Chromosomal abnormalities are described according to the International System for Human Cytogenetic Nomenclature (Table 10-3).10 To describe the chromosomal complement, the total chromosome number is listed first, followed by the sex chromosomes, then by numerical and structural abnormalities in ascending order. The observation of at least two cells with the same structural rearrangement, e.g., a translocation, deletion, inversion, or gain of the same chromosome, or of three cells showing loss of the same chromosome, is considered evidence for the presence of an abnormal clone. However, one cell with a normal karyotype is considered evidence for the presence of a normal cell line. Patients whose cells show no alteration or nonclonal (single cell) abnormalities are considered to be normal. An exception to this is a single cell characterized by a recurring structural abnormality. In such instances it is likely that this represents the karyotype of the malignant cells in that particular patient.

TABLE 10-3 GLOSSARY OF CYTOGENETIC TERMINOLOGY

SPECIFIC DISORDERS
CHRONIC MYELOGENOUS LEUKEMIA
The first consistent chromosome abnormality in any malignant disease was identified in CML. The Philadelphia, or Ph chromosome, was initially thought to be a deletion of chromosome 22 but was later shown to be a translocation involving chromosomes 9 and 22 [t(9;22)(q34;q11.2)], Fig. 10-1). The t(9;22) occurs in a pluripotent stem cell that gives rise to both lymphoid and myeloid lineage cells. The standard t(9;22) is identified in about 92 percent of CML patients; another 6 percent have variant translocations that involve a third chromosome in addition to numbers 9 and 22. The genetic consequences of the t(9;22) or the complex translocations is to move a portion of the Abelson (ABL) proto-oncogene on chromosome 9 adjacent to a portion of the BCR gene on 22. Analyses of leukemia cells from rare patients with typical CML who lack the t(9;22) has revealed a rearrangement involving ABL and BCR that is detectable only at the molecular level (1–2% of cases).12

FIGURE 10-1 Partial karyotypes from trypsin-Giemsa-banded metaphase cells depicting recurring chromosomal rearrangements observed in myeloid leukemias. (a) t(9;22)(q34;q11), CML. (b) t(8;21)(q22;q22), AML-M2. (c) inv(16)(pl3q22), AMMoL-M4Eo. (d) t(15;17)(q22;q11–12), APL. (e) t(9;11) (p22;q23), AMoL-M5. (f) del(5)(ql3q33), t-AML. The rearranged chromosomes are identified with arrows.

Marrow cells from rare “CML” patients lack both a Ph chromosome and the BCR/ABL fusion but often have a normal karyotype or trisomy 8.13 These patients have a substantially shorter survival than do those whose cells have the t(9;22). Most of these patients have a myelodysplastic syndrome (MDS), most commonly chronic myelomonocytic leukemia or refractory anemia with excess blasts, or the poorly understood disorder of “atypical CML.” Thus, the t(9;22) and resultant BCR/ABL fusion is the sine qua non of CML.13
The BCR/ABL protein is located on the cytoplasmic surface of the cell membrane and acquires a novel function in transmitting growth-regulatory signals from cell surface receptors to the nucleus via the RAS signal transduction pathway.13,14 The BCR/ABL fusion gene can be detected with standard Southern blot analysis of DNA or with RT-PCR analysis of mRNA for diagnosis and detection of residual disease. FISH can also be used to detect the Ph chromosome in both metaphase and interphase cells (Plate XXIV-C).
As they enter the terminal acute phase, most CML patients (80%) show karyotypic evolution, with the appearance of new chromosomal abnormalities in very distinct patterns in addition to the Ph chromosome.11 A change in the karyotype is considered to be a grave prognostic sign.11 With the exception of an isochromosome of the long arm of chromosome 17, i(17)(q10), which is usually associated with myeloid blast transformation, there is no association of a particular karyotype with lymphoid or myeloid blast transformation. The additional abnormalities are not correlated with the response to therapy during the acute phase.11 The most common changes, a gain of chromosomes 8 or 19, or a second Ph (by gain of the first), or an i(17q), frequently occur in combination to produce modal chromosome numbers of 47 to 50.11
OTHER MYELOPROLIFERATIVE DISORDERS
POLYCYTHEMIA VERA
A cytogenetically abnormal clone is present in 14 percent of untreated polycythemia vera patients compared with 39 percent of treated patients.2,11 When the disease transforms to acute myeloid leukemia (AML), 85 percent have an abnormal clone. The presence of a chromosome abnormality at diagnosis does not necessarily predict a short survival or the development of leukemia. A change in the karyotype may be an ominous sign. Marrow cells frequently contain additional chromosomes (+8 or +9); these abnormalities also occur together, which is otherwise rare.11 Structural rearrangements most often involve a deletion of the long arm of chromosome 20, noted in 30 percent of patients, or a duplication of the long arm of chromosome 1, in 20 percent of patients. Loss of chromosome 7 (20 percent of patients) and del(5q) (40 percent of patients) are often observed in the leukemic phase. These changes may be related to the prior cytotoxic treatment received by these patients.
IDIOPATHIC MYELOFIBROSIS
Cytogenetic analysis of myeloid metaplasia with myelofibrosis has revealed clonal abnormalities in 35 percent of patients.2 These abnormalities are similar to those noted in other myeloid disorders; the most common anomalies are +8, –7, del(7q), del(11q), del(13q), and del(20q).1,2 A change in the karyotype may signal evolution to acute leukemia.
THROMBOCYTHEMIA
Fewer than 5 percent of patients with essential thrombocytosis have an abnormal clone. No consistent abnormalities have been identified.
MYELODYSPLASTIC SYNDROMES
The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hemopathies including chronic myelomonocytic leukemia (CMMoL), refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), and RAEB in transformation (RAEB-T). Clonal chromosome abnormalities can be detected in marrow cells of 40 to 70 percent of patients with MDS at diagnosis (RA, 30%; RARS, 27%; RAEB, 70%; RAEB-T, 70%; CMMoL, 30%).2,15,16 The proportion varies with the risk that a subtype will transform to AML, which is highest for RAEB and RAEB-T. The common chromosome changes, +8, –5/del(5q), –7/del(7q), and del(20q), are similar to those seen in AML. The recurring abnormalities that are closely associated with the distinct morphologic subsets of AML de novo are almost never seen in MDS. With the exception of the 5q– syndrome, the chromosome changes show no close association with the specific subtypes of MDS. The 5q– syndrome occurs in a subset of older patients, frequently women, with RA, generally low blast counts, and normal or elevated platelet counts.17 These patients have an interstitial deletion of 5q, typically as the sole abnormality. The deletions vary in size but are similar to those noted in AML. These patients can have a relatively benign course which extends over several years.17
Cytogenetic abnormalities in MDS are predictive of survival and progression to AML.15,16 Patients with a “good outcome” have normal karyotypes, –Y alone, del(5q) alone, or del(20q) alone; those with an “intermediate outcome” have other abnormalities; and those with a “poor outcome” have complex karyotypes (³3 abnormalities, typically with abnormalities of chromosome 5 and/or 7) or chromosome 7 abnormalities.16
ACUTE MYELOID LEUKEMIA DE NOVO
THE 8;21 TRANSLOCATION
With initial banding analyses, clonal abnormalities were detected in 50 percent of patients with AML. This percentage has increased with improved banding and culture techniques; many laboratories are currently finding that at least 80 percent of patients have an abnormal karyotype.2,4 The most frequent abnormalities are a gain of chromosome 8 and a loss of chromosome 7, which are seen in most subtypes of AML.2,4 Specific rearrangements are closely associated with particular subtypes of AML as defined by the FAB classification (Table 10-4).2,4,18,19 The 8;21 translocation [t(8;21)(q22;q22)] was described in 1973 and was the first translocation identified in AML (see Fig. 10-1). The t(8;21) is common and is observed in 18 percent of all AML cases with an abnormal karyotype and in 30 percent of those patients with AML with maturation (AML-M2).21 This translocation is the most frequent abnormality in children with AML, and occurs in 15 to 20 percent of karyotypically abnormal cases. Loss of a sex chromosome (–Y in males, –X in females) accompanies the t(8;21) in 75 percent of cases. Most cases with the t(8;21) are classified as AML with maturation, but some cases have been diagnosed as acute myelomonocytic leukemia. Although AML-M2 is heterogeneous, the presence of the t(8;21) identifies a morphologically and clinically distinct subset. AML-2 with the t(8;21) has a favorable prognosis in adults, but the outcome in children is poor.19

TABLE 10-4 RECURRING CHROMOSOME ABNORMALITIES IN MALIGNANT MYELOID DISEASES

At the molecular level, the t(8;21) involves the AML1 gene, which encodes a transcription factor that is critical in hematopoiesis.20,21 The AML1 gene on chromosome 21 is fused to the ETO gene on chromosome 8 and results in an AML1/ETO chimeric protein.20,21 Transformation by AML1/ETO likely results from altered transcriptional regulation of normal AML1 target genes, combined with the activation of new target genes that prevent programmed cell death or cause aberrant proliferation.
THE 15;17 TRANSLOCATION
The t(15;17)(q22;q11.2–12) (see Fig. 12) is highly specific for acute promyelocytic leukemia (APL) and has not been found in any other disease. Although the t(15;17) was believed to be present in all cases of APL initially, it is now recognized that there are rare variant translocations, which occur in less than two percent of cases. These include the t(11;17)(q23;q11.2–12) and t(5;17)(q34;q11.2–12).22 Establishing the diagnosis of APL with the typical t(15;17) is important, because this disease is sensitive to therapy with all-trans-retinoic acid and arsenic trioxide, whereas other cases of AML and the APL-like disorders associated with the variant translocations do not respond to retinoic acid. The t(15;17) results in a fusion retinoic-acid-receptor-alpha protein (PML/RARA), which is thought to interfere with the retinoic acid receptor pathway that regulates terminal differentiation of myeloid precursors.22
ABNORMALITIES OF CHROMOSOME 16
Another clinical-cytogenetic association involves acute myelomonocytic leukemia with abnormal eosinophils (AMMoL-M4Eo), including large and irregular basophilic granules and positive reactions with periodic-acid-Schiff and chloroacetate esterase. Most patients have an inversion of chromosome 16, inv(16)(pl3q22) (see Fig. 10-1), but some have a t(16;16)(pl3;q22); these aberrations are relatively common, occurring in 25 percent of AMMoL patients.2,4,19 They have a good response to intensive chemotherapy, with a complete remission rate of approximately 90 percent and an overall 5-year survival of 75 percent.19 The breakpoint at 16q22 occurs within the CBFB gene, which encodes one subunit of the AML1/CBFB transcription factor. Thus, like the t(8;21), the inv(16) disrupts the AML1 pathway regulating hematopoiesis.20,21
ABNORMALITIES OF CHROMOSOME 11
Recurring translocations involving 11q23 are seen in approximately 35 percent of AML-M5 patients, and are of interest for at least three reasons.2,23 First, there are over 30 different recurring rearrangements that involve 11q23, and thus, along with 14q32, 11q23 is one of the bands most frequently involved in rearrangements in human tumor cells.2,3,23 The breakpoints in the translocation partners include 1p32, 4q21, and 19p13.3 in acute lymphoblastic leukemia (ALL), and 1q21, 2q21, 6q27, 9p22, 10p11, 17q25, 19p13.3, and 19p13.1 in AML. Second, these translocations occur in both lymphoid and myeloid leukemias. One common translocation in infants, t(4;11)(q21;q23), has a lymphoblastic phenotype, whereas other translocations, such as the t(9;11) (q22;q23) (see Fig. 12) and t(11;19)(q23;p13.1), are common in monoblastic leukemias. Translocations involving 11q23 have a very unusual age distribution; they comprise about three-quarters of the chromosome abnormalities in leukemia cells of children under one year of age.23 With the exception of the t(9;11) which may have an intermediate outcome, translocations of 11q23 are associated with a poor outcome.19 Translocations of 11q23 involve MLL, a very large gene (>100 Kb) with multiple transcripts of 12 to 15 Kb.20 All breakpoints fall within an 8.3-Kb breakpoint cluster region encompassing exons 5–11; thus, MLL translocations can be detected by Southern blot analysis of DNA using a small cDNA probe containing these exons.23 The MLL protein is likely to be a transcription factor but may also function in chromatin remodeling; the translocations result in fusion proteins.
Trisomy 11 is a rare abnormality, noted as a sole aberration in one to two percent of all AML or MDS cases,2,20 and has an unfavorable outcome. Trisomy 11 is notable in that duplications of the MLL gene are detected in 90 percent of AMLs with +11 as the sole abnormality, and in 10 percent of AML cases with an apparently normal karyotype.20 The rearrangement is the result of a partial tandem duplication of MLL exons 2–6 or 2–8, mediated by recombination between Alu repetitive elements, and may produce a partially duplicated protein.
OTHER CHROMOSOME ABNORMALITIES
Each of the other recurring rearrangements in AML occurs in less than three percent of patients.3 A unique feature of abnormalities involving the long arm of chromosome 3, inv(3)(q2l;q26) or t(3;3) (q2l;q26) is the presence of platelet counts above 100,000/µl, sometimes over 106/µl, and an increase in bone marrow megakaryocytes, especially micromegakaryocytes.4 It is noteworthy that most of the structural rearrangements described above occur in younger patients with a median age in the thirties, whereas some of the other abnormalities, such as –5/del(5q) or –7/del(7q) occur in patients with a median age over 50. Moreover, many of the latter patients have occupational exposure to mutagenic agents such as chemicals, including solvents, petroleum, and pesticides.18
ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES ASSOCIATED WITH PRIOR CYTOTOXIC TREATMENT (T-MDS AND T-AML)
Therapy-related MDS and AML (t-MDS/t-AML) has been recognized as a late complication of cytotoxic therapy used in the treatment of both malignant and nonmalignant diseases.24 In patients who received alkylating agents the characteristic recurring chromosome abnormalities observed are loss of part or all of chromosomes 5 and/or 7 (–5/del[5q] or –7/del[7q]) (see Fig. 12). In our experience, 93 percent of t-MDS/t-AML patients had an abnormal karyotype and 75 percent had an abnormality of chromosome 5, chromosome 7, or both (Refs. 4 and 24 and unpublished data); this has been confirmed in other series. In contrast, only about 16 percent of patients with AML de novo have a similar abnormality of chromosomes 5 or 7 or both.2,18
By cytogenetic analysis of 177 patients with malignant myeloid diseases and a del(5q) we identified a small segment of 5q, consisting of band 5q31, that was deleted in each patient.25 This segment has been termed the commonly deleted segment. By molecular analyses we have narrowed the commonly deleted segment to a region of approximately 1 Mb containing the EGR1 tumor suppressor gene and the CDC25C G2 checkpoint gene.25 Five hematopoietic growth factor genes mapped to 5q31 (GM-CSF, IL3, IL4, IL5, and IL9) are excluded from this region. We have detected no mutations of the remaining EGR1 or CDC25C alleles, suggesting that a novel tumor suppressor gene in 5q31 is involved in the pathogenesis of t-AML.25
A second subtype of t-AML has been identified that is distinctly different from the more common leukemia that follows alkylating agents or irradiation. This type of t-AML is seen in patients receiving drugs known to inhibit topoisomerase II, e.g., etoposide, teniposide, and doxorubicin.24,26 Clinically, these patients have a shorter latency period (1–2 years); present with overt leukemia, usually with monocytic features rarely present with MDS; and have a more favorable response to intensive remission induction therapy. Balanced translocations involving the MLL gene at 11q23 or the AML1 gene at 21q22 are common in this subgroup.26
MALIGNANT LYMPHOPROLIFERATIVE DISORDERS
ACUTE LYMPHOBLASTIC LEUKEMIA
The most useful prognostic indicators in acute lymphoblastic leukemia (ALL)—the most frequent leukemia in children—are age, white cell count, immunophenotype, karyotype (including ploidy), and CNS status.2,27,28 Children who are between 2 and 10 years old, with a white cell count of less than 10,000/µl, and whose leukemia cells express the common ALL antigen (CALLA, CD10) have the best prognosis. A number of recurring cytogenetic abnormalities are associated with distinct immunologic phenotypes of ALL (Table 10-5) with distinct outcomes.2,3 and 4

TABLE 10-5 CYTOGENETIC-IMMUNOPHENOTYPIC CORRELATIONS IN MALIGNANT LYMPHOID DISEASES

The 9;22 Translocation The incidence of the t(9;22) in ALL is 30 percent in adults and 5 percent in children. Thus, the Ph chromosome is the most frequent rearrangement in adult ALL. About one-half of the patients show additional abnormalities, a frequency that is substantially higher than that observed in CML. Monosomy 7 is a common secondary abnormality in Ph+ ALL and is associated with a poorer outcome.27 A chromosomally normal cell line is frequently noted in the bone marrow of Ph+ ALL patients (70 percent), but is rare in untreated CML. Most cases have a precursor B phenotype; however, some cases have had both B cell and myeloid markers.29 The disease in both adults and children is characterized by high WBC counts, a high percentage of circulating blasts, and a poor prognosis. As in CML, the t(9;22) in ALL results in a BCR/ABL fusion gene; however, in over half of the patients the break in BCR is more proximal, resulting in a smaller fusion protein.29
Abnormalities of Chromosome 11 Translocations involving the MLL gene at 11q23 are observed in 5 to 7 percent of ALL patients.2,4,23 Of these, the most common is the t(4;11)(q21;q23) (see Fig. 10-2). The t(11;19)(q23;p13.3) is second in frequency; however, this rearrangement is not limited to ALL, in that approximately 50 percent of these cases have AML, usually AML-M5. Of note is the high frequency of translocations involving 11q23 in infant ALL (60–80%). A recent study using RT-PCR detected the t(4;11) in 80 percent of infant ALL cases.27,29 Patients with the t(4;11) have high leukocyte counts (median WBC 183,000/µl), L1 or L2 morphology, an immature precursor B phenotype (CD10–, CD19+), with coexpression of monocytic or, less commonly, T cell markers.27,28 Clinically, they have aggressive features with hyperleukocytosis, extramedullary disease, and a poor response to conventional chemotherapy. Adults with the t(4;11) have a CR rate of 75 percent, but a median event-free survival (EFS) of only 7 months.28 Children with the t(4;11) have a similar poor outcome.27 Rearrangements affecting MLL represent a major class of mutations in acute leukemia and identify patients with a poor outcome.

FIGURE 10-2 (A–D) Photomicrographs of metaphase and interphase cells following FISH. In panels A-C, the cells are counterstained with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). (A) Hybridization of a directly labeled centromere-specific probe for chromosome 8 (CEP8TM Spectrum Green, Vysis Inc.) to metaphase and interphase cells from an AML with trisomy 8. Centromere-specific probes hybridize to the repetitive DNA sequences that are present at the centromeres of human chromosomes. The chromosome 8 homologs are identified with arrows. (B) Hybridization of a directly labeled chromosome 8-specific painting probe (WCP8TM Spectrum Green, Vysis Inc.) to a metaphase cell with trisomy 8 from an AML. (C) Hybridization of a locus-specific probe for the detection of a recurring translocation, the t(9;22)(q34;q11.2) in CML. The probe is a mixture of digoxigenin-labeled DNA probes (detected with rhodamine-labeled antibodies) for the major breakpoint cluster region of the BCR gene at 22q11.2, and biotin-labeled probes (detected with FITC-avidin) for the ABL gene at 9q34 (M-bcr/abl probe, Oncor). In cells with the t(9;22), only one green signal (arrowhead) and one red signal (short arrow) is observed on the normal 9 and 22 homologs, and a yellow fusion signal (long arrow) is observed on the Ph chromosome as a result of the juxtaposition of the ABL and BCR sequences. (D) Spectral karyotyping analysis of a metaphase cell from an AML-M7. Twenty-four differentially labeled probes, each representing one human chromosome, are cohybridized, and imaging analysis software assigns a unique color to each. A complex karyotype was identified by conventional cytogenetic analysis, including a derivative chromosome 1 with additional material of unknown origin on 1p, a deletion of 8p, a derivative chromosome 11 resulting from an unbalanced translocation involving 1 and 11, and a derivative chromosome 12, consisting of 11q and 12q. The results of spectral karyotyping confirmed the identity of the rearranged chromosome 12 (arrowhead) and clarified the other abnormalities. The additional material on 1p was derived from chromosome 8 (long arrow, blue signal), and the der(11) actually consisted of material from chromosomes 1, 11, and 12 (short arrow, 11p white signal; chromosome 12 brown signal; 1p blue-pink signal).

The 12;21 Translocation A translocation, t(12;21)(p12;q22), has been identified in a high proportion (~25 percent) of childhood precursor B leukemia.27,30 The translocation is not easily detected by cytogenetic analysis due to the similarity in size and banding pattern of 12p and 21q. However, the rearrangement can be detected reliably using RT-PCR or FISH analysis. The t(12;21) defines a distinct subgroup of patients characterized by an age between 1 and 10 years, B lineage immunophenotype (CD10+, CD19+), and a favorable outcome.27,30 It is not seen in T cell ALL and is uncommon in adults (approximately four percent of ALL cases). In a recent series, patients with the t(12;21) had a 5-year EFS of 91 percent, as compared with 65 percent for patients without this rearrangement. One-half of these patients would have fallen into a high-risk group using standard risk factors; thus, the t(12;21) may identify a subset of patients within the high-risk group who would benefit from well-tolerated, less toxic antimetabolite therapy.
Hyperdiploidy The leukemia cells of some patients with ALL are characterized by a gain of many chromosomes. Two distinct subgroups are recognized: a group with one to four extra chromosomes (47–50), and the more common group with >50 chromosomes. Chromosome numbers usually range from 51 to 60, and a few patients may have up to 65 chromosomes. Hyperdiploidy (>50 chromosomes) is common in children (~30%) but is rarely observed in adults (<5%). Certain additional chromosomes are common (X chromosome and chromosomes 4, 6, 10, 14, 17, 18, and 21). Chromosome 21 is gained most frequently (100 percent of cases).2,4 Patients who have hyperdiploidy with >50 chromosomes have all of the previously recognized clinical factors that indicate a good prognosis, including age between one and nine years, low WBC count (median 6,700/µl), and favorable immunophenotype (early pre-B or pre-B).27,28 In a recent analysis of 186 children with hyperdiploidy ALL, it was observed that ALL defined by 51 to 55 chromosomes and ALL defined by 56 to 65 chromosomes may be distinct clinical entities.31 The 105 patients in the first group (51 to 55 chromosomes) had an EFS at 5 years of 72 percent, compared with 86 percent (P = 0.04) for those patients with >56 chromosomes (63 patients).
The 1;19 and 8;14 Translocations The t(1;19)(q23;p3) has been identified in about 25 percent of patients with a pre-B phenotype; that is, the leukemia cells have cytoplasmic immunoglobulin and are CD10+ (Fig. 10-2).27,30 A reciprocal translocation involving the long arms of chromosomes 8 and 14 [t(8;14)(q24;q32)] is observed in B cell ALL (L3) (see Fig. 10-2).2 These patients have a high incidence of CNS involvement and/or abdominal nodal involvement at diagnosis. Although the outcome for both children and adults with a t(8;14) has been poor, the use of high intensity chemotherapy has markedly improved the outcome (EFS of 80 percent in children).27
T CELL ACUTE LYMPHOBLASTIC LEUKEMIA
A distinct pattern of recurring karyotypic abnormalities in T cell neoplasms has emerged.2,29 Rearrangements involving 14q11 (see Fig. 10-2) and two regions of chromosome 7 (7q34–35 and 7p15) are particularly frequent in T cell malignancies (see Table 10-5). The most common are the t(11;14) (p13;q11) (~3%), t(10;11)(q24;q11) (~3%), and t(7;9)(q34–35;q34) (~2%).1,3,29 In addition to their occurrence in T cell leukemia, these abnormalities have also been observed in lymphomas of T cell origin. The genes that are located at the breakpoints of a number of these abnormalities have been identified (see also Table 10-5). Patients with T cell ALL are most often young males and often have a mediastinal tumor mass, high WBC count, and leukemia cells in the cerebro-spinal fluid. These same clinical characteristics are associated with lymphoblastic lymphoma, another T cell malignancy.
CHRONIC LYMPHOCYTIC LEUKEMIA
Unfortunately, only half of CLL patients have an adequate number of metaphase cells in unstimulated cultures for thorough evaluation. Trisomy 12 is the most common cytogenetic abnormality reported in patients with B cell CLL; it is found in 20 to 60 percent of those with a cytogenetic abnormality.32 Abnormalities involving band 14q32 are also common, e.g., t(14;19)(q32;q13) (see Table 10-5). FISH is a sensitive alternative method for detecting abnormalities in interphase CLL cells. Using FISH, 30 percent of patients are found to have trisomy 12, and this abnormality is associated with a poorer survival.32 Similarly, a del(13q) can be detected in 30 percent of CLL cases using FISH. T cell CLL and large granular lymphocytic leukemia are uncommon disorders in which the malignant mature lymphocytes have a T cell immunophenotype. Rearrangements involving band 14q11, with or without an accompanying break in 14q32, have been reported in T-CLL as well as in T cell lymphomas (Table 10-5).1,2 and 3 The most common of these rearrangements is an inv(14)(q11q32) (see Fig. 10-2).
LYMPHOMA
Cytogenetic analyses of lymphoma have been reported in a number of large series.2,4,33,34 and 35 These investigations have demonstrated that over 90 percent of cases are characterized by clonal chromosomal abnormalities and, more importantly, many of the recurring abnormalities correlate with histology and immunophenotype (see Table 10-5). For example, the t(14;18) is observed in a high proportion of follicular small cleaved cell lymphomas (70–90%), most patients with a t(3;22)(q27;q11) or t(3;14)(q27;q32) have diffuse large cell lymphomas (B cell), and patients with a t(8;14)(q24;q32) have either small noncleaved cell or diffuse large cell lymphomas (DLCL). Band 14q32, the location of the Ig heavy chain gene (IGH) is frequently involved in translocations in B cell neoplasms (~70%). In contrast, a large proportion of T cell neoplasms are characterized by rearrangements that involve 14q11, 7q34–35, or 7p15, the locations of the T cell receptor genes.
In 1972 a consistent abnormality (14q+) was identified in the cells of fresh Burkitt lymphomas (BL) and in cultured cell lines. Several years later the rearrangement was shown to be a reciprocal translocation involving chromosomes 8 and 14, t(8;14)(q24;q32) (see Fig. 10-2). The t(8;14) is characteristic of both endemic and nonendemic Burkitt tumors, as well as Epstein-Barr virus (EBV) negative and EBV-positive tumors. The t(8;14) has also been observed in other lymphomas, particularly small noncleaved cell (non-Burkitt) and large-cell immunoblastic lymphomas, AIDS-associated BL (100%), and AIDS-related DLCL (30%).33,34 and 35 As additional Burkitt tumors were examined, it became apparent that at least two other related translocations occur, t(2;8)(p12;q24) and t(8;22)(q24;q11). All three translocations involve chromosome band 8q24. As discussed earlier, these same translocations have been seen in some patients with B cell ALL. The t(8;14) involves a break within the IGH locus on chromosome 14 and a break either 5′ or within MYC on chromosome 8, and it relocates the MYC coding exons to chromosome 14.5,33 MYC is a transcription factor that plays a role in a number of cellular processes including proliferation and apoptosis, and its oncogenic properties are due to its constitutive expression.
Between 70 and 90 percent of follicular lymphomas and 20 percent of diffuse B cell lymphomas have the t(14;18) (see Fig. 10-2), in which the BCL2 gene at 18q21 is juxtaposed to the IGH J segment, leading to the deregulated expression of BCL2.33,34 Other malignancies which overexpress BCL2 but do not harbor the t(14;18) include hairy cell leukemia and CLL. The BCL2 gene encodes a 26 kDa membrane protein that functions to increase cell survival (antiapoptosis). Thus, this class of oncogene contributes to the development of a neoplastic state by preventing programmed cell death, rather than by promoting proliferation.
The t(11;14) (q13;q32) is observed in a relatively new pathologic entity known as mantle cell lymphoma.36 In most series, the percentages of cases having the t(11;14) by molecular analysis have varied between 30 and 55 percent of cases, but these may be underestimates, as only the major breakpoint cluster regions have been looked for. Besides mantle cell lymphomas, the t(11;14) has also been reported in 3 percent of multiple myeloma and up to 20 percent of prolymphocytic leukemias.36 Mantle cell lymphomas are currently regarded as a poor prognostic group, with a median survival from diagnosis of three years. This translocation results in the activation of the cyclin D1 (CCND1) gene by the IGH gene.33 Interestingly, the CCND1 gene is located 100 to 130 Kb away from the breakpoint on 11q13. The D-type cyclins act as growth factor sensors, causing cells to go through the restriction start point of the cell cycle at G1 and committing them to divide.
The BCL6 gene was cloned from the recurring breakpoint at 3q27 in cells characterized by a t(3;22)(q27;q11), t(3;14)(q27;q32), or rarely t(2;3)(p12;q27).33 BCL6 rearrangements occur in 40 percent of DLCL and, in some series, up to 10 percent of follicular lymphomas. The translocations lead to the truncation of the BCL6 gene within the first exon or the first intron, substitution of its promoter sequences with an IG promoter, and deregulated expression. The BCL6 gene product is a 96 kDa nuclear protein that acts as a potent transcriptional repressor. It is predominantly expressed in the B cell lineage, particularly in mature B cells, but not in immature bone marrow precursors or the more mature plasma cell.
A number of recurring chromosomal abnormalities have been recognized in T cell leukemias and lymphomas (see Table 10-5). Similar to B cell neoplasms, in which rearrangements frequently involve the chromosomal bands containing the immunoglobulin gene loci, T cell neoplasms often have rearrangements involving band q11 of chromosome 14, the site of the T cell receptor a-chain and d-chain genes (TCRA, TCRD) or, less often, one of two regions of chromosome 7 (7q34–35 or 7p15) to which the T cell receptor b-chain (TCRB) and g-chain (TCRG) genes have been localized respectively.1,3 These translocations result from aberrant recombination events during V-D-J recombination. With few exceptions, the involved gene on the partner chromosome encodes a transcription factor, whose expression is deregulated or activated as a result of the rearrangement (see Table 10-5).3,4 A chromosomal rearrangement which brings an oncogene under the controlling influence of promoters or enhancers active for immunoglobulin synthesis in B cells or T cell–receptor synthesis in T cells may, as a consequence, impart a proliferative advantage to the affected cell and result in malignant clonal expansion.
A distinctive subtype of lymphoma, namely, Ki-1(CD30)-positive anaplastic large cell lymphoma (Ki-1+ ALCL) has been characterized during the past few years. Patients with Ki-1+ ALCL tend to be young, and they present with skin and/or lymph node infiltration by large, often bizarre lymphoma cells, which preferentially involve the paracortical areas and lymph node sinuses. The majority of such tumors express one or more T cell antigens, a minority express B cell antigens, and some express both T and B cell antigens (the null phenotype). A reciprocal translocation, t(2;5)(p23;q35), appears to be restricted to ALCL of either T cell or null phenotype and is present in a high percentage of these cases.33 This translocation has also been found in CD30+ primary cutaneous lymphomas.
CHAPTER REFERENCES

1.
Mitelman F: Catalog of Chromosome Aberrations in Cancer, 5th ed. Wiley-Liss, New York, 1994.

2.
Heim S, Mitelman F: Cancer Cytogenetics, 2d ed. New York, Wiley-Liss, 1995.

3.
Mitelman F, Mertens F, Johansson B: A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat Genet 15:417, 1997.

4.
Le Beau MM, Larson RA: Cytogenetics and neoplasia, in Hematology: Basic Principles and Practices, 3d ed, edited by R Hoffman, EJ Benz, SJ Shattil, B Furie, HJ Cohen, LE Silbersein, P McGlave. Churchill Livingstone, New York, 1998.

5.
Rabbitts T: Chromosomal translocations in human cancer. Nature 372:143, 1994.

6.
Look AT: Oncogenic transcription factors in the human acute leukemias. Science 278:1059, 1997.

7.
Brown MA: Tumor suppressor genes and human cancer. Adv Hum Genet 36:45, 1997.

8.
Le Beau MM: Fluorescence in situ hybridization in cancer diagnosis, in Important Advances in Oncology, edited by VT De Vita, S Hellman, SA Rosenberg. Lippincott, Philadelphia, 1993:29.

9.
Le Beau MM: One FISH, two FISH, red FISH, blue FISH. Nat Genet 12:341, 1996.

10.
ISCN: An International System for Human Cytogenetic Nomenclature, edited by F Mitelman. Karger, Basel, 1995.

11.
Rowley JD, Testa JR: Chromosome abnormalities in malignant hematologic diseases. Adv Cancer Res 36:103, 1982.

12.
Ganesan TS, Rassool F, Guo A-P, et al: Rearrangement of the bcr gene in Philadelphia chromosome-negative chronic myeloid leukemia. Blood 68:957, 1986.

13.
Gordon MY, Goldman JM: Cellular and molecular mechanisms in chronic myeloid leukemia. Br J Haematol 95:10, 1996.

14.
Witte ON: Role of the BCR/ABL oncogene in human leukemia. Cancer Res 53:485, 1993.

15.
Morel P, Hebbar M, Lai JL, et al: Cytogenetic analysis has strong prognostic value in de novo myelodysplastic syndromes and can be incorporated in a new scoring system: a report on 408 cases. Leukemia 7:1315, 1993.

16.
Greenberg P, Cox C, Le Beau MM, et al: International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89:2079, 1997.

17.
Boultwood J, Lewis S, Wainscoat JS: The 5q– syndrome. Blood 84:3253, 1994.

18.
Fourth International Workshop on Chromosomes in Leukemia. Cancer Genet Cytogenet 71:249, 1984.

19.
Mrozek K, Heinonen K, de la Chapelle A, et al: Clinical significance of cytogenetics in acute myeloid leukemia. Sem Oncol 24:17, 1997.

20.
Caligiuri MA, Strout MP, Gilliland DG: Molecular biology of acute myeloid leukemia. Semin Oncol 24:32, 1997.

21.
Nucifora G, Rowley JD: AML1 and the 8;21 and 3;21 translocations in acute and chronic myeloid leukemia. Blood 86:1, 1995.

22.
Pandolfi PP: PML, PLZF, and NPM genes in the molecular pathogenesis of acute promyelocytic leukemia. Haematologica 81:472, 1996.

23.
Rowley JD: Rearrangements involving chromosome band 11q23 in acute leukemia. Semin Cancer Biol 4:377, 1993.

24.
Thirman M, Larson RA: Therapy-related myeloid leukemia. Hematol Oncol Clin North Am 10:293, 1996.

25.
Zhao N, Stoffel A, Wang PW, et al: Molecular delineation of the smallest commonly deleted region of chromosome 5 in malignant myeloid diseases to 1–1.5 Mb and preparation of a PAC-based physical map. Proc Natl Acad Sci USA 94:6948, 1997.

26.
Pedersen-Bjergaard, Rowley JD: The balanced and the unbalanced chromosome aberrations of acute myeloid leukemia may develop in different ways and may contribute differently to malignant transformation. Blood 83:2780, 1994.

27.
Camitta BM, Pullen J, Murphy S: Biology and treatment of acute lymphoblastic leukemia in children. Semin Oncol 24:83, 1997.

28.
Bloomfield CD, Goldman AI, Alimena G, et al: Chromosomal abnormalities identify high-risk and low-risk patients with acute lymphoblastic leukemia. Blood 67:415, 1986.

29.
Thandla S, Aplan PD: Molecular biology of acute lymphocytic leukemia. Sem Oncol 24:45, 1997.

30.
Rubnitz JE, Downing JR, Pui C-H, et al: TEL gene rearrangement in acute lymphoblastic leukemia: a new genetic marker with prognostic significance. J Clin Oncol 15:1150, 1997.

31.
Raimondi SC, Pui C-H, Hancock ML, et al: Heterogeneity of hyperdiploid (51–67) childhood acute lymphoblastic leukemia. Leukemia 10:213, 1996.

32.
Döhner H, Stilgenbauer S, Fischer K, et al: Cytogenetic and molecular cytogenetic analysis of B cell chronic lymphocytic leukemia: specific chromosome aberrations identify prognostic subgroups of patients and point to loci of candidate genes. Leukemia 11:S19, 1997.

33.
Ong ST, Le Beau MM: Chromosomal abnormalities and molecular genetics of non-Hodgkin’s lymphoma. Sem Oncol 25:447, 1998.

34.
Fifth International Workshop on Chromosomes in Leukemia-Lymphoma: Correlation of chromosome abnormalities in non-Hodgkin’s lymphoma and adult T-cell leukemia lymphoma. Blood 70:1554, 1987.

35.
Offit K, Wong G, Filippa DA, et al: Cytogenetic analysis of 434 consecutively ascertained specimens of non-Hodgkin’s lymphoma: Clinical correlations. Blood 77:1508, 1991.

36.
Raffeld M, Jaffe ES: bcl-1, t(11;14), and mantle cell-derived lymphomas. Blood 78:259, 1991.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

CHAPTER 9 GENETIC PRINCIPLES AND MOLECULAR BIOLOGY

CHAPTER 9 GENETIC PRINCIPLES AND MOLECULAR BIOLOGY
Williams Hematology

CHAPTER 9 GENETIC PRINCIPLES AND MOLECULAR BIOLOGY

ERNEST BEUTLER

The Pattern of Inheritance

X Linkage and X Inactivation

Mitochondrial Inheritance
The Family History
Linkage
DNA and the Genetic Code
Gene Expression

Transcription

RNA Processing

Translation
Regulation of Gene Expression

Normal Regulatory Processes

Experimental Interference with Gene Expression
The Methods of Molecular Biology

Cloning DNA

The Polymerase Chain Reaction

Cutting DNA with Restriction Endonucleases

Sequencing

Detecting Mutations in Individual Patients

Transgenic Animals

Gene Duplication

Pseudogenes

Types of Mutations

Mutation Nomenclature

Genotype-Phenotype Correlations
Chapter References

The understanding of hematology is more than ever dependent upon an appreciation of genetic principles and the tools that can be used to study genetic variation. All of the genetic information that makes up an organism is encoded in the DNA. This information is transcribed into RNA, and then the triplet code of the RNA is translated into protein. Mutations that change the DNA code, either present in the germline or acquired after birth, can cause a variety of hematologic disorders. A variety of changes in DNA occur, including single base changes, deletions, and insertions. The detection of defined mutations that cause a variety of diseases is now possible and has become a routine method for the diagnosis of some disorders, particularly prenatally. Inheritance patterns depend upon the characteristics of the disorder and the chromosomal location of the mutation. Common autosomal recessive hematologic diseases include sickle cell disease, the thalassemias, and Gaucher disease. Hereditary spherocytosis, thrombophilia due to factor V Leiden, most forms of von Willebrand disease, and acute intermittent porphyria are characterized by autosomal dominant inheritance. Mutations that cause glucose-6-phosphate dehydrogenase deficiency, hemophilia A and B, and the most common form of chronic granulomatous disease are all carried on the X-chromosome and therefore manifest sex-linked inheritance, with transmission of the disease state from mother to son. Understanding of the genetics of a disorder is necessary for accurate genetic counselling.

Acronyms and abbreviations that appear in this chapter include: BAC, bacterial artificial chromosome; G-6-PD, glucose-6-phosphate dehydrogenase; PAC, P1-derived artificial chromosome; PCR, polymerase chain reaction; poly(A), polyriboadenylic acid; RFLP, restriction fragment length polymorphism; RT, reverse transcription; YAC, yeast artificial chromosome.

Many of the hematologic diseases described in this text have a genetic basis. Often the disease is caused by a mutation in a single gene. Some of these disorders, such as sickle cell disease (Chap. 47), thalassemia (Chap. 46), glucose-6-phosphate dehydrogenase deficiency (Chap. 45), and factor V Leiden (Chap. 127) are extremely common. Others, such as congenital dyserythropoietic anemia type I (Chap. 35), chronic granulomatous disease (Chap. 72), or afibrinogenemia (Chap. 124) are rare, but all are due to mutations in a gene that results in the formation of a defective protein or an insufficient amount of a normal protein. The principal focus of this chapter is such genetic disorders. However, a number of acquired hematologic diseases, including lymphomas, leukemias, and paroxysmal nocturnal hemoglobinuria, are now understood to result from damage to the genetic apparatus that is not inherited but rather occurs in a cell at some time during the lifetime of the patient. Understanding these diseases requires an appreciation of how the genetic apparatus functions.
All of the information required for the development of a complete adult organism is encoded in the DNA of a single cell, the zygote. This information, designated the genome, includes the data needed for the synthesis of all enzymes; of all the plasma proteins, including clotting factors, complement components, and transport proteins; of all the membrane proteins, including receptors; and of all of the cytoskeletal proteins. The units of information into which the genome is organized are the genes. Some of these genes direct the formation of ribosomal RNA and of proteins that regulate the function of genes. The remainder encode the proteins involved in the structure and function of the body. Genetic diseases are the result of changes, or mutations, in these genes.
THE PATTERN OF INHERITANCE
The inheritance of each genetic disease follows a distinctive pattern. The concept of dominant and recessive inheritance is one of the most deeply ingrained in our genetic thinking. It has long played a primary role in the introduction of every high school student of biology to genetics and is used extensively in the classification of genetic disease. A dominant disease is one that is expressed when the patient has only a single copy of the mutant gene, i.e., in the heterozygous state. A recessive disease, on the other hand, is expressed only when both copies of the gene are abnormal. If the mutations on both alleles are the same, as is the case with some very common diseases, and with some less common diseases when the parents are related, then the patient is said to be homozygous. If two different abnormal alleles have been inherited, then the patient is designated as being a compound heterozygote (or less accurately, a mixed heterozygote or double heterozygote). It is often implied that genes are dominant or recessive. This is incorrect. It is disease states, or phenotypes, that are dominant or recessive. The gene for sickle cell hemoglobin is expressed in the heterozygous state, so that the carrier of this gene has sickle cell trait. Sickle cell trait is therefore dominant, but sickle cell disease, which occurs in the homozygote, is recessive. By definition, the heterozygous phenotype of a recessive disease does not differ from the homozygous normal state, but it can usually be identified by biochemical means.
X LINKAGE AND X INACTIVATION
The principles of dominant and recessive disease can be readily applied to mutations occurring on the autosomes (chromosomes other than the X chromosome), but the situation is somewhat different in the case of genes on the X chromosome. Although the X chromosome is involved, at least indirectly, in the sex determination process, most of the genes on the X chromosome have nothing whatsoever to do with sex determination. Hematologically, some of the more important of these “sex-linked” genes include those which code for G-6-PD, phosphoglycerate kinase, factor VIII, factor IX, Bruton-type agammaglobulinemia, and one form of chronic granulomatous disease.
The chromosomal complement of males differs from that of females in that males have one X chromosome and one Y chromosome, while females have two X chromosomes. However, early in embryonic development one of the two X chromosomes of somatic cells of female mammals becomes genetically inactive: in some cells the paternally derived chromosome is inactivated; in others, the maternally derived chromosome is inactivated.1,2 Inactivation remains fixed, so that all the progeny of the cell in which the maternally derived X chromosome is inactive show only the gene products from the paternal X. Female heterozygotes for sex-linked genes such as G-6-PD deficiency, phosphoglycerate kinase deficiency, factor VIII deficiency, or factor IX deficiency are therefore a mosaic of cells, some of which manifest the full-blown deficiency, as it is found in affected males, and some of which are normal. The final proportion of cells with one or the other X chromosome active depends upon random factors, i.e., the binomial probability distribution, and on selection between cell populations, which may occur following the inactivation process.3,4 The process of X inactivation is not only useful in understanding the expression of X-linked diseases in women but has been valuable in studying the possible clonal origin of a variety of disorders. As shown in Fig. 9-1, the progeny of a single cell of a female heterozygous for an X-linked gene will manifest only the phenotype of the original cell. Examination of electrophoretically distinguishable variants of G-6-PD has made it possible to demonstrate in this way that the red cells are a clone in chronic myelogenous leukemia,5 in paroxysmal nocturnal hemoglobinuria,6 and probably in acute myelogenous leukemia.7,8 This indicates that each of these disorders arises through transformation of a single cell and that in the case of the leukemias erythroid cells as well as leukocytes are part of the malignant clone.

FIGURE 9-1 At fertilization, the female zygote inherits one maternal chromosome (Xm) and one paternal X chromosome (Xp). At some time early in embryogenesis, one X in each cell is inactivated at random and condenses to form the Barr body. The active X remains active not only for the lifetime of that cell but for the lifetime of all of its progeny. A tumor with a clonal origin will consist entirely of cells in all of which either Xm or Xp are active. A tumor with a multicentric origin may contain both Xm and Xp cells.

With the development of DNA-based technology it has been possible to use X-linked genes as a clonal marker even when there is not a different protein product from the two alleles. A different pattern of methylation of cytidines distinguishes the active from the inactive X-chromosome.9 This fact, together with the existence of restriction endonucleases that distinguish methylated from unmethylated cytidine, has made it possible to utilize restriction fragment length polymorphisms to determine the clonal origin of neoplasms,10,11 even when no polymorphism involving an X-linked enzyme is available. The existence of polymorphisms involving the coding region of genes also makes possible the detection of clones by reverse transcription and amplification of mRNA.12,13
The pattern of genetic transmission of sex-linked genes is characteristic: a father cannot transmit a sex-linked gene to his son, since the offspring is a boy by virtue of the fact that he inherited the father’s Y chromosome, not his X chromosome. Conversely, it is a truism that males always inherit sex-linked genes from their mother and that the mother must therefore be either heterozygous or homozygous for the gene. Because X inactivation is random, however, the degree of expression of X-linked genes in females is highly variable. This is why, even with the most sophisticated methodology, it is not always possible to detect the heterozygous state in the mother of an affected individual. It also explains why even identical twin carriers of diseases such as factor VIII deficiency can have very different levels of the clotting factor.
MITOCHONDRIAL INHERITANCE
The vast majority of the genetic material in cells is encoded in the chromosomal DNA. However, mitochondria have their own replicating DNA. Apparently having arisen from symbiotic bacteria over a billion years ago, the DNA of mitochondrial DNA (mtDNA) exists as a closed circular molecule of 16,569 nucleotides. This DNA encodes 13 polypeptides, all of which are subunits of the mitochondrial energy-producing pathway; a small and a large ribosomal RNA; and 22 transfer RNAs.14 Some proteins found in mitochondria are, however, encoded in nuclear DNA. Mitochondria are transmitted through the egg; thus, inheritance is entirely maternal.15,16 Cells contain several hundred mitochondria, each with its own circle of mtDNA. To become clinically significant, mitochondrial mutations must confer some selective advantage upon the mitochondrion with the mutation; mutations that affect only a few of the hundreds of mitochondria in each cell are unlikely to produce a phenotype. Mitochondrial mutations, often consisting of deletions, are responsible for a number of neurologic diseases.15 Some of the childhood myelodysplastic syndromes,17,18 particularly Pearson marrow-pancreas syndrome,19 are hematologic manifestations of mitochondrial mutations.
THE FAMILY HISTORY
A carefully taken family history can give a physician considerable insight into the nature of a hematologic disorder. One should ascertain whether another member of the family has had a similar disease. In the case of patients with anemia, this is often difficult, since so many women have a history of anemia, usually due to iron deficiency. To estimate the severity of anemia it is particularly germane to inquire whether transfusion was required. A history of gallstones, particularly at an early age, may indicate that a hemolytic disorder was present. Similarly, episodes of jaundice in family members may be the only clue to the existence of familial hemolytic anemia.
Presence of the disease in one of the parents strongly suggests a dominant mode of transmission. If neither parent is affected, but several siblings have the disease, an autosomal recessive transmission is more likely. Consanguinity of the patient’s parents makes it highly probable that a disease is an autosomal recessive disorder. Occurrence primarily in male siblings and maternal uncles, with mild or absent manifestations of the disease in the mother, suggests a sex-linked mode of inheritance. Father-to-son transmission rules out sex linkage.
Lack of any family history does not rule out the genetic basis of a disease. In some instances the disease may be so mild in other family members that it is not recognized. Whenever possible, the physician should examine the family members, rather than relying solely on history. In some instances, of course, the gene mutation causing the disorder may have arisen in the generation in which the disease presents.
Once the mode of genetic transmission is clear, the diagnostic alternatives have been narrowed considerably. For example, methemoglobinemia transmitted as an autosomal dominant disorder is due to hemoglobin M, while methemoglobinemia transmitted as an autosomal recessive disorder is due to NADH diaphorase deficiency. Hemolytic anemia with autosomal dominant transmission is likely to be due to hereditary spherocytosis, but sex-linked transmission of the hemolytic state suggests a deficiency of G-6-DP or, more rarely, phosphoglycerate kinase. A bleeding disorder that is transmitted in a sex-linked fashion may be due to a deficiency of factor VIII or factor IX, but autosomal recessive inheritance should suggest to the physician a deficiency of other clotting factors, such as X, XI, or V. Careful analysis of the family history not only will make possible more appropriate genetic counseling to the patient and family but also will shorten the road to a correct diagnosis.
LINKAGE
In human somatic cells chromosomes are present in pairs—one pair of sex chromosomes (two X chromosomes in females and an X and a Y in males) and 22 pairs of autosomes. One chromosome of each pair is distributed into the gametes, so that eggs and sperm of humans each contain 23 chromosomes.
If two genes are located on different chromosomes or are far apart on the same chromosome they are said to be unlinked: the offspring of a carrier of these two genes has one chance in two of inheriting either of the genes, and the probability of inheriting one or the other, both, or neither is governed by the laws of chance. For example, if a woman is a carrier of pyruvate kinase deficiency and of sickle cell trait, two genes that are on different autosomes, the probabilities of inheritance of pyruvate kinase deficiency, on the one hand, and sickle cell trait, on the other, are entirely independent. One-fourth the offspring will inherit both pyruvate kinase deficiency and sickle cell trait, one-fourth the offspring will inherit neither, one-fourth will inherit only sickle cell trait, and one-fourth will inherit only pyruvate kinase deficiency.
If the two genes in question are close together on the same chromosome, however, the situation may be quite different. For example, the genes for hemophilia A and for G-6-PD deficiency are both sex-linked. If a woman carries both these genes on one of her X chromosomes, the probability of her child’s inheriting either both of the abnormal genes or neither of the abnormal genes is much greater than the probability of its inheriting one or the other. Yet the inheritance of only one of these two genes is not an impossibility, because of the phenomenon of crossing-over during meiosis. In the course of the formation of germ cells, homologous pairs of chromosomes come into side-by-side apposition and regularly exchange chromosomal material. Thus, two genes that were originally on the same X chromosome may find themselves on separate chromosomes after germ cell formation (Fig. 9-2). The probability of their being separated during meiosis is a function of their distance from one another on the chromosome, and this distance is expressed in terms of map units, or morgans. One-hundredth of a morgan, a centimorgan, represents the genetic distance that gives a one percent probability per generation of a crossover between the two genes. A rule of thumb is that this corresponds to a physical distance of 1,000,000 base pairs, but the actual physical distance represented by a centimorgan varies a great deal from one location in the genome to another; the tendency to cross over varies greatly from place to place. It is not unusual for genes on the same chromosome to be so far apart that the probability of finding them in separate germ cells is just as great as though they had been on separate chromosomes. For this reason, genes on the same chromosome may be linked but may also be unlinked; in the latter case they are referred to as syntenic. G-6-PD and hemophilia A are both on the X chromosome, with a map distance estimated at approximately 0.04 morgans, or 4 centimorgans.20 Therefore, if two mutant genes at this locus are on the same X chromosome in a female, there is a four percent chance of the genes being in separate gametes. The genes for both G-6-PD and the Xg blood group are also on the X chromosome, but are apparently unlinked.21

FIGURE 9-2 Schematic representation of equal crossing-over during meiosis. There has been an exchange of chromosomal material between the maternally derived and paternally derived chromosome, but all genes are represented on the products of the crossover.

DNA AND THE GENETIC CODE
Understanding how the massive amount of information required to allow a complex organism to grow and survive is coded has been one of the major advances of modern biology. The information is all contained in polynucleotides, deoxyribonucleic acid (DNA). DNA contains only four different bases—adenine (A), guanine (G), thymine (T), and cytosine (C). DNA exists as a double helix in which A is always paired with T, and G is always paired with C.
The two ends of a strand of DNA are not the same. The nucleosides that make up each strand are linked to each other through a molecule of phosphoric acid attached to the 3′ carbon of the deoxyribose of one nucleoside and to the 5′ carbon of the next one. A linear strand of DNA thus has one end in which the hydroxyl group attached to the 5′ carbon is free; at the other end it is the hydroxyl group attached to the 3′ carbon that is not involved in a link. These ends are designated the 5′ and 3′ ends respectively, and by convention the 5′ end is drawn at the left and is called the “upstream” end. The 3′ end, then, is designated as “downstream”. In the pairing of two complementary strands of DNA the polarity of the two strands is opposite, i.e., the 5′ end of each strand is paired with the 3′ end of the other. By convention, the strand shown at the top is the coding, or “sense” strand, but the strand at the bottom is the one that actually serves as a template for RNA synthesis. Thus, the sequence of the mRNA is that of the top strand, and the triplet code may be read from this strand.
It is the faithful pairing of A with T and C with G in double-stranded DNA that makes possible the accurate replication of the genetic code. When cells divide, the two DNA strands separate. As this occurs the bases of the separate strands pair with the complementary purine or pyrimidine nucleotide, which become linked to each other, forming a complementary strand of nucleotides. In this way the cell forms two double strands that are identical with the original double strand.
The sequence of base pairs in the DNA strand specifies the sequence of amino acids in proteins. Each base cannot represent a single amino acid, since only four bases are found in DNA and there are 20 commonly occurring amino acids in proteins. Similarly, pairs of bases are not sufficient; they could code for only 16 amino acids. A triplet code is therefore the minimum number of bases that is required to code for 20 amino acids. The genetic code has been found in fact to consist of triplets: each amino acid is specified by one or more sequences of three bases. Long stretches of the triplet code are colinear with the amino acid sequence of the protein the synthesis of which the gene specifies, but these stretches are separated by intervening sequences, or introns, that do not code for the amino acid sequence of the protein (see Fig. 47-2). Moreover, DNA does not directly assemble amino acids into protein. This is achieved through a mechanism that involves another polynucleotide, ribonucleic acid (RNA). There are two differences between DNA and RNA. First of all, the nucleotide units contain ribose instead of deoxyribose. Secondly, in RNA uridine (U) is used instead of the thymidine (T) component of DNA. Messenger ribonucleic acid (mRNA) is synthesized with a base sequence determined by the nuclear DNA, which serves as a template in a copying process that is designated as transcription.
GENE EXPRESSION
TRANSCRIPTION
The transcription of DNA into mRNA is the first step in gene expression. In order for a gene to be transcribed a promoter must be located “upstream” (i.e., in the 5′ direction) from the DNA. Typical promoters have certain sequences in common. These include a “CAT box,” the cytosine- and guanine-rich CCAAT sequence, and a “TAATA box,” an adenine- and thymine-rich sequence. Mutations in these regions impair transcription of a gene; such lesions have been identified as causes of the thalassemias and are discussed in greater detail in Chap. 46. The effectiveness of a promoter may be increased by more distant DNA sequences, known as enhancers, which may be either upstream or downstream from the gene. The identification of sequences that enhance expression of the globin genes has been of particular importance in designing vectors for gene transfer to remedy the hemoglobinopathies22,23 (see Chap. 19).
RNA PROCESSING
The mRNA that is formed on the DNA template by RNA polymerase is not ready to be translated to a polypeptide. First it must be processed, by adding a cap to the 5′ end and a poly-A tail to the 3′ end and by removing introns. Capping consists of formation of an atypical 5′ to 5′ triphosphate bond between the 5′ terminus of the mRNA and a molecule of 7-methylguanosine. The addition of a poly-A tail serves to stabilize the mRNA. Recognition of a sequence (AAUAAA) serves as a signal that a poly-A tail should be added at a point that is approximately 15 bases downstream from the signal when another consensus sequence, YGUGUUYY (where Y stands for a pyrimidine, i.e. uridine or cytidine), is present further downstream. Sometimes more than one adenylation signal is present, and then additional species of mRNA with 3′ portions differing in length are formed.
Excision of introns is particularly important, since they interrupt the coding sequence. The first 5′ bases of the intron are always GpU and the last 3′ bases always ApG (the p represents the phosphate bond between the nucleotides). But there are many such couplets in the RNA, and additional information is required for an actual splice site to exist. The nature of this information has not been clearly defined, but a “consensus” sequence has been defined that most splice sites resemble closely. Removal of the intron is a complex enzymatic process involving the prior formation of a “lariat” structure.24 Splicing of a given normal mRNA does not always occur in the same manner. Sometimes “alternative splicing” occurs, so that after mRNA is processed some of the molecules contain an exon that is missing from other messenger molecules. This is a powerful mechanism that allows a single gene to direct the synthesis of more than one polypeptide. Potentially the type of polypeptide made can be modulated according to need, and different tissues and different developmental stages may utilize different splice sites to make tissue-specific polypeptides. Alternative splicing has been important, for example, in producing different forms of erythrocyte membrane band 4.125 and different forms of pyruvate kinase for the liver and for the erythrocyte.26
TRANSLATION
Processed mRNA contains the code for the synthesis of proteins, and an elaborate mechanism has evolved for the translation of the triplet code in the mRNA into protein. A ribosomal complex, consisting of ribosomal RNA (rRNA) subunits and protein components, attaches to the 5′ end of the mRNA. The transport of the needed amino acids to the ribosomal complex is achieved by clover-shaped RNA molecules designated transfer RNA (tRNA). tRNA molecules contain a recognition site which binds to a triplet on mRNA and a site that carries the amino acid appropriate for that triplet to the mRNA, where the ribosomal complex creates the peptide bond between it and the amino acid that is immediately 5′ to it. The initiation of protein synthesis is always at a AUG codon, usually one quite near the 5′ end of the messenger RNA. A consensus sequence27 around this codon marks it for the starting point of protein synthesis. The ribosome moves down the mRNA, adding amino acids to the nascent protein chain as it goes, until it reaches a termination codon, which serves as the signal to stop protein synthesis. The ribosome is then released and can begin the synthesis of another protein molecule. This complex process requires the presence of initiation factors (IF-1 through IF-6) and elongation factors (EF-1 through EF-3), as well as a releasing factor (RF). Both ATP and GTP are required.28 The cycle through which the peptide is formed on the ribosome is illustrated schematically in Fig. 9-3.

FIGURE 9-3 The elongation of a polypeptide as the ribosome moves down the mRNA. Each amino acid (aa) is added to the preceding one by the coordinated activity of elongation factors (EF). From Merrick,28 by permission.

Since the initiation codon AUG codes for methionine, the amino terminus of the primary translated protein is always a methionine, but this is usually cleaved from the protein during processing. Modification of the protein may include changes such as the removal of a leader sequence that directs the protein to a membrane, the addition of sugars to glycoproteins, the addition of fatty acids, and the formation of internal sulfhydryl bonds.
REGULATION OF GENE EXPRESSION
NORMAL REGULATORY PROCESSES
Many genes are highly specialized in their function. Hemoglobin is made only by erythrocyte precursors, crystallin only by the lens, and immunoglobulins only by lymphoid cells. Such genes must be silenced in other types of cells. On the other hand, so-called housekeeping genes produce their products in all cells. The latter include the enzymes of the basic metabolic processes that provide energy to all cells, such as hexokinase, phosphoglycerate kinase, and G-6-PD, or that provide basic structural proteins.
Clearly, an elaborate system for the regulation of protein production exists in all organisms, and this system is only beginning to be understood. Regulation of transcription determines to a large extent whether a protein will be synthesized.29 Promoters and enhancers are activated by transcription factors that are produced by the cell. Such factors, in turn, may be activated or inactivated by phosphorylation and by other processes. How enhancers act at a distance to increase the activity of promoters is not well understood, and the locus control region of the globin genes is serving as a paradigm in gaining understanding of possible interactions between transcription factors, enhancers, and promoters. Regulation also occurs at the translational level. The mRNA of ferritin contains an iron-responsive element that binds to a 87-kDa regulatory protein in the absence of iron, effectively shutting off translation.30 The same type of binding site in the 3′ untranslated region of the transferrin receptor mRNA serves to stabilize the message by allowing the protein to bind in the absence of iron.30 Similarly, a UA-rich portion in the 3′ untranslated portion of the tumor necrosis factor gene serves to inhibit translation of that mRNA.31 It is also likely that the stability of the mRNA itself is regulated by nucleases.32,33
EXPERIMENTAL INTERFERENCE WITH GENE EXPRESSION
It is possible to interdict the expression of a gene at several different levels. Genes can be interrupted in murine embryonic stem cells by the process of targeted disruption, destroying their function.34,35 The resulting “knockout mice” (a subset of transgenic mice, see “Transgenic Animals,” below) can provide valuable insights into the function of genes and serve as animal models of human disease (see Chap. 10).
The translation of mRNA can be inhibited and the RNA degraded by placing antisense RNA or DNA into cells. These molecules have a sequence complementary to the mRNA that is to be inactivated. When such oligonucleotides are present they inhibit gene expression through a variety of mechanisms. For example, they form a double strand with the RNA, just as two complementary strands of DNA will hybridize to form the normal double-stranded form of DNA. Because the double-stranded form cannot be translated and is probably degraded rapidly, the production of its protein product is inhibited specifically. Since antisense RNA can be produced in vivo by transcribing the complementary strand of a gene, it may represent a natural regulatory mechanism.36,37 and 38 In experimental systems, antisense DNA or stable DNA analogs such as the methyl phosphonates39 can be transfected directly into cells, or the RNA can be made by a plasmid with the appropriate DNA template and a promotor. Some of the uses of this approach include the suppression of lymphoma growth with DNA oligonucleotides antisense to introns of the oncogene c-myc,40 the suppression of the growth of marrow cells from patients with chronic myelogenous leukemia by antisense DNA directed at the BCR-ABL junction,41 the down-regulation of growth of BCL-2-positive lymphoma cells in culture by BCL-2 antisense,42 and the inhibition of Friend murine erythroleukemia cell growth by transfection with a plasmid that produces antisense to c-jun.43
The discovery of the enzymatic activity of certain forms of RNA represents a major advance in our understanding of how life may have originated on earth. Cleaving RNA at defined sequences, much as restriction endonucleases cleave DNA, is one of the known enzymatic functions of RNA, and this function provides a means by which the expression of a gene can be interdicted in experimental systems. This ribozyme approach has been used, for example, in preventing replication of the HIV-1 virus44,45 and for cleaving BCR-ABL with a view to developing a treatment for chronic myelogenous leukemia.46
THE METHODS OF MOLECULAR BIOLOGY
CLONING DNA
The sequencing of DNA and the preparation of probes requires that a fragment of DNA is amplified manyfold to provide a relatively pure sample for study. The classical method by which this is achieved, cloning, is one of the central techniques of molecular biology. It is generally accomplished by inserting the DNA into a vector, a bacteriophage or plasmid that normally replicates within a bacterial cell. When such a phage or plasmid contains a foreign DNA fragment, the fragment too undergoes replication and can then be purified in greatly amplified form.
If the DNA is not available in pure form to begin with it must be purified from a collection of DNA fragments that is designated a “library”. An adequate genomic library consists of millions of fragments of the genetic material of a cell that have been ligated into a suitable vector. Another valuable type of library is made by transcribing mRNA from a tissue into cDNA (“complementary” DNA) using the enzyme reverse transcriptase. Such a cDNA library is particularly useful for the isolation of genes because in it are represented only the intron-free portions of genes that are being actively transcribed in a tissue. In contrast, a genomic library represents all of the genetic material, coding and noncoding, transcribed and nontranscribed.
A large number of vectors that have the capacity to replicate fragments of DNA of widely differing sizes have been designed. The largest of these are yeast artificial chromosomes, which may incorporate a million or more base pairs of DNA into a vector that is grown in a yeast host.47,48 Such vectors are very useful in mapping genes because of their very large size, but there is a tendency for the DNA in YACs to be rearranged, which can lead to errors. Other vectors that also incorporate large fragments of DNA, ranging to about 100,000 bp in length, are bacterial artificial chromosomes, P1-derived artificial chromosomes, and cosmids (20,000 to 30,000 bp). Much smaller inserts, ranging in size from about 3,000 to 12,000 bp, can be cloned into bacteriophages. A library consisting of a large collection of the vector containing many different inserts is plated on a confluent layer (“lawn”) of micro-organisms; bacteria transfected with a plasmid library are plated on a semisolid culture medium. It is then necessary to detect the amplified wanted DNA fragment. If the exact sequence of at least 17 nucleotides is known, a probe consisting of a radioactively labeled synthetic complementary sequence can be used to detect the clone that is wanted. The precise base sequence cannot be deduced from the amino acid sequence, because there is more than one codon for most amino acids. However, if an appropriate portion of the amino acid sequence is selected, several different complementary sequences, encompassing all of the possibilities, may be used as probes.
Antibodies against the gene product may also serve as probes by using an “expression vector” in which a promotor is present upstream from the cloned DNA. When the fragment is in the correct orientation and when it is “in frame” so that the triplets are read correctly, sufficient gene product may be formed to allow immunologic detection. Colonies (or, in the case of phage vectors, plaques) that react with the probe are picked and subcultured at lower density until a single reactive colony or plaque is isolated.
THE POLYMERASE CHAIN REACTION
Amplification of the desired part of the genome may be achieved when some of the sequence is already known by using the polymerase chain reaction, a technique that is much simpler than cloning. For example, one may wish to determine the sequence of a portion of a gene for diagnostic purposes, but cloning the gene(s) of interest is too time-consuming and labor intensive to be practical. Two primers, matching opposite strands of DNA on either side of the region of interest, are used to amplify the intervening segment of DNA more than a millionfold. Successive cycles of DNA synthesis from the primers, and chain separation by heating between the cycles, are the basis of this powerful technique.49,50 The polymerase chain reaction is so sensitive that under optimal conditions the DNA from a single cell may be amplified. Moreover, the stability of DNA is such that very old preserved material may be used. Thus, it is possible to amplify the DNA from blood smears,51 from mummies, and even from insects preserved in amber for over 25 million years.52 Amplifying by PCR complementary DNA (cDNA) produced by reverse-transcribing mRNA in tissue extracts (RT-PCR) provides a very sensitive means for measuring the expression of genes in tissues.
CUTTING DNA WITH RESTRICTION ENDONUCLEASES
The discovery that many bacteria elaborate enzymes that cleave double-stranded DNA at the sites of very specific sequences greatly facilitated the study of DNA. Such enzymes generally recognize palindromes, i.e., DNA sequences that read the same in one direction on the upper strand and in the opposite direction in the lower strand. Fig. 9-4 illustrates how one such palindrome is cleaved by the commonly used restriction endonuclease Eco RI. Several hundred restriction endonucleases are now commercially available. Some recognize sequences of only four nucleotides and some as many as eight. The average size of fragments produced by the former is, of course, smaller than the average size of those produced by the latter.

FIGURE 9-4 A schematic representation of ECO R1 cleaving its recognition sequence, which is outlined by the rectangle. Whenever this restriction endonuclease encounters the palindromic sequence GAATTC, it cleaves DNA at the position shown by the arrows.

Restriction endonucleases are useful both for cloning DNA and for analyzing its structure. By digesting DNA with various endonucleases and combinations of endonucleases one may construct a restriction map, i.e., a linear representation of the fragment of DNA with the location of the various restriction sites that have been identified. Maps can be constructed from uncloned genetic DNA, provided that probes for the detection of the relevant fragments are available. Many of the restriction endonucleases produce fragments with overlapping ends (see, for example, Eco RI in Fig. 9-4). Such “sticky ends” may be used for the ligation (i.e., splicing) of DNA fragments into a vector by using a vector with complementary sticky ends. The seal is made permanent with the enzyme DNA ligase.
The size of restriction fragments produced after digesting whole genomic DNA with restriction endonucleases may be appreciated using the technique of Southern blotting, a useful procedure named after the investigator who developed it.53 The DNA is digested with one or more restriction endonucleases and then subjected to electrophoresis in a gel that separates fragments by size. It is then transferred to a membrane that binds DNA, and the appropriate DNA fragments are detected using labeled probes. Alternatively, the segment of DNA that is of interest may be amplified using the PCR technique and digested by a restriction endonuclease to determine whether or not target sites are present.
One of the most powerful uses of restriction endonucleases is in the detection of genetic variability. Changes in nucleotides may create or abolish restriction sites. Thus, they change the size of fragments that are formed when the DNA is digested. Such areas of variability represent restriction fragment length polymorphisms (RFLP). In some cases the changes in nucleotide sequence may be the ones that cause the disease itself. For example, the sickle cell mutation causes disappearance of a restriction site recognized by the enzyme Mst II,54 and the G-6-PD A– mutation causes formation of a restriction site recognized by Nla III; such changes have proved valuable in diagnosis (see Chap. 45 and Chap. 47).
Deletions of chromosomal material, as occur in a-thalassemia, also produce changes in fragment sizes. Larger fragments may appear if the deleted fragment contains a restriction site, or smaller fragments if it does not. If the area covered by the probe is deleted in its entirety, as occurs in hydrops fetalis, no band will be seen at all. Even when the lesion that causes the disease does not directly affect a restriction site, RLFPs may be valuable in disease detection by virtue of close linkage of the restriction site to a disease-causing gene. Multiple restriction sites near the gene of interest produce haplotypes that may unequivocally identify a chromosome. Such haplotypes have been particularly useful in the prenatal diagnosis of the thalassemias (see Chap. 46).
SEQUENCING
The chain termination technique55 is most commonly used to determine the sequence of DNA. It depends upon synthesizing a labeled strand of DNA, with the DNA to be sequenced serving as the template. The mixture of nucleotides used contains, in addition to the native deoxynucleotides, a nucleotide analog that results in chain termination when incorporated. The normal nucleotides are present in excess, and therefore chain termination occurs only sporadically, but always when the analog is incorporated. Four different incubation mixtures are used, each with an analog of one of the four nucleotides. Gel electrophoresis of the labeled products produces “ladders” of polynucleotides. The size of each fragment depends on the point at which there exists a nucleotide corresponding to the chain-terminating analog in the mixture (Fig. 9-5). Sequencing can now be carried out rapidly and accurately by automated methods.56

FIGURE 9-5 Radioautograph of a gel being used to determine the sequence of the glucocerebrosidase gene by the chain termination method. Four reaction mixtures are used. Each mixture contains a polynucleotide primer (Pr) that has a sequence complementary to the beginning of the strand to be sequenced, and all four normal deoxynucleoside triphosphates labeled with 32P. The “G” mixture also contains dideoxyguanosine triphosphate to act as a chain terminator when a guanine is reached. The “A” mixture contains the adenine chain terminator, and so on. Each mixture is placed in a slot: the “G” mixture in G, and so on. Upon electrophoresis the gel separates polynucleotides by size. Thus, the positions to which polynucleotides move in the gel correspond to the positions at which the indicated nucleotides are added to the end of the DNA strand as it is being synthesized. The sequence of the DNA can then be deduced. The apparent sequence of some of the bands are shown at the left.

While DNA sequencing formerly required cloning of the fragment to be studied, amplification by PCR serves as a simpler alternative when the surrounding sequences are known.57,58
DETECTING MUTATIONS IN INDIVIDUAL PATIENTS
The cloning and sequencing of DNA is too time-consuming to permit application for diagnostic purposes to individual patients. Fortunately, there are shortcuts that can be used when the nature of the lesion is known and a yes-or-no answer is sought with regards to the existence of a certain substitution. The value of restriction sites in this regard has been discussed above, but since many substitutions neither abolish nor create restriction sites the use of restriction endonucleases is not feasible in every case. However, a mismatch in one of the amplifying primers used in amplifying DNA by PCR, selected so as to create a restriction site where none existed before, is a technique that has been used successfully to detect mutations.59 Using amplifying primers that fit one genotype but not the other has been used in “color PCR”60 and in the amplification refractory mutation system (ARMS).61 The failure of fragments of DNA to ligate when aligned on a template in which there is a misfit of the terminal nucleotide also has been used to detect mutations.62,63 and 64 The hybridization of labeled oligonucleotide probes with a defined sequence to an amplified DNA target, but not to a DNA target harboring even a single nucleotide change, a method designated allele-specific oligonucleotide hybridization (ASOH), is also very useful.65,66 Probes containing approximately 17 nucleotides fitting either the normal or the mutant sequence are hybridized to PCR-amplified DNA. A single mismatch in an oligonucleotide of this size produces a sufficient change in melting temperature (i.e., the temperature at which the strands of DNA separate) that the two sequences can be distinguished from one another.
When the mutation is not known, other techniques may prove useful. Single-stranded conformation polymorphism (SSCP) analysis takes advantage of the fact that a single base substitution will usually change the conformation of single-stranded DNA and change its migration in a gel when it is subjected to electrophoresis. This technique has been found to be particularly powerful, revealing most mutations in segments of DNA between 200 and 400 bases in length.67,68 Alternatively single base mismatches may be detected by hybridizing mRNA with a known sequence to the DNA and cleaving the duplexes with ribonuclease,69 or by measuring the denaturation of mismatched double-stranded DNA (heteroduplexes) in a gradient.70
TRANSGENIC ANIMALS
The mechanical insertion of DNA fragments into the nucleus of a fertilized ovum provides a means for altering the genetic constitution of animals. Animals that have been engineered in this manner are referred to as transgenic. The use of promotors that are inducible or tissue specific permits studies of the effect of a gene product that might be lethal if expressed in all tissues or at all times during embryogenesis. Transgenic mice that carry the human sickle b-globin gene and in which the murine globin genes have been “knocked out” have been produced71,72 and produce high enough levels of human hemoglobin S to have potential as an animal model of sickle cell disease.
GENE DUPLICATION
Crossing-over during meiosis usually occurs with great precision. Homologous genes pair with each other, and although genes which were together on one chromosome before meiosis may now be on opposite chromosomes of the pair, each chromosome still contains a complete set of genes (see Fig. 9-2). Occasionally, however, an error occurs and pairing during meiosis is imperfect. Under these circumstances—unequal crossing-over (see Fig. 46-10)—one of the daughter chromosomes contains a duplicated gene, while the other one exists with a gene deleted.
Once a duplication has occurred, further duplications occur more readily because pairing of the first of the duplicate genes on one chromosome with the second gene of the duplicate on the other produces one chromosome with a triplicated gene and one with a single gene. Duplication has probably played a very important role in the course of evolution73 because the presence of two genes with the same function allows experiments of nature, mutations, to occur on one of the genes without totally losing the original function, which is still carried out by the duplicate. Examples of the results of gene duplication abound in hematology, particularly with respect to the hemoglobin loci. The a-chain loci are duplicated, and there are also two nearly identical copies of the g-chain locus (see Chap. 46). Furthermore, the close similarity of their amino acid sequence and the fact that they are tightly linked indicate that the b, g, and d loci represent the result of duplication of a single ancestral gene. The process of unequal crossing-over takes place not only between genes, but also within genes. When this occurs, one would anticipate that a portion of the amino acid sequence of a protein is represented twice on one chromosome and is missing on the other. The Lepore hemoglobins, leading to a thalassemic clinical state, are an example of this type of unequal crossing-over (see Fig. 46-6). These abnormal hemoglobins have the amino acid sequence of the d chain at the amino end, and the sequence of the b chain at the carboxyl end. The complement to this kind of abnormality, the “anti-Lepore” hemoglobin, also has been found (see Chap. 46). Similarly, a mutation of the glucocerebrosidase gene causing Gaucher disease has been found to be the result of a crossover between the active gene and the pseudogene.74
PSEUDOGENES
Pseudogenes are DNA sequences that resemble the corresponding functional genes, but do not result in the production of a gene product. Pseudogenes exist, for example, for the b-globin chain, von Willebrand factor, ferritin, and glucocerebrosidase. These pseudogenes apparently arose by gene duplication and simulate the true gene even in having introns. They have apparently lost their ability to function, through mutations either in the coding region or in their promoter. Some pseudogenes are devoid of introns. They may well have arisen in evolution as a result of the reverse transcription of a processed mRNA by retroviral reverse transcriptase. Unlike genes that arose by tandem duplication as a result of unequal crossover, such pseudogenes can be found anywhere in the genome. For example, a functional glutathione-S-transferase gene is on chromosome 11 and a pseudogene is located on chromosome 12.75
TYPES OF MUTATIONS
Mutations can occur in structural genes (the part of the DNA that specifies the amino acid sequence of protein), in the poorly understood regulatory apparatus that determines whether or not a gene will be available for transcription, in introns, or in portions of the DNA between genes that have no known function. As shown in Table 9-1, hematologic diseases provide examples of every known mechanism for causing mutations.

TABLE 9-1 EXAMPLES OF GENETIC MECHANISMS IN HEMATOLOGIC DISEASE

A change of one nucleotide to another without a change in the number of nucleotides in the sequence is called a point mutation. Other types of mutations are deletions and insertions (e.g., duplication of stretches of DNA in a gene). Mutations do not occur at random. Changes in the dinucleotide CpG to TpG are particularly common because invertebrate DNA cytidines followed by guanine are readily methylated and the methylcytosine formed is susceptible to oxidation to thymine. Thus, in both hemophilia A76 and G-6-PD deficiency77 an unusually high proportion of point mutations are found in CpG dinucleotides. Deletions or duplications of portions of genes tend to occur in areas in which the same sequence is repeated more than once. Thus, there are “hot spots” in the genome in which, for one reason or another, mutations are particularly likely to occur.
Another mechanism by which mutation appears to occur is that of gene conversion. This poorly understood phenomenon results in the sequence of one gene being transferred en bloc to another. This phenomenon is thought to account for the maintenance of identical sequence between duplicated genes.78,79
Many mutations affect the amount of processed mRNA that is formed. For example, mutations that cause abnormal splicing may produce a messenger that cannot be translated. Regulatory mutations that impair the rate at which a gene is transcribed into mRNA can be the consequence of mutations in promoter or enhancer elements. Mutations that cause thalassemia by impairing transcription of the hemoglobin b locus are the best characterized of these (see Chap. 46). However, most mutations causing hematologic disease seem to be structural mutations, those in which the sequence of the coding region of the gene is altered.
Errors in the base sequence of the structural gene result in failure to form any protein, in the formation of a very unstable protein that may never appear in the fully assembled form, or in the formation of an abnormal protein. The latter circumstance appears to be the most common. The abnormal protein may maintain all, some, or none of the functional properties of the normal protein. Even when it has lost the functional properties of the original protein it may retain the antigenic properties, and it is then designated cross-reacting material (CRM). Its stability may be normal or reduced. Mutations that result in the formation of stable proteins with normal functional properties are not clinically significant, but they may be very valuable from the point of view of population and family studies, or as genetic markers for various types of biologic investigations. Some “deficiencies” of enzymes are also clinically harmless. For example, genetic absence of the glycosyl transferases that convert the H antigen to the A or B antigen (see Chap. 137) results in the appearance of blood group O, surely a clinical state that cannot be considered a disease. Genetic variants that reach a frequency of more than one percent in a population are known as polymorphisms. Sometimes genetic variants such as the sickle cell gene or the G-6-PD deficiency gene reach polymorphic levels because the deleterious effects that they may have are counterbalanced by beneficial effects on survival, such as increased resistance to malaria. They are known as balanced polymorphisms.
All cells receive the same complement of genes. Nonetheless some mutations are tissue-specific. Several circumstances can account for this. Some enzymes that appear to perform the same function are encoded by different genes in different tissues. For example, the pyruvate kinase of leukocytes and that of erythrocytes are under separate genetic control (see Chap. 45). In other cases, alternative splicing of the primary mRNA can produce different polypeptides.80,81 Differences in posttranslational processing, including proteolysis and glycosylation of the same polypeptide by different enzymes in different tissues, can lead to different final products. However, in most instances a mutation that affects an enzyme in one type of blood cell will also affect the same enzyme in other blood cells, in liver, in brain, and in other tissues.
The types of enzyme deficiencies encountered clinically are limited by the ability of the affected individual to survive. Thus, complete absence of a key glycolytic enzyme from all tissues is incompatible with the basic process of energy metabolism and would almost surely be lethal long before birth. In contrast, the inheritance of enzyme deficiencies that are manifested only in erythrocytes is apparently quite compatible with survival, and thus many of the enzyme defects that are observed in humans are ones that only affect the red blood cell.
MUTATION NOMENCLATURE
Historically, mutations were first detected by sequencing the protein, usually hemoglobin. Indeed, the mutation in sickle cell disease was described before the genetic code had been deciphered. Thus, mutations were designated by indicating the amino acid change. Amino acid-based nomenclature does not unambiguously define the mutation, since the same amino acid substitution can be caused by different nucleotide substitutions. Further ambiguity is introduced by the fact that three different starting points for the numbering of amino acids in protein are commonly employed: (1) the methionine start codon; (2) the amino acid after the methionine start codon; and (3) the amino terminal amino acid of the processed protein. Finally, there are many mutations, such as those that change splice sites or promoters, that cannot be designated by an amino acid substitution. Nonetheless, designations based on amino acid mutation have been so widely used that they serve as useful “nicknames” for mutations; the nucleotide-based designation would simply not be recognized by workers in the field. Moreover, knowing the amino acid change sometimes provides valuable information regarding the effect of the mutation at the protein level. Therefore, while the more robust nucleotide-based mutation is preferred in this text, the amino acid–based notation is used when it is the one that is generally recognized by workers in the field. Standards have been established for the different notations that are in use.82,83,84 and 85
GENOTYPE-PHENOTYPE CORRELATIONS
Even before detection of mutations at the DNA level was feasible, clinicians could deduce that the same genotype did not always produce the same clinical disease picture (phenotype). Sibs inheriting autosomal recessive disorders from their parents often have been observed to have discordant clinical presentations—one severely affected, one mildly so—even though the same pair of disease-producing genes was inherited. With the development of the ability to define genotypes directly, the great degree of genotype-phenotype dissociation has become even more evident. Thus, persons inheriting the same sickle cell, G-6-PD, factor VIII, or glucocerebrosidase mutations may have mild or severe sickle cell disease, hemolytic anemia, hemophilia A, or Gaucher disease respectively. The factors that modify disease expression are usually not understood. In the case of G-6-PD deficiency, a second mutation, one in the UDP-glucuronosyltransferase-1 gene, has been shown to determine whether severe jaundice will be present.86,87
CHAPTER REFERENCES

1.
Beutler E, Yeh M, Fairbanks VF: The normal human female as a mosaic of X-chromosome activity: Studies using the gene for G-6-PD deficiency as a marker. Proc Natl Acad Sci USA 48:9, 1962.

2.
Lyon MF: Sex chromatin and gene action in the mammalian X-chromosome. Am J Hum Genet 14:135, 1962.

3.
Gartler SM, Linder D: Developmental and evolutionary implications of the mosaic nature of the G-6-PD system. Cold Spring Harb Symp Quant Biol 29:253, 1964.

4.
Beutler E: The distribution of gene products among populations of cells in heterozygous humans. Cold Spring Harb Symp Quant Biol 29:261, 1964.

5.
Fialkow PJ, Gartler SM, Yoshida A: Clonal origin of chronic myelocytic leukemia in man. Proc Natl Acad Sci USA 58:1468, 1967.

6.
Oni SB, Osunkoya BO, Luzzatto L: Paroxysmal nocturnal hemoglobinuria: Evidence for monoclonal origin of abnormal red cells. Blood 36:145, 1970.

7.
Beutler E, West C, Johnson C: Involvement of the erythroid series in acute myeloid leukemia. Blood 53:1203, 1979.

8.
Fialkow PJ, Singer JW, Raskind WH, et al: Clonal development, stem-cell differentiation, and clinical remissions in acute nonlymphocytic leukemia. N Engl J Med 317:468, 1987.

9.
Lindsay S, Monk M, Holliday R, et al: Differences in methylation on the active and inactive human X chromosomes. Ann Hum Genet 49:115, 1985.

10.
Gilliland DG, Blanchard KL, Bunn HF: Clonality in acquired hematologic disorders. Annu Rev Med 42:491, 1991.

11.
Gilliland DG, Blanchard KL, Levy J, Perrin S, Bunn HF: Clonality in myeloproliferative disorders: analysis by means of the polymerase chain reaction. Proc Natl Acad Sci USA 88:6848, 1991.

12.
Curnutte JT, Hopkins PJ, Kuhl W, Beutler E: Studying X-inactivation. Lancet 339:749, 1992.

13.
Prchal JT, Guan YL, Prchal JF, Barany F: Transcriptional analysis of the active X-chromosome in normal and clonal hematopoiesis. Blood 81:269, 1993.

14.
Wallace DC: Mitochondrial DNA sequence variation in human evolution and disease. Proc Natl Acad Sci USA 91:8739, 1994.

15.
Graeber MB, Muller U: Recent developments in the molecular genetics of mitochondrial disorders. J Neurol Sci 153:251, 1998.

16.
Ohno S: The one ancestor per generation rule and three other rules of mitochondrial inheritance. Proc Natl Acad Sci USA 94:8033, 1997.

17.
Bader-Meunier B, Rotig A, Mielot F, et al: Refractory anaemia and mitochondrial cytopathy in childhood. Br J Haematol 87:381, 1994.

18.
Superti-Furga A, Schoenle E, Tuchschmid P, et al: Pearson bone marrow-pancreas syndrome with insulin-dependent diabetes, progressive renal tubulopathy, organic aciduria and elevated fetal haemoglobin caused by deletion and duplication of mitochondrial DNA. Eur J Pediatr 152:44, 1993.

19.
Cormier V, Rötig A, Quartino AR, et al: Widespread multi-tissue deletions of the mitochondrial genome in the Pearson marrow-pancreas syndrome. J Pediatr 117:599, 1990.

20.
Boyer SH, Graham JB: Linkage between the X chromosome loci for glucose-6-phosphate dehydrogenase electrophoretic variation and hemophilia A. Am J Hum Genet 17:320, 1965.

21.
Siniscalco M, Filippi G, Latte B, et al: Failure to detect linkage between Xg and other X-borne loci in Sardinians. Ann Hum Genet 29:231, 1966.

22.
Jarman AP, Wood WG, Sharpe JA, et al: Characterization of the major regulatory element upstream of the human alpha-globin gene cluster. Mol Cell Biol 11:4679, 1991.

23.
Orkin SH: Globin gene regulation and switching: circa 1990. Cell 63:665, 1990.

24.
Keller W: The RNA lariat: A new ring to the splicing of mRNA precursors. Cell 39:423, 1984.

25.
Conboy JG, Chan J, Mohandas N, Kan YW: Multiple protein 4.1 isoforms produced by alternative splicing in human erythroid cells. Proc Natl Acad Sci USA 85:9062, 1988.

26.
Noguchi T, Yamada K, Inoue H, Matsuda T, Tanaka T: The L- and R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. J Biol Chem 262:14366, 1987.

27.
Kozak M: Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucl Acids Res 12:857, 1984.

28.
Merrick WC: Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev 56:291, 1992.

29.
Maniatis T, Goodbourn S, Fischer JA: Regulation of inducible and tissue-specific gene expression. Science 236:1237, 1987.

30.
Rouault T, Klausner R: Regulation of iron metabolism in eukaryotes. Curr Top Cell Regul 35:1–19, 1997.

31.
Han J, Brown T, Beutler B: Endotoxin-responsive sequences control cachectin/tumor necrosis factor biosynthesis at the translational level. J Exp Med 171:465, 1990.

32.
Han J, Beutler B, Huez G: Complex regulation of tumor necrosis factor mRNA turnover in lipopolysaccharide-activated macrophages. Biochim Biophys Acta 1090:22, 1991.

33.
Beutler E, Gelbart T, Han J, Koziol JA, Beutler B: Evolution of the genome and the genetic code: Selection at the dinucleotide level by methylation and polyribonucleotide cleavage. Proc Natl Acad Sci USA 86:192, 1989.

34.
Gridley T: Insertional versus targeted mutagenesis in mice. New Biol 3:1025, 1991.

35.
Waldman AS: Targeted homologous recombination in mammalian cells. Crit Rev Oncol Hematol 12:49, 1992.

36.
Weintraub HM: Antisense RNA and DNA. Sci Am 262:40, 1990.

37.
Simons RW: Naturally occurring antisense RNA control—a brief review. Gene 72:35, 1988.

38.
Weintraub LR, Goral A, Grasso J, et al: Pathogenesis of hepatic fibrosis in experimental iron overload. Br J Haematol 59:321, 1985.

39.
Smith CC, Aurelian L, Reddy MP, Miller PS, Ts’o POP: Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5. Proc Natl Acad Sci USA 83:2787, 1986.

40.
McManaway ME, Neckers LM, Loke SL, et al: Tumour-specific inhibition of lymphoma growth by an antisense oligodeoxynucleotide. Lancet 335:808, 1990.

41.
Szczylik C, Skorski T, Nicolaides NC, et al: Selective inhibition of leukemia cell proliferation by BCR-ABL antisense oligodeoxynucleotides. Science 253:562, 1991.

42.
Cotter FE, Johnson P, Hall P, et al: Antisense oligonucleotides suppress B-cell lymphoma growth in a SCID-hu mouse model. Oncogene 9:3049, 1994.

43.
Smith MJ, Prochownik EV: Inhibition of c-jun causes reversible proliferative arrest and withdrawal from the cell cycle. Blood 79:2107, 1992.

44.
Chen CJ, Banerjea AC, Harmison GG, Haglund K, Schubert M: Multitarget-ribozyme directed to cleave at up to nine highly conserved HIV-1 env RNA regions inhibits HIV-1 replication—potential effectiveness against most presently sequenced HIV-1 isolates. Nucl Acids Res 20:4581, 1992.

45.
Heidenreich O, Eckstein F: Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1. J Biol Chem 267:1904, 1992.

46.
Kuwabara T, Warashina M, Tanabe T, et al: Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA. Nucl Acids Res 25:3074, 1997.

47.
Burt MJ, Smit DJ, Pyper WR, Powell LW, Jazwinska EC: A 4.5-megabase YAC contig and physical map over the hemochromatosis gene region. Genomics 33:153, 1996.

48.
Schuler GD, Boguski MS, Stewart EA, et al: A gene map of the human genome. Science 274:540, 1996.

49.
Amplification of nucleic acid sequences: The choices multiply. J NIH Res 3:81, 1991.

50.
Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds): PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

51.
De Melo MB, Sales TSI, Lorand-Metze I, Costa FF: Rapid method for isolation of DNA from glass slide smears for PCR. Acta Haematol (Basel) 87:214, 1992.

52.
DeSalle R, Gatesy J, Wheeler W, Grimaldi D: DNA sequences from a fossil termite in Oligo-Miocene amber and their phylogenetic implications. Science 257:1933, 1992.

53.
Southern E: Gel electrophoresis of restriction fragments. Methods Enzymol 68:152, 1979.

54.
Chang JC, Kan YW: Antenatal diagnosis of sickle cell anaemia by direct analysis of the sickle mutation. Lancet 2:1127, 1981.

55.
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463, 1977.

56.
Rosenthal N: Molecular medicine. Recognizing DNA. N Engl J Med 333:925, 1995.

57.
Winship PR: An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucl Acids Res 17:1266, 1989.

58.
Beutler E, Kuhl W, Gelbart T, Forman L: DNA sequence abnormalities of human glucose-6-phosphate dehydrogenase variants. J Biol Chem 266:4145, 1991.

59.
Kumar R, Dunn LL: Designed diagnostic restriction fragment length polymorphisms for the detection of point mutations in ras oncogenes. Oncogene Res 4:235, 1989.

60.
Chehab FF, Kan YW: Detection of specific DNA sequences by fluorescence amplification: A color complementation assay. Proc Natl Acad Sci USA 86:9178, 1989.

61.
Mistry PK, Smith SJ, Ali M, et al: Genetic diagnosis of Gaucher’s disease. Lancet 339:889, 1992.

62.
Landegren U, Kaiser R, Sanders J, Hood L: A ligase-mediated gene detection technique. Science 241:1077, 1988.

63.
Barany F: Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci USA 88:189, 1991.

64.
Kalin I, Shephard S, Candrian U: Evaluation of the ligase chain reaction (LCR) for the detection of point mutations. Mutat Res 283:119, 1992.

65.
Beutler E, Gelbart T, West C, et al: Mutation analysis in hereditary hemochromatosis. Blood Cells Mol Dis 22:187, 1996.

66.
Cai SP, Zhang JZ, Huang DH, Wang ZX, Kan YW: A simple approach to prenatal diagnosis of beta-thalassemia in a geographic area where multiple mutations occur. Blood 71:1357, 1988.

67.
Mashiyama S, Murakami Y, Yoshimoto T, Sekiya T, Hayashi K: Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene 6:1313, 1991.

68.
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T: Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766, 1989.

69.
Myers RM, Larin Z, Maniatis T: Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science 230:1242, 1985.

70.
Abrams ES, Murdaugh SE, Lerman LS: Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp. Genomics 7:463, 1990.

71.
Ryan TM, Ciavatta DJ, Townes TM: Knockout-transgenic mouse model of sickle cell disease. Science 278:873, 1997.

72.
Paszty C, Brion CM, Manci E, et al: Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease. Science 278:876, 1997.

73.
Ohno S: Evolution by Gene Duplication. Springer Verlag, Berlin, 1970.

74.
Zimran A, Sorge J, Gross E, et al: A glucocerebrosidase fusion gene in Gaucher disease. Implications for the molecular anatomy, pathogenesis and diagnosis of this disorder. J Clin Invest 85:219, 1990.

75.
Board PG, Coggan M, Woodcock DM: The human Pi class glutathione transferase sequence at 12q13-q14 is a reverse-transcribed pseudogene. Genomics 14:470, 1992.

76.
Youssoufian H, Kazazian HH Jr, Phillips DG, et al: Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots. Nature 324:380, 1986.

77.
Vulliamy TJ, D’Urso M, Battistuzzi G, et al: Diverse point mutations in the human glucose 6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia. Proc Natl Acad Sci USA 85:5171, 1988.

78.
Baltimore D: Gene conversion: Some implications for immunoglobulin genes. Cell 24:592, 1981.

79.
Hess JF, Schmid CW, Shen CK: A gradient of sequence divergence in the human adult alpha-globin duplication units. Science 226:67, 1984.

80.
Amara SG, Jonas V, Rosenfeld MG, Ong ES, Evans RM: Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298:240, 1982.

81.
Pihlajaniemi T, Myllyla R, Seyer J, Kurkinen M, Prockop DJ: Partial characterization of a low molecular weight human collagen that undergoes alternative splicing. Proc Natl Acad Sci USA 84:940, 1987.

82.
Ad Hoc Committee on Mutation Nomenclature: Update on nomenclature for human gene mutations. Hum Mutat 8:197, 1996.

83.
Antonarakis SE: Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat 11:1, 1998.

84.
Beaudet AL, Tsui LC: A suggested nomenclature for designating mutations. Hum Mutat 4:245, 1993.

85.
Beutler E, McKusick VA, Motulsky AG, Scriver CR, Hutchinson F: Mutation nomenclature: Nicknames, systematic names, and unique identifiers. Hum Mutat 8:203, 1996.

86.
Kaplan M, Renbaum P, Levy-Lahad E, et al: Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia. Proc Natl Acad Sci USA 94:12128, 1997.

87.
Sampietro M, Lupica L, Perrero L, et al: The expression of uridine diphosphate glucuronosyltransferase gene is a major determinant of bilirubin level in heterozygous beta-thalassaemia and in glucose-6-phosphate dehydrogenase deficiency. Br J Haematol 99:437, 1997.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

CHAPTER 8 HEMATOLOGY IN THE AGED

CHAPTER 8 HEMATOLOGY IN THE AGED
Williams Hematology

CHAPTER 8 HEMATOLOGY IN THE AGED

MARSHALL A. LICHTMAN
WILLIAM J. WILLIAMS

Aging and Hematopoiesis

Marrow Cellularity

Chromosome Studies

Erythrocytes

Serum Iron, Iron-Binding Capacity, and Ferritin Levels

Serum Erythropoietin Concentration

Serum Vitamin B12 and Folate Levels

Leukocytes

Immune Responses

Platelets

Plasma Coagulation Factors

Erythrocyte Sedimentation Rate and C Reactive Protein

The Incidence of Clonal Hemopathies
Chapter References

The hematopoietic system is modestly affected by aging, and these effects become particularly notable after age 65. There is a continuous decrease in the volume of the hematopoietic marrow with age, which does not cause significant alterations in either granulocyte, monocyte, or platelet counts, although a slight (£1.0 g/dl) decrease in population mean hemoglobin concentration in men occurs. The recruitment of neutrophils in response to exogenous stimuli is slightly decreased, but the response to infection does not appear impaired. Neutrophil function is not significantly decreased with age of the subject. Although the population mean vitamin B12 and folate levels decrease with age, these changes do not result in decreased hematopoiesis as judged by blood counts, except in individual patients with significant deficiencies. Anemia in older individuals should be evaluated in the same manner as anemia in younger individuals. Certain coagulation proteins are altered significantly with aging, and a propensity to accelerated coagulation and compensatory fibrinolysis is present, leading to a new steady state. Decreased immune cell function is the most consistent change in older persons and perhaps the most important functionally. Although there is a tendency to decreased lymphocyte counts in the blood, the major effects are mediated by dysregulation of T lymphocyte function, perhaps as a result of the prolonged period since thymic atrophy in older subjects. This change affects both cellular immune functions and antibody responses to antigens because of the T helper cell function required. Many studies of aging have to be interpreted in the light of inadequate population samples for study, the difficulty and therefore the rarity of using longitudinal as contrasted with cross-sectional analyses, the small sample sizes after stratification for gender and decade of age, and the need to study smaller age intervals in the 8th through 10th decades of life because of more dramatic changes over short intervals at these ages.

Acronyms and abbreviations that appear in this chapter include: BPG, bisphosphoglycerate; EPO, erythropoietin; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-monocyte colony stimulating factor; IL, interleukin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; SEER, National Cancer Institute Surveillance Epidemiology End Results.

In 1998, individuals 65 years of age or older accounted for 12.7 percent of the population of the United States; this group is expected to grow to 23.0 percent of the population by the year 2040. Currently, there are 4.0 million people in the United States who are 85 years old or older.1 Data from 1985 through 1989 indicate that life expectancy at age 65 is 14 years for males and 18 years for females in most developed countries.2 As a result, physicians are increasingly caring for older patients and are being called upon to interpret hematologic data in the context of the age of the patient. Age-related effects on cellular DNA results in a dramatic increase in the incidence of clonal hematopoietic diseases, especially leukemia, lymphoma, myeloma, and closely related diseases in the decades after age 50. In addition, the decrease in immune function has an impact on vaccine use and resistance to infection in older individuals.
AGING AND HEMATOPOIESIS
Throughout embryogenesis and early infancy nearly all cells of the body have mitotic capacity. Subsequently, certain cells of the body lose their ability to divide (e.g., nervous tissue, muscles).3 Others continue to divide until full growth has been achieved. Thereafter, cells usually do not divide at a significant rate except under conditions of stress, when they become capable of rapid cell division. These cells are said to be “potentially mitotic” or “discontinuous replicators,” as exemplified by hepatic cells and renal tubular cells.3 Cells of organs that require continuous self-renewal, such as the marrow, the scalp hair follicles, and the gastrointestinal mucosa, are continuously mitotic throughout life.3
Studies of diploid human cells maintained in continuous culture have led to the assertion that there is a limit to the number of divisions a cell may undergo,4,5 and 6 a state of replication senescence, which may be related in part to telomere shortening.7 However, there is no evidence of exhaustion of marrow stem cells with extreme aging. The proliferative capacity of marrow cells from older animals and humans has been studied by a variety of techniques, both in vivo and in vitro.8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40 and 41 Most studies indicate that marrow can sustain normal blood cell counts in older animals,27,28,29 and 30,32,35,42 but the reserve capacity may be limited during periods of exaggerated demand.11,14,15,19,22,23 and 24,29,199 The hematopoietic limitations observed in older animals could be intrinsic to marrow stem cells or to cells of the hematopoietic stroma and/or their cytokine production.17,28,32,33,35 The short-term hematopoietic responses to the growth factors granulocyte-monocyte colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and erythropoietin (EPO) are well maintained in older subjects,39,40 although the response of multipotential (CD34+) cells to granulocyte colony-stimulating factor (G-CSF) in culture in older patients is decreased, and the mobilization of neutrophils by G-CSF in vivo is diminished.40,41 There is no evidence, however, that the effects of aging on marrow proliferative capacity, or, ultimately on steady-state blood cell levels, are clinically significant within existing life-spans.12,14,15 and 16
MARROW CELLULARITY
The cellularity of the marrow decreases with aging, as estimated from studies of histologic sections.43,44 and 45 Magnetic resonance imaging confirms an age-related reduction in marrow cellularity.46 Studies of marrow from the anterior iliac crest demonstrate a progressive decrease in cellularity from 80–100 percent to about 50 percent over the first 30 years of life.47 Cellularity of about 40 percent has been found in sternal marrow from normal adults.48 In iliac crest marrow there is a plateau of about 50 percent cellularity to age 65, after which a decrease in cellularity to about 30 percent occurs over the succeeding decade.44 This latter decline may be due to an increase in fat related to osteoporosis, with reduction of the volume of cancellous bone, rather than to a decrease in hematopoietic cells.49 These changes may account for the more pronounced marrow hypocellularity in the subcortical zone.
CHROMOSOME STUDIES
Three principal cytogenetic changes in hematopoietic cells have been identified in relationship to human aging: loss of chromosomes, increased micronucleus formation, and telomere shortening. There is an exponential increase during aging in the proportion of adult women whose phytohemagglutinin-stimulated blood lymphocytes display X chromosome aneuploidy as a result of X chromosome loss. Thus, the proportion of women with X chromosome aneuploidy increases from about 1 percent of women under age 25 years to about 15 percent of women over 45 years of age.50,51 This alteration is not evident, however, in marrow erythroid or granulocytic cells. Loss of the Y chromosome also increases with age and is a feature of marrow hematopoietic cells. Y chromosome loss is very unusual under age 50 years but occurs in about 10 percent of men beyond age 50 years, with a continuously increasing frequency each decade between 50 and 90 years of age.52 Loss of the Y chromosome in men with clonal hemopathies occurs at a rate expected in unaffected males of the same age and thus is an aging rather than a neoplastic phenomenon. Autosome loss increases in frequency with age. Smaller chromosomes are lost more frequently than larger chromosomes.51 An increase in stable aberrations of chromosomes, including insertions, translocations, and dicentric and acentric chromosome fragments, are evident with aging.53,54 An increase in somatic mutations occurs with age when studied in blood lymphocytes, but this may reflect an accumulation with time rather than an age-dependent increase in mutation rate.55
An increase in micronuclei is evident in the blood lymphocytes of older as compared with younger individuals.54 In women this phenomenon is directly correlated with X chromosome aneuploidy, since fluorescence in situ hybridization demonstrates lost portions of the X chromosome within micronuclei.51,56
The termini of chromosomes contain telomeres consisting of specific proteins and tandemly repeated sequences of DNA that have the base structure TTAGGG. Telomeres shorten during “aging” of cells in culture and in the cells of humans (and other species) as they age. Aging of hematopoietic tissues is complex because of the potential lengthy dormancy and the self-renewal capability of stem cells, whereas their derivative cells die and are replaced in relatively short periods of time. Telomere length has been examined in blood leukocytes. A shortening of telomere length with age of the host occurs but does so in a complex, not linear, fashion, which may have to do with the relative proliferative rate at the time of study.57,58 and 59
ERYTHROCYTES
HEMOGLOBIN LEVEL
Many population studies of hematologic variables in aging subjects suffer from several limitations: sampling is often done by convenience rather than random selection of a free-living, defined population; cross-sectional rather than cohort studies are conducted; and small sample sizes, especially after stratification for gender and decade of age, permit undue influence by a few deviant values. Most studies have shown that the mean hemoglobin level or hematocrit60,61,62,63 and 64 for a population of men falls slightly after middle age. Although statistically significant in some cases, mean hemoglobin levels decrease by less than 1.0 g/dl in the sixth through eighth decades.60,61,62,63,64,65 and 66 In a group of men age 96 to 106 years the mean hemoglobin level was 12.4 g/dl,67 but a later report of centenarians did not find a decrease in mean hemoglobin as compared with other men.68 In a group of men aged 84 to 98 years the mean hemoglobin level was 14.8 g/dl, only 0.8 g/dl less than that of a younger comparison group.62 The lowest levels, however, are found in the oldest patients.63,64,69 The hemoglobin levels in women may increase slightly with age60,65,70 or remain unchanged.71 Small mean decreases in hemoglobin levels in older women have been reported.61,63,64,66,67 In studies that have identified a decrease in hemoglobin level of both men and women, the decrease is less in women than in men. The narrowing of the difference in hemoglobin level between older men and woman may be the result of decreased androgen levels in older men and decreased estrogen levels in older women.
Iron deficiency and the anemia of chronic disease have usually been responsible for low hemoglobin levels in the majority of asymptomatic elderly people.61,69,73,74 Iron absorption is not impaired in the elderly, but utilization of orally administered iron for hemoglobin production is reduced.75 Since hemoglobin concentration does not decrease significantly with age, elderly patients with anemia should be evaluated for a cause (e.g., iron, folate, or vitamin B12 deficiency or underlying malignancy or renal disease, etc.) before ascribing it to age.76,77 and 78
Unexplained anemia is also frequently observed in studies of elderly people.61,69,74 One set of studies found that the red cells of older individuals separated in vitro had a greater proportion of dense cells in each density fraction, a greater proportion of reticulocytes, and an increase in autologous IgG antibodies per cell. In vitro erythrophagocytosis by macrophages was increased when red cells from older individuals were the target particles.79,80 The inference drawn was that shortened red cell survival may play a role in the unexplained mild decrease in hemoglobin concentration in some older individuals.
ERYTHROCYTE 2,3-BISPHOSPHOGLYCERATE CONCENTRATION (2,3-BPG)
The erythrocyte 2,3-bisphosphoglycerate (2,3-BPG) level has been reported to fall with age from a mean value of 14.9 µmol/g hemoglobin at ages 18 to 24 to 13.9 µmol/g hemoglobin at ages 75 to 84.81,82 This decrease is statistically significant. It could account for a slight increase in oxygen affinity of hemoglobin, but is of doubtful physiologic significance.
OSMOTIC FRAGILITY
Erythrocyte osmotic fragility is increased in older individuals in comparison with younger subjects.83,84 This phenomenon is associated with an increased mean corpuscular volume (MCV) and decreased mean corpuscular hemoglobin concentration (MCHC) of the red cells of older people.84
SERUM IRON, IRON-BINDING CAPACITY, AND FERRITIN LEVELS
In individuals of both sexes with normal hemoglobin levels, and presumably with normal iron stores, the serum iron level falls after the ages of 20 to 30.70,85 In one study the values fell from a mean of about 130 µg/dl (28 µmol/liter) in males and 116 µg/dl (21 µmol/liter) for females to a mean at age 71 to 80 of about 75 µg/dl (13 µmol/liter) in men and 66 µg/dl (12 µmol/liter) in women.85 Levels of 50 µg/dl (9 µmol/liter) or less were found in 40 percent of men and women above the age of 50.86 The iron-binding capacity also falls in the elderly.70,87,88
Serum ferritin levels rise from a median of 25 µg/liter to 94 µg/liter in males in the third decade and then to a median of 124 µg/liter above age 45.89 Ferritin levels in females remain low until middle age and then increase from a median of 25 µg/liter to 89 µg/liter in women after menopause.89 Serum ferritin levels appear to reflect iron stores in elderly people.73,90
SERUM ERYTHROPOIETIN CONCENTRATION
Serum erythropoietin levels in nonanemic elderly individuals appear to be the same as those found in younger people,91,92 and 93 although elevated levels were found in one study94 and lower levels in another.95 Serum erythropoietin levels are generally inversely related to hemoglobin levels,91,92 and 93 suggesting that the erythropoietin response in the elderly is similar to that in younger individuals. The peak and trough of the diurnal variation in erythropoietin levels is the same in younger and older individuals.90
SERUM VITAMIN B12 AND FOLATE LEVELS
Low serum vitamin B12 levels are found in a significant number of older individuals who do not have clinical findings of vitamin B12 deficiency (i.e., anemia or a neurologic disorder).47,96,97,98,99,100,101 and 102 They are very nonspecific screening measurements. The absorption of pure vitamin B12 (“Schilling test”) is normal in older individuals,88 but absorption of protein-bound vitamin B12 may be reduced103 in such patients and also in apparently healthy adults over 55 years of age.104 Reexamination of this question, however, showed normal absorption of free and protein-bound cobalamin in older subjects.105 On the other hand, untreated patients with pernicious anemia may have only a moderate reduction in the serum vitamin B12 level and not have anemia or macrocytosis.106 These data require that reductions in the serum vitamin B12 level be evaluated carefully.106,107 and 108 Some individuals with low serum vitamin B12 levels have been followed for a 4-year period without developing anemia or other signs of vitamin B12 deficiency.109 Serum and urine methylmalonic acid and serum homocysteine assays may be helpful in assessing such patients. Patients with metabolically significant decreases in plasma vitamin B12 concentration will usually have elevated levels of methylmalonic acid and homocysteine, and their levels decrease to normal after vitamin B12 replacement (see Chap. 25).
Both serum66,101,107 and red cell66 folate levels were below the usual lower limit of normal (3 µg/liter) in a small proportion (3–7%) of both males and females over age 65. Low median values compared to those in young subjects were found for the plasma folate levels of a group of individuals in the eighth decade.108 Similarly low levels were also found, however, in young people who were clinically well and apparently on a normal diet,110,112 creating uncertainty regarding the “normal” level of serum folate and making the interpretation of these results difficult. None of the patients with low serum folate levels were anemic, and the significance of these findings is uncertain.
The MCV increases slightly but significantly with age.62,70,71,72,73,113,114 and 115 Cigarette smoking may also cause an increase in the MCV,114,115 and it has been reported that older persons who smoke may have a MCV of 100 fl or more in the absence of any demonstrable cause of macrocytosis.115
LEUKOCYTES
TOTAL AND DIFFERENTIAL LEUKOCYTE COUNT
There is no consistent, significant variation in the total leukocyte count in older subjects. Normal leukocyte and neutrophil counts were found in nonagenarian67 and centenarian populations.68 Some investigators have found that above age 65 the total leukocyte count tends to be lower in both sexes,69 due primarily to a decrease in the lymphocyte count.116,117,118,119,120 and 121 Others have reported a decrease in the leukocyte count due to a fall in the lymphocyte and the neutrophil count in women, but not in men, over age 50.22,123 The absolute lymphocyte count has also been reported to be unchanged in the aged.124,125 and 126
LEUKOCYTE RESPONSE TO INFECTION
Medical lore has it that the leukocyte count does not rise as high in response to infection in elderly individuals as in young people and that often the principal manifestation of a leukocyte response is an increase in the number of band forms in an otherwise normal leukocyte count.127,128 However, in two series of cases of acute appendicitis and one of pneumonia, the leukocytosis of patients over age 60 was the same as that found in younger patients.129,130 The leukocyte count and the proportion of neutrophils rise much less in response to bacterial pyrogen in individuals over age 70 than in young adults.131 Similarly, the neutrophilic leukocytosis that occurs 5 h after the oral administration of 40 mg prednisolone is diminished in patients over 55 years of age.132 These observations suggest a diminished marrow granulocyte reserve in the elderly and/or a decrease in hematopoietic growth factor release.133 The decreased responsiveness of older individuals to granulocyte colony-stimulating factor-induced release of neutrophils from the marrow supports these suppositions.40,41 Leukocyte function and serum opsonic capacity is well preserved in elderly individuals,134,135 but defects in phagocytic ability136,137 and diminished responses to chemotactic peptides138,139 and to oxidative stress140 have been documented. Defects in neutrophil function in elderly subjects may be due to inhibitory substances detected in plasma.141 Splenic function in elderly subjects may be impaired, as evidenced by an increase in the percentage of pitted erythrocytes in the blood.142
IMMUNE RESPONSES
There is compelling experimental evidence that a decrease in immune function mediated by lymphocytes is the most significant change with aging.200 Thymus involution occurs after puberty, and total thymic atrophy occurs by late middle age. With these changes, thymic-mediated T lymphocyte development disappears, and older individuals are dependent on their existing T lymphocyte pool to mediate T cell–dependent immune responses.124,125 and 126,143,144
T cells in older subjects have impaired responsiveness to mitogens and antigens,145,146 in part due to a decrease in expression of CD28 costimulator on the cell surface.146 The clonal expansion of T cells in culture is decreased, suggesting an inadequate response to antigen stimulation. Clones do not reach full development because of fewer doublings when T cells are obtained from older individuals.147,148 In the absence of thymic function, the number of naive T cells decreases in older individuals and memory T cells are the predominant type.149 Spontaneous T cell clonal expansion is a feature of older individuals and may occur among CD4+150,151 and CD8+ cell subsets.151 Although likened to benign monoclonal gammopathy, the T cell clones may be stable and less prone to malignant progression.151
B lymphocyte function is dependent on T cell accessory roles, and the decreased ability to generate antibody responses, especially to primary antigens,126,152,153 may be the result of T cell inadequacies rather than an intrinsic fault of B lymphocytes. The response to T cell–dependent antigens is characterized by the formation of low-affinity antibodies and anti-idiotypic autoantibodies.153 Although variable from study to study, total B lymphocyte,68,154 T lymphocyte,68,154 and T lymphocyte subset68,155,156 concentrations in the blood have been found to be decreased in older individuals. Natural killer cells are increased in number, but their function is disturbed.68,154,157,201 Not unexpectedly, delayed hypersensitivity reactions are reduced in the elderly.158,159,160 and 161 These immunologic deficits are correlated with overall mortality in individuals over age 60.162
Serum immunoglobulin M and G concentrations do not change significantly in older subjects. Serum IgA levels increase with age.163 An increased prevalence of autoantibodies (e.g., anti-IgG rheumatoid factor) occurs in older people.118,152,163 Monoclonal plasma immunoglobulins (essential monoclonal gammopathy) are found with increasing frequency with age, reaching three percent in people over age 70 and nearly six percent in those from 80 to 89164,165 (see Chap 105).
PLATELETS
The platelet count does not change with age.68,69 Increased plasma levels of two platelet a-granule constituents, b-thromboglobulin and platelet factor 4, have been found in individuals over 65 years of age in comparison with younger individuals.166,167 Enhanced in vitro reactivity to platelet-aggregating agents has been observed.168,169,170,171,172 and 173 Decreased platelet membrane protein kinase C activity and translocation to the cytosol after platelet activation was noted in platelets from older subjects.174
PLASMA COAGULATION FACTORS
Several studies have emphasized the changes in the level of proteins involved in the formation or dissolution of fibrin.175,176 and 177 Plasma concentrations of factor VII coagulant activity and antigen,175,176,177,178 and 179 and factor VIIIC,175,176,180 as well as von Willebrand factor,175,180 fibrinogen,175,176,178,181 fibrinopeptide A,175,176,182 and tissue plasminogen activator antigen175,183 are increased with age. Fibrinogen level has been found to be a risk factor for thrombotic vascular disease.181 In healthy centenarians, levels of activated factor VII, activation peptides of prothrombin, factors IX and X, and thrombin-antithrombin complex concentration were increased, signs of higher-than-expected coagulation enzyme activity.176 Higher D-dimer and plasmin-antiplasmin complexes indicate an accompanying increase in fibrinolytic activity.176 Thus, coagulant and fibrinolytic activities appear to be increased in the older subjects by both in vitro,176,184,185,186 and 187 and in vivo studies.182,188 Older patients may show an exaggerated anticoagulant response to warfarin.189
ERYTHROCYTE SEDIMENTATION RATE AND C REACTIVE PROTEIN
The erythrocyte sedimentation rate increases significantly with age.62,190,191,192 and 193 Mean values of 14 mm/h (Westergren) and individual values as high as 69 mm/h were found in apparently well women age 70 to 89 years who were followed for 3 to 11 years.193 The erythrocyte sedimentation rate is of limited value in detecting disease in elderly patients. Estimation of levels of acute-phase proteins appears to offer no advantage over the erythrocyte sedimentation rate.195,196 The C-reactive protein content of serum also is mildly elevated in older individuals without an apparent inflammatory process.197,198
THE INCIDENCE OF CLONAL HEMOPATHIES
Several hematologic diseases are increased in frequency with age; for example, pernicious anemia. The notable increase in clonal (neoplastic) diseases of hematopoiesis is shown in Figure 8-1, which depicts the rate of occurrence of the leukemias (the aggregate of the four major types), lymphoma, and myeloma at 5-year intervals. The inclusion of acute lymphocytic leukemia, which has a mode at about 3.5 years and then increases in frequency again after middle age, does not dampen the dramatic age-dependent incidence rate. The curves do not provide insight into the cause of the relationship, which could reflect the accumulated injury resulting from external factors, the accumulated effects of spontaneous somatic mutations, or some combination of these events.

FIGURE 8-1 The abscissa depicts age in intervals of 5 years. The ordinate represents the incidence per 100,000 Americans of myeloma, lymphoma, and leukemia. The rates for each of the four major leukemias and the various subtypes of lymphoma are aggregated. The increment at 0 to 4 years among the leukemias reflects a mode in acute lymphocytic leukemia at that age. These data were obtained from the National Cancer Institute Surveillance Epidemiology End Results (SEER) Program.

CHAPTER REFERENCES

1.
WWW.census.gov/population/estimates/nation/intfile2-1.txt

2.
Kinsella KG: Changes in life expectancy 1900–1990. Am J Clin Nutr 55:1196S, 1992.

3.
Lansdorp PM: Self-renewal of stem cells. Biol Blood Marrow Transplantation 3:171, 1997.

4.
Hayflick L: Mortality and immortality at the cellular level. A review. Biochemistry 62:1180, 1997.

5.
Perillo NL, Walford RL, Newman MA, Effros RB: Human T lymphocytes possess a limited in vitro life span. Exp Gerontol 24:177, 1989.

6.
Kirkland JL: The biochemistry of mammalian senescence. Clin Biochem 25:61, 1992.

7.
Rubin H: Cell aging in vivo and vitro. Mech Ageing Dev 100:209, 1998.

8.
Cudkowicz G, Upton AC, Shearer GM, Hughes WL: Lymphocyte content and proliferative capacity of serially transplanted mouse bone marrow. Nature 201:165, 1964.

9.
Siminovitch L, Till JE, McCulloch EA: Decline in colony-forming ability of marrow cells subjected to serial transplantation into irradiated mice. J Cell Comp Physiol 64:23, 1964.

10.
Yuhas JM, Storer JB: The effect of age on two modes of radiation death and on hematopoietic cell survival in the mouse. Radiat Res 32:596, 1967.

11.
Davis ML, Upton AC, Satterfield LC: Growth and senescence of the bone marrow stem cell pool in RFM/Un mice. Proc Soc Exp Biol Med 137:1452, 1971.

12.
Lajtha LB, Schofield R: Regulation of stem cell renewal and differentiation of possible significance in aging. Adv Gerontol Res 3:131, 1971.

13.
Vos O, Dolans MJAS: Self-renewal of colony forming units (CFU) in serial bone marrow transplantation experiments. Cell Tissue Kinet 5:371, 1972.

14.
Harrison DE: Normal production of erythrocytes by mouse marrow continuous for 73 months. Proc Natl Acad Sci USA 70:3184, 1973.

15.
Relucke U, Burlington H, Cronkite EP, Laissue J: Hayflick’s hypothesis: an approach to in vivo testing. Fed Proc 34:71, 1975.

16.
Harrison DE: Normal function of transplanted marrow cell lines from aged mice. J Gerontol 30:279, 1975.

17.
Harrison DE: Defective erythropoietic responses of aged mice not improved by young marrow. J Gerontol 30:286, 1975.

18.
Mauch P, Botnick LE, Hannon EC, et al: Decline in bone marrow proliferative capacity as a function of age. Blood 60:245, 1982.

19.
Inoui T, Cronkite EP: The influence of in vivo incubation of aged murine spleen colony-forming units on their proliferative capacity. Mech Ageing Dev 23:177, 1983.

20.
Boggs DR, Saxe DF, Boggs SS: Aging and hematopoiesis. II. The ability of bone marrow cells from young aged mice to cure and maintain cure in W/W. Transplantation 37:300k, 1984.

21.
Lipschitz DA, Udupa KB, Milton KY, Thompson CO: Effect of age on hematopoiesis in man. Blood 63:502, 1984.

22.
Udupa KB, Lipschitz DA: Erythropoiesis in the aged mouse. II. Response to stimulation in vitro. J Lab Clin Med 103:581, 1984.

23.
Boggs DR, Patrene KD: Hematopoiesis and aging. III. Anemia and a blunted erythropoietic response to hemorrhage in aged mice. Am J Hematol 19:327, 1985.

24.
Williams LH, Udupa KB, Lipschitz DA: Evaluation of the effect of age on hematopoiesis in the C57BL/6 mouse. Exp Hematol 14:827, 1986.

25.
Stead NW: Defective mononuclear cell support of erythropoiesis in the elderly. Am J Med Sci 293:85, 1987.

26.
Hirota Y, Okamura S, Kimura N, et al: Haematopoiesis in the aged as studied by in vitro colony assay. Eur J Haematol 40:83, 1988.

27.
Schofield R, Dexter TM, Lord BI, Tasta NG: Comparison of haemopoiesis in young and old mice. Mech Ageing Dev 34:1, 1986.

28.
Boggs D, Patrene K, Steinberg H: Aging and hematopoiesis. VI. Neutrophilia and other leukocyte changes in aged mice. Exp Hematol 14:372, 1986.

29.
Williams LH, Udupa KB, Lipschitz DA: Evaluation of the effect of age on hematopoiesis in the C57BL/6 mouse. Exp Hematol 14:827, 1986.

30.
Maggio-Price L, Wolf NS, Priestley GV, et al: Evaluation of stem cell reserve using serial bone marrow transplantation and competitive repopulation in a murine model of chronic hemolytic anemia. Exp Hematol 16:653, 1988.

31.
Baldwin JG: Hematopoietic function in the elderly. Arch Intern Med 148:2544, 1988.

32.
Harrison DE, Astle CM, Stone M: Numbers and functions of transplantable primitive immunohematopoietic stem cells: effects of age. J Immunol 142:3833, 1989.

33.
Lee MA, Segal GM, Bagby GC: The hematopoietic microenvironment in the elderly: defects in IL-1-induced CSF expression in vitro. Exp Hematol 17:952, 1989.

34.
Tejero C, Testa NG, Hendry JH: Decline in cycling of granulocyte-macrophage colony-forming cells with increasing age in mice. Exp Hematol 17:66, 1989.

35.
Sharp A, Zipori D, Toledo J, et al: Age related changes in hemopoietic capacity of bone marrow cells. Mech Ageing Dev 48:91, 1989.

36.
Morrison SJ, Wandycz AM, Akashi K, et al: The aging of hematopoietic stem cells. Nat Med 2:1011, 1996.

37.
Boggs SA, Patrene KD, Austin CA, et al: Latent deficiency of the hematopoietic microenvironment of aged mice as revealed in W/Wv mice given +l/+ cells. Exp Hematol 19:683, 1991.

38.
Keating A: The hematopoietic stem cell in elderly patients with leukemia. Leukemia 10(suppl 1):530, 1996.

39.
Shank WA Jr, Balducci L: Recombinant hemopoietic growth factors: comparative hemopoietic response in younger and older subjects. J Am Geriatr Soc 40:151, 1992.

40.
Chatta GS, Andrews RG, Rodger E, et al: Hematopoietic progenitors and aging: alterations in granulocyte precursors and responsiveness to recombinant human G-CSF, GM-CSF, and IL-3. J Gerontol 48:M207, 1993.

41.
Chatta GS, Price TH, Dale DC, et al: The effects of in vivo rhG-CSF on the neutrophil response in healthy young and elderly volunteers. Blood 84:2923, 1994.

42.
Egusa Y, Fujiwara Y, Syahrrudin E, et al: Effect of age on human peripheral blood stem cells. Oncol Rep 5:398, 1998.

43.
Custer RP, Ahlfeldt FE: Studies on the structure and function of the bone marrow. J Lab Clin Med 17:960, 1932.

44.
Hartsock RJ, Smith EB, Petty CS: Normal variations with aging on the amount of hematopoietic tissue in bone marrow from the anterior iliac crest. Am J Clin Pathol 43:326, 1965.

45.
Kricun ME: Red-yellow marrow conversion: its effect on location of some solitary bone lesions. Skeletal Radiol 14:10, 1985.

46.
Ricci C, Cova M, Kang YS, et al: Normal age-related patterns of cellular and fatty bone marrow distribution in the axial skeleton: MR imaging study. Radiology 177:83, 1990.

47.
Callander ST, Spray GH: Latent pernicious anemia. Br J Haematol 8:230, 1962.

48.
Beutler E, Drennan W, Block M: The bone marrow and liver in iron-deficiency anemia: a histopathologic study of sections with special reference to the stainable iron content. J Lab Clin Med 43:427, 1954.

49.
Mangolas SC, Jilka RL: Bone marrow, cytokines, and bone remodelling. N Engl J Med 332:305, 1995.

50.
Nowinski GP, Van Dyke DL, Tilley BC, et al: The frequency of aneuploidy in cultured lymphocytes is correlated with age and gender but not with reproductive history. Am J Hum Genet 46:1101, 1990.

51.
Stone JF, Sandberg AA: Sex chromosome aneuploidy and aging. Mutat Res 338:107, 1995.

52.
United Kingdom Cancer Cytogenetics Group: Loss of Y chromosome from normal and neoplastic bone marrow. Genes, Chromosomes, Cancer 5:83, 1992.

53.
Ramsey MJ, Moore DH II, Briner JF, et al: The effects of age and lifestyle factors on the accumulation of cytogenetic damage as measured by chromosome painting. Mutat Res 338:95, 1995.

54.
Bolognesi C, Abbondandolo A, Barale R, et al: Age-related increase of baseline frequencies of sister chromatid exchanges, chromosome aberrations, and micronuclei in human lymphocytes. Cancer Epidemiol Biomark Prevent 6:249, 1997.

55.
Grist SA, McCarron M, Kutlaca A, et al: In vivo human somatic mutation: frequency and spectrum with age. Mutat Res 266:189, 1992.

56.
Catalan J, Autio K, Wessman M, et al: Age-associated micronuclei containing centromeres and the X chromosome in lymphocytes of women. Cytogen Cell Genet 68:11, 1995.

57.
Frenck RW Jr, Blackburn EH, Shannon KM: The rate of telomere sequence loss in human leukocyte varies with age. Proc Natl Acad Sci USA 95:5607, 1998.

58.
Notario R, Cimmino A, Tabarini D, et al. In vivo telomere dynamics of human hematopoietic stem cells. Proc Natl Acad Sci USA 94:13782, 1997.

59.
Batliwalla F, Monteiro J, Serrano D, Gregorson PK: Oligoclonality of CD8+ T cells in health and disease: aging, infection, or immune regulation. Hum Immunol 48:68, 1996.

60.
McDonough JR, Hames CG, Garrison GE, et al: The relationship of hematocrit to cardiovascular states of health in the negro and white population of Evans County, Georgia. J Chron Dis 18:243, 1965.

61.
McLennan WJ, Andrews GR, Macleod C, Caird FI: Anaemia in the elderly. Q J Med 42:1, 1973.

62.
Zauber NP, Zauber AG: Hematologic data of healthy very old people. JAMA 257:2181, 1987.

63.
Nilsson-Ehle H, Jagenburg R, Landahl S, et al: Decline of blood haemoglobin in the aged: a longitudinal study of an urban Swedish population from age 70 to 81. Br J Haematol 71:437, 1989.

64.
Salive ME, Cornoni-Huntley J, Guralnik JM, et al: Anemia and hemoglobin level in older persons: relationship with age, gender, and health status. J Am Geriatr Soc 40:489, 1992.

65.
Cruickshank JM: Some variations in the normal haemoglobin concentration. Br J Haematol 18:523, 1970.

66.
Elwood PC, Shinton NK, Wilson CD, et al: Haemoglobin, vitamin B12 and folate levels in the elderly. Br J Haematol 21:557, 1971.

67.
Zaino EC: Blood counts in the nonagenarian. NY State J Med 81:1199, 1981.

68.
Sansoni P, Cossarizza A, Brianti V, et al: Lymphocyte subsets and natural killer cell activity in healthy old people and centenarians. Blood 82:2767, 1993.

69.
Nilsson-Ehle H, Jagenburg R, Landahl S, et al: Haematological abnormalities and reference intervals in the elderly: a cross-sectional comparative study of three Swedish population samples aged 70, 75 and 81 years. Acta Med Scand 224:595, 1988.

70.
Yip R, Johnson C, Dallman PR: Age-related changes in laboratory values used in the diagnosis of anemia and iron deficiency. Am J Clin Nutr 39:427, 1984.

71.
Jernigan JA, Gudat JC, Blake JL, et al: Reference values for blood findings in relatively fit elderly persons. J Am Geriatr Soc 28:308, 1980.

72.
Kelly A, Munan L: Haematologic profile of natural populations: red cell parameters. Br J Haematol 35:153, 1977.

73.
Htoo MSH, Kofkoff RL, Freedman ML: Erythrocyte parameters in the elderly: an argument against new geriatric normal values. J Am Geriatr Soc 27:547, 1979.

74.
Lipschitz DA, Mitchell CO, Thompson C: The anemia of senescence. Am J Hematol 11:47, 1981.

75.
Marx JJM: Normal iron absorption and decreased red cell iron uptake in the aged. Blood 53:204, 1979.

76.
Garry PJ, Goodwin JS, Hunt WC: Iron status and anemia in the elderly: new findings and a review of previous studies. J Am Geriatr Soc 31:389, 1983.

77.
Timiras ML, Brownstein H: Prevalence of anemia and correlation of hemoglobin with age in a geriatric screening clinic population. J Am Geriatr Soc 35:639, 1987.

78.
Baldwin JG: Hematopoietic function in the elderly. Arch Intern Med 148:2544, 1988.

79.
Glass GA, Gershon D, Gershon H: Some characteristics of the human erythrocyte as a function of donor and cell age. Exp Hematol 13:1122, 1978.

80.
Sheibon E, Gershon H: Recognition and sequestration of young and old erythrocytes from young and elderly human donors: in vitro studies. J Lab Clin Med 121:493, 1993.

81.
Purcell Y, Brozovic B: Red cell 2,3-diphosphoglycerate concentration in man decreases with age. Nature 241:511, 1974.

82.
Kalofoutis A, Paterakis S, Koutselenis A, Spanos V: Relationship between erythrocyte 2,3-diphosphoglycerate and age in a normal population. Clin Chem 22:1918, 1976.

83.
Detraglia M, Cook FB, Stasiw DM, Cerny LC: Erythrocyte fragility in aging. Biochim Biophys Acta 345:213, 1974.

84.
Araki K, Rifkind JM: Age dependent changes in osmotic hemolysis of human erythrocyte. J Gerontol 35:499, 1980.

85.
Pirrie R: The influence of age upon serum iron in normal subjects. J Clin Path 5:10, 1952.

86.
Powell DEB, Thomas JH, Mills P: Serum iron in elderly hospital patients. Gerontol Clin (Basel) 10:21, 1968.

87.
Rechenberger J: über die Eisenbildungskapazität des Blutserums in den verscheidenen Lebensaltern. Z Alternsforsch 9:98, 1955.

88.
Powell DEB, Thomas JH: The iron-binding capacity of serum in elderly hospital patients. Gerontol Clin (Basel) 11:36, 1969.

89.
Cook JD, Finch CA, Smith NJ: Evaluation of the iron status of a population. Blood 48:449, 1976.

90.
Guyatt GH, Patterson C, Ali M, et al: Diagnosis of iron deficiency anemia in the elderly. Am J Med 88:205, 1990.

91.
Mori M, Murai Y, Hirai M, et al: Serum erythropoietin titers in the aged. Mech Ageing Dev 46:105, 1988.

92.
Powers JS, Lichtenstein MJ, Collins JC, et al: Serum erythropoietin in healthy older persons. J Am Geriatr Soc 37:388, 1989.

93.
Powers JS, Krantz SB, Collins JC, et al: Erythropoietin response to anemia as a function of age. J Am Geriatr Soc 39:30, 1991.

94.
Kario K, Matsuo T, Nakao K: Serum erythropoietin levels in the elderly. Gerontology 37:345, 1991.

95.
Pasqualetti P, Casale R: No influence of aging on the circadian rhythm of erythropoietin in healthy subjects. Gerontology 43: 206, 1997.

96.
Henderson JG, Strachen RW, Swanson Beck J, et al: The antigastrin-antibody test as a screening procedure for vitamin B12 deficiency in psychiatric practice. Lancet 2:809, 1966.

97.
Schilling RF, Fairbanks VF, Miller R, et al: “Improved” vitamin B12 assays: a report on two commercial kits. Clin Chem 29:582, 1983.

98.
Cooper BA, Fehedy V, Blanshay P: Recognition of deficiency of vitamin B12 using measurement of serum concentration. J Lab Clin Med 107:447, 1986.

99.
Thompson WG, Babitz L, Cassino C, et al: Evaluation of current criteria used to measure vitamin B12 levels. Am J Med 82:291, 1987.

100.
Nilsson-Ehle H, Landahl S, Lindstedt G, et al: Low serum cobalamin levels in a population study of 70- and 75-year-old subjects: gastrointestinal causes and hematologic effects. Dig Dis Sci 34:716, 1989.

101.
Lindenbaum J, Rosenberg IH, Wilson PWF, et al: Prevalence of cobalamin deficiency in the Framingham elderly population. Am J Clin Nutr 60:2, 1994.

102.
Carmel R: Cobalamin, the stomach and aging. Am J Clin Nutr 66:750, 1997.

103.
Carmel R, Sinow RM, Siegel ME, Samloff IM: Food cobalamin malabsorption occurs frequently in patients with unexplained low serum cobalamin levels. Arch Intern Med 148:1715, 1988.

104.
Scarlett JD, Read H, O’Dea K: Protein-bound cobalamin absorption declines in the elderly. Am J Hematol 39:79, 1992.

105.
van Asselt DZ, van den Broek MJ, Lamers CB, et al: Free and protein-bound cobalamin absorption in healthy middle-aged and older subjects. J Amer Geriatr Soc 44:949, 1996.

106.
Carmel R: Pernicious anemia. The expected findings of very low serum cobalamin levels, anemia, and macrocytosis are often lacking. Arch Intern Med 148:1712, 1988.

107.
Herbert V: Don’t ignore low serum cobalamin (vitamin B12) levels. Arch Intern Med 148:1705, 1988.

108.
Carmel R, Sinow RM, Karnaze DS: Atypical cobalamin deficiency: subtle biochemical evidence of deficiency is commonly demonstrable in patients with megaloblastic anemia and is often associated with protein-bound cobalamin malabsorption. J Lab Clin Med 109:454, 1987.

109.
Pathy MS, Newcombe RG: Temporal variation of serum levels of vitamin B12, folate, iron, and total iron-binding capacity. Gerontology 26:34, 1980.

110.
Girdwood RH, Thompson AD, Williamson J: Folate status in the elderly. Br Med J 2:670, 1967.

111.
Osterlind PO, Alafuzoff I, Lofgren A-C, et al: Blood components in an elderly population. Gerontology 30:247, 1984.

112.
Hall CA, Bardwell SA, Allen ES, Rappazzo ME: Variation in plasma folate levels among groups of healthy persons. Am J Clin Nutr 28:854, 1975.

113.
Okuno T: Red cell size as measured by the Coulter model S. J Clin Pathol 25:599, 1972.

114.
Okuno T: Smoking and blood changes. JAMA 225:1387, 1973.

115.
Helman N, Rubenstein LS: The effects of age, sex, and smoking on erythrocytes and leukocytes. Am J Clin Pathol 63:35, 1975.

116.
Caird FI, Andrews GR, Gallie TB: The leukocyte count in old age. Age and Ageing 1:239, 1972.

117.
Conrad RA, Demoise CF, Scott WA, Makar M: Immunohematological studies of Marshall Islanders sixteen years after fallout radiation exposure. J Gerontol 26:28, 1971.

118.
Diaz-Jouanen E, Strickland RG, Williams RC Jr: Studies of human lymphocytes in the newborn and the aged. Am J Med 58:620, 1975.

119.
MacKinney AA Jr: Effect of aging on the peripheral blood lymphocyte count. J Gerontol 33:213, 1978.

120.
Jamil NAK, Millard RE: Studies of T, B, and “null” blood lymphocytes in normal persons of different age groups. Gerontology 27:79, 1981.

121.
Polednak AP: Age changes in differential leukocyte count among female adults. Hum Biol 50:30, 1978.

122.
Allan RN, Alexander MK: A sex difference in the leukocyte count. J Clin Pathol 21:691, 1968.

123.
Cruickshank JM, Alexander MK: The effect of age, sex, parity, haemoglobin level, and oral contraceptive preparations on the normal leukocyte count. Br J Haematol 18:541, 1970.

124.
Globerson A: T lymphocytes and aging. Int Arch Allergy Immunol 107:491, 1995.

125.
Miller RA: The aging immune system. Science 273:70, 1996.

126.
Wick G, Grubeck-Lobenstein B: The aging immune system: primary and secondary alterations of immune reactivity in elderly. Exp Gerontol 32:401,1997.

127.
Thomas JH, Powell DEB: Blood Disorders in the Elderly, p 18. John Wright and Sons, Bristol, UK, 1971.

128.
Thorbjarharson B, Loehr WJ: Acute appendicitis in patients over the age of sixty. Surg Gynecol Obstet 125:1277, 1967.

129.
Peitokallio P, Jauhiainen K: Acute appendicitis in the aged patients: study of 300 cases after the age of 60. Arch Surg 100:140, 1970.

130.
Sasso RD, Hanna EA, Moore DL: Leukocyte and neutrophil counts in acute appendicitis. Am J Surg 120:563, 1970.

131.
Fedullo AJ, Swinburne AJ: Relationship of patient age to clinical features and outcome for in-hospital treatment of pneumonia. J Gerontol 40:29, 1985.

132.
Timaffy M: A comparative study of bone marrow function in young and old individuals. Gerontol Clin (Basel) 4:13, 1962.

133.
Cream JJ: Prednisolone-induced granulocytosis. Br J Haematol 15:259, 1968.

134.
Buchanan JP, Peters CA, Rasmussen CJ, Rothstein G: Impaired expression of haematopoietic growth factors: a candidate mechanism for the hematopoietic defect of aging. Exp Gerontol 31:135, 1996.

135.
Corberand JX, Laharrague PF, Fillola G: Neutrophils of healthy aged humans are normal. Mech Ageing Dev 36:57, 1986.

136.
Nagel JE, Han K, Coon PJ, et al: Age differences in phagocytosis by polymorphonuclear leukocytes measured by flow cytometry. J Leukoc Biol 39:399, 1986.

137.
Emanuelli G, Lanzio M, Anfossi T, et al: Influence of age on polymorphonuclear leukocytes in vitro: phagocytic activity in healthy human subjects. Gerontology 32:308, 1986.

138.
Lipschitz DA, Udupa KB, Boxer LA: The role of calcium in the age-related decline of neutrophil function. Blood 71:659, 1988.

139.
Rao KMK, Curie MS, Padmanabhan J, Cohen HJ: Age-related alterations in actin cytoskeleton and receptor expression in human leukocytes. J Gerontol 47:B37, 1992.

140.
Niwa Y, Ishimoto K, Kanoh T: Induction of superoxide dismutase in leukocytes by paraquat: correlation with age and possible predictor of longevity. Blood 76:835, 1990.

141.
Dantew B, Spagnuolo PJ, Goldsmith GGH, Marino JA: Neutrophil adhesion in the elderly: inhibitory effects of plasma from elderly patients. Clin Immunol Immunopathol 54:247, 1990.

142.
Markus HS, Toghill PJ: Impaired splenic function in elderly people. Age and Ageing 20:287, 1991.

143.
Grubeck-Lobenstein: Changes in the aging immune system. Biologicals 25:205, 1997.

144.
Yoshikawa TT: Perspective: aging and infectious diseases: past, present and future. J Infect Dis 176:1053, 1997.

145.
Song L, Kim YH, Chopra RK, et al: Age-related effects in T cell activation and proliferation. Exp Gerontol 28:313, 1993.

146.
Effros RB, Boucher N, Porter V, et al: Decline in CD2 T cells in centenarians and in long-term T cell cultures: a possible cause for both in vivo and in vitro immunosenescence. Exp Gerontol 29:60, 1994.

147.
Grubeck-Lobenstein B, Lechner H, Trieb K: Long-term in vitro growth of human T cell clones: can postmitotic senescent cell population be defined? Int Arch Allergy Immunol 110:278, 1996.

148.
Lechner H, Amort M, Steger MM, et al: Regulation of CD 95 (APO-1) expression and the induction of apoptosis in human T cells: changes in old age. Int Arch Allergy Immunol 110:238, 1996.

149.
Cossarizza A, Ortolani C, Paganelli R, et al: CD4 isoform expression on CD4+ and CD8+ T cells throughout life, from newborn to centenarians. Implication for T cell memory. Mech Aging Dev 86:173, 1996.

150.
Posnett DN, Sinka R, Kabak S, Russo C: Clonal populations of T cells in normal elderly humans: the T cell equivalent to “benign monoclonal gammopathy.” J Exp Med 179:609, 1994.

151.
Schwab R, Szabo P, Manavalan JS, et al: Expanded CD4+ and CD8+ T cell clones in elderly humans. J Immunol 158:4493, 1997.

152.
Schulze DH, Goidl EA: Age-associated changes in antibody-forming cells (B cells). Proc Soc Exp Biol Med 196:253, 1991.

153.
Powers DC: Immunological principles and emerging strategies of vaccination for the elderly. J Am Geriatr Soc 40:81, 1992.

154.
McArthur WP, Bloom K, Taylor M, et al: Peripheral blood leukocyte populations in the elderly with and without periodontal disease. J Clin Periodontol 23:846, 1996.

155.
Miyaji C, Watanabe H, Minagawa M, et al: Numerical and functional characteristics of lymphocyte subsets in centenarians. J Clin Immunol 17:420, 1997.

156.
Ruiz M, Esparza B, Perez C, et al: CD8+ T cell subsets in aging. Immunol Invest 24:891, 1995.

157.
McNerlan SE, Rea IM, Alexander HD, Morris TCM: Changes in natural killer cells, the CD57CD8 subset, and related cytokines in health aging. J Clin Immunol 18:31, 1998.

158.
Moesgaard F, Nielsen ML, Larsen N, et al: Cell-mediated immunity assessed by skin testing (Multitest®). I. Normal values in healthy Danish adults. Allergy 42:591, 1987.

159.
Stead WW, To T: Significance of the tuberculin skin test in elderly patients. Ann Intern Med 107:837, 1987.

160.
Marrie TJ, Johnson S, Durant H: Cell-mediated immunity of healthy adult Nova Scotians in various age groups compared with nursing home and hospitalized senior citizens. J Allergy Clin Immunol 81:836, 1988.

161.
Castle SC, Norman DC, Perls TT, et al: Analysis of cutaneous delayed-type hypersensitivity reaction and T cell proliferative response in elderly nursing home patients: an approach to identifying immunodeficient patients. Gerontology 36:217, 1990.

162.
Wayne SJ, Rhyne RL, Garry PJ, Goodwin JS: Cell-mediated immunity as a predictor of morbidity and mortality in subjects over 60. J Gerontol 45:M45, 1990.

163.
Lichtman MA, Vaughn JH, Hames CG: The distribution of serum immunoglobulins, anti-gamma G globulins (“rheumatoid factors”), and antinuclear antibodies in Evans County, Georgia. Arthr Rheum 10:204, 1967.

164.
Axelsson U, Bachmann R, Hällén J: Frequency of pathological proteins (M-components) in 6995 sera from an adult population. Acta Med Scand 179:235, 1966.

165.
Hällén J: frequency of “abnormal” serum globulins (M-components) in the aged. Acta Med Scand 173:737, 1963.

166.
Van Rensburg EJ, Heyns A du P: The effect of age, arteriosclerosis and hypercholesterolemia on platelet function tests, in Thrombotic and Haemorrhagic Disorders, Springer-Verlag, 1990.

167.
Zahavi J, Jones NRG, Leyton J, et al: Enhanced in vivo platelet “release reaction” in old healthy individuals. Thromb Res 17:329, 1980.

168.
Fetkovska N, Amstein R, Ferraein F, et al: 5HT-kinetics and sensitivity of human blood platelets: variations with age, gender and platelet number. Thromb Haemost 60:486, 1988.

169.
Kasjanovova D, Balaz V: Age-related changes in human platelet function in vitro. Mech Ageing Dev 37:175, 1986.

170.
Winther K, Naesh O: Aging and platelet b-adrenoceptor function. Eur J Pharmacol 136:219, 1987.

171.
Vericel E, Croset M, Sedivy P, et al: Platelets and aging. I. Aggregation, arachidonate metabolism and antioxidant status. Thromb Res 49:331, 1988.

172.
Winther K, Naesh O: Platelet alpha-adrenoreceptor function and aging. Thromb Res 46:677, 1987.

173.
Bastyr EJ, Kadrofske MM, Vinik AI: Platelet activity and phosphoinositide turnover increase with advancing age. Am J Med 88:601, 1990.

174.
Wang H-Y, Bashore TR, Friedman E: Exercise reduces age-dependent decrease in platelet protein kinase C activity and translocation. J Gerontol 50A:M12, 1995.

175.
Kario K, Matsuo T, Kobayashi H, et al: Close relationship between hemostatic factors and acute-phase reaction as normal aging process. J Am Geriatr Soc 44:614, 1996.

176.
Mari D, Mannucci PM, Coppola R, et al: Hypercoagulability in centenarians: the paradox of successful aging. Blood 85:3144, 1995.

177.
Haverkate F, Thompson SG, Duckert F: Haemostasis factors in angina pectoris: relation to gender, age and acute phase reaction: results from the ECAT Angina Pectoris Study Group. Thromb Haemost 73:561, 1995.

178.
Balleisen L, Bailey J, Epping P-H, et al: Epidemiological study on factor VII, factor VIII and fibrinogen in an industrial population. I. Baseline data on the relation to age, gender, body weight, smoking, alcohol, pill-using, and menopause. Thromb Haemost 54:475, 1985.

179.
Scarabin PY, Van Dreden P, Bonithon-Kop C, et al: Age-related changes in factor VII activation in healthy women. Clin Sci 75:341, 1988.

180.
Conlan MG, Folsom AR, Finch A, et al: Associations of Factor VIII and von Willebrand factor with age, race, sex, and risk factors for atherosclerosis. Thromb Haemost 70:380, 1993.

181.
Ernst E, Rosch KL: Fibrinogen as a cardiovascular risk factor: a meta-analysis and review of the literature. Ann Intern Med 118:956, 1993.

182.
Bauer KA, Weiss LM, Sparrow D, et al: Aging associated changes in indices of thrombin generation and protein C activation in humans: normative aging study. J Clin Invest 80:1527, 1987.

183.
Sundell B, Nilsson TK, Rainby M, et al: Fibrinolytic variables are related to age, sex, blood pressure, and body build measurements: a cross-sectional study in Norsjo, Sweden. J Clin Epidemiol 42:719, 1989.

184.
Eliasson M, Evrin PE, Lundblad D, et al: Influence of gender, age, sampling time on fibrinolytic variables and fibrinogen. A population study. Fibrinolysis 7:316, 1993.

185.
Siegert G, Bergmann S, Jaross W: Influence of age, gender and lipoprotein metabolism parameters on the activity of plasminogen activator inhibitor and the fibrinogen concentration. Fibrinolysis 6 (suppl 3):47, 1992.

186.
Cawkwell RC: Patient’s age and the activated partial thromboplastin time test. Thromb Haemost 39:780, 1978.

187.
Ibbotson SH, Tate GM, Davies JA: Thrombin activity by intrinsic activation of plasma in-vitro accelerates with increasing age of the donor. Thromb Haemost 67:377, 1992.

188.
Kario K, Matsuo T, Kobayashi H: Which factors affect high D-dimer levels in the elderly? Thromb Res 62:501, 1991.

189.
Gurwitz JH, Avorn J, Ross-Oegnan D, et al: Aging and the anticoagulant response to warfarin therapy. Ann Intern Med 116:901, 1992.

190.
Boyd RV, Hoffbrand BI: Erythrocyte sedimentation rate in elderly hospital in-patients. Br Med J 1:901, 1966.

191.
Böttiger LE, Svedberg CA: Normal erythrocyte sedimentation rate and age. Br Med J 2:85, 1967.

192.
Sharland DE: Erythrocyte sedimentation rate: the normal range in the elderly. J Am Geriatr Soc 28:346, 1980.

193.
Sparrow D, Rowe JW, Silbert JE: Cross-sectional and longitudinal changes in the erythrocyte sedimentation rate in man. J Gerontol 36:180, 1981.

194.
Shearn MA, Kang IY: Effect of age and sex on the erythrocyte sedimentation rate. J Rheumatol 13:297, 1986.

195.
Katz PR, Gutman SJ, Richman G, et al: Erythrocyte sedimentation rate and C-reactive protein compared in the elderly. Clin Chem 35:466, 1989.

196.
Katz PR, Karuza J, Gutman SI, et al: A comparison between erythrocyte sedimentation rate (ESR) and selected acute-phase proteins in the elderly. Am J Clin Pathol 94:637, 1990.

197.
Ballou SP, Lozanski GP, Hadder S, et al: Quantitative and qualitative alterations of acute phase proteins in healthy elderly persons. Age and Ageing 25:224, 1996.

198.
Caswell M, Pike LA, Bull BS: Effect of patients’ age on tests of the acute-phase response. Arch Pathol Lab Med 117:906, 1993.

199.
Globerson A: Hematopoietic stem cells and aging. Exp Gerontol 34:137, 1999.

200.
Pawelec G, Solana R, Remarque E, Mariani E: Impact of aging on innate immunity. J Leuk Biol 64:703, 1998.

201.
Solana R, Alonso MC, Pena J: Natural killer cells in healthy aging. Exp Gerontol 34:435, 1999.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

CHAPTER 7 HEMATOLOGY OF THE NEWBORN

CHAPTER 7 HEMATOLOGY OF THE NEWBORN
Williams Hematology

CHAPTER 7 HEMATOLOGY OF THE NEWBORN

GEORGE B. SEGEL
JAMES PALIS

Fetal Hematopoiesis

Production of Embryonic and Fetal Hematopoietic Cells

Ontogeny of Hematopoietic Stem Cells

Synthesis of Fetal Hemoglobins

Fetal Blood
Neonatal Hematopoiesis

Red Cells

White Cells

Platelets

Neonatal Lymphopoiesis

Coagulation in the Neonate

Hematologic Effects of Maternal Drugs on the Fetus and Newborn
Chapter References

A newborn represents the culmination of developmental events from conception and implantation through organogenesis. The embryo requires red cells for the transport of maternal oxygen to permit this growth and development. Birth brings dramatic changes in circulation and oxygenation, which affect hematopoiesis, as the newborn makes the transition to a separate biological existence. This chapter discusses the ontogeny of hematopoiesis and focuses on hematopoiesis of the normal newborn.

Acronyms and abbreviations that appear in this chapter include: AGM, aorta-gonad-mesonephros; BFU-E, burst forming unit–erythroid; BMP, bone morphogenetic protein; BPG, bisphosphoglycerate; CFU-E, colony forming unit–erythroid; CFU-GEMM, colony forming unit–granulocyte-erythroid-monocyte-macrophage; CFU-GM, colony forming unit–granulocyte-monocyte; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-monocyte colony stimulating factor; IL, interleukin; MCV, mean cell volume; NBT, nitroblue tetrazolium; SIDS, sudden infant death syndrome; TNF, tumor necrosis factor

FETAL HEMATOPOIESIS
PRODUCTION OF EMBRYONIC AND FETAL HEMATOPOIETIC CELLS
During embryogenesis, hematopoiesis occurs in spatially and temporally distinct sites, including the extraembryonic yolk sac, the fetal liver, and the preterm bone marrow. Erythropoiesis is established soon after implantation of the blastocyst, with primitive erythroid cells appearing in yolk sac blood islands by day 18 of gestation.1,2 The origin of hematopoietic cells in mammals is tied closely to gastrulation and the formation of mesoderm. Inducers of mesoderm, including transforming growth factor b (TGF-b), fibroblast growth factor, and bone morphogenetic protein-4 (BMP-4), likely are important molecules regulating the onset of hematopoiesis.3 Yolk sac erythroblasts arise in close association with the first embryonic blood vessels, suggesting that endothelial cells and blood cells arise from a common hemangioblast precursor.
YOLK SAC HEMATOPOIESIS
The development of primitive erythroblasts in the yolk sac is critical for embryonic survival. In the mouse, targeted disruption of the murine transcription factors SCL (TAL1), LMO2 (RBTN2), and GATA-1 each abrogates primitive erythropoiesis in the yolk sac and leads to early embryonic death.4,5 and 6 Yolk sac erythroblasts have several characteristics distinguishing them from their later definitive counterparts. Primitive erythroblasts differentiate within the vascular network rather than in the extravascular space and remain nucleated as they circulate. Primitive erythroblasts are characterized by more rapid maturation, increased sensitivity to erythropoietin, and a shortened life span compared to fetal and adult erythroblasts.7 Yolk sac erythroblasts are extremely large red cells (megaloblasts) with an estimated mean cell volume (MCV) of >450 fl/cell.
The erythroid progenitors, burst-forming units-erythroid (BFU-E), and the later erythroid progenitors, colony-forming units-erythroid (CFU-E), are present in the yolk sac at 4 weeks gestation.8 Primitive erythroblasts and erythroid progenitors then enter the embryo proper through the circulation. BFU-E appear in the fetal liver as early as 5 weeks of gestation, and CFU-E are evident soon thereafter.8 Erythroid and nonerythroid progenitors are evident also in the nonliver regions of the embryo proper.9 After 7 weeks gestation, hematopoietic progenitors are no longer detected in the yolk sac.10 Yolk sac derived primitive erythroblasts continue to circulate until approximately 12 weeks of gestation.
HEPATIC HEMATOPOIESIS
The liver serves as the primary source of red cells from the 9th to the 24th weeks of gestation. Between 7 and 15 weeks gestation, 60 percent of the liver cells are hematopoietic.11 Erythroid cells differentiate in close association with macrophages and extrude their nuclei prior to entering the blood stream. These fetal liver-derived definitive “macrocytes” are smaller than yolk sac megaloblasts and contain one-third the amount of hemoglobin. Differentiation of erythroid cells in the fetal liver is dependent on erythropoietin signaling through its receptor and the JAK2 kinase.12,13 Fetal liver-derived erythroid progenitors will differentiate in vitro with erythropoietin alone, in contrast to adult bone marrow-derived BFU-E that require erythropoietin plus interleukin-3 (IL-3).14,15 Erythropoietin transcripts are present during the first trimester in the liver.9 The liver remains the primary site of erythropoietin transcription throughout fetal life.16 Erythropoietin transcripts also are present in the developing human kidney as early as 17 weeks of gestation and increase after 30 weeks.16 Erythropoietin is expressed both in the fetal liver and in the postnatal kidney.16 Like primitive erythropoiesis in the yolk sac, definitive erythropoiesis in the fetal liver is necessary for continued survival of the embryo. Targeted disruption of the c-myb and EKLF transcription factors in the mouse each blocks fetal liver erythropoiesis and leads to fetal death.17,18 These mutations do not effect yolk sac erythropoiesis, indicating fundamental differences in the transcriptional regulation of these distinct forms of erythropoiesis.
In contrast to the yolk sac, where hematopoiesis is restricted to erythroid and macrophage cells, hematopoiesis in the fetal liver also includes other myeloid as well as lymphoid lineages. Megakaryocytes are present in the liver by 6 weeks of gestation. Platelets are first evident in the circulation at 8–9 weeks gestation.11 Small numbers of circulating leukocytes are present at the 11th week of gestation.2 Granulopoiesis is present in the liver parenchyma and in some areas of connective tissue as early as 7 weeks gestation. Despite the low number and immature appearance of hepatic neutrophils, the fetal liver contains abundant hematopoietic progenitor cells, including colony-forming unit–granulocyte-erythroid-monocyte-macrophage (CFU-GEMM) and colony-forming unit–granulocyte-monocyte (CFU-GM).19,20 CFU-GM growth depends upon several cytokines, including granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), and interleukins.21 When compared to adult bone marrow-derived myeloid progenitors, these fetal liver derived myeloid progenitors have a similar dose response in vitro to G-CSF.22 G-CSF is expressed by hepatocytes at 14 weeks gestation.23
MARROW HEMATOPOIESIS
Hematopoietic cells are first seen in the marrow of the 10- to 11- week embryo,1,2 and they remain confined to the diaphyseal regions of long bones until 15 weeks gestation.24 Initially there are approximately equal numbers of myeloid and erythroid cells in the fetal marrow. However, myeloid cells predominate by 12 weeks gestation, and the myeloid to erythroid ratio approaches the adult level of 3 to 1 by 21 weeks gestation.11 Macrophage cells in the fetal marrow, but not in the fetal liver, express the lipopolysaccharide receptor CD14.23 The marrow becomes the major site of hematopoiesis after the 24th week of gestation.
LYMPHOPOIESIS
Lymphopoiesis is present in the lymph plexuses and the thymus beginning at 9 weeks gestation.11 B cells with surface IgM are present in the liver, and circulating lymphocytes also are seen at 9 weeks gestation.2 Lymphocyte subpopulations are detected by 13 weeks gestation in fetal liver.25 Absolute numbers of major lymphoid subsets in 20–26-week-old fetuses, as defined by the antigens CD2, CD3, CD4, CD8, CD19, CD20 and CD16 (see Chapter 14 for functional significance of these phenotypes), are similar to those in newborns (see “Neonatal Lymphopoiesis”).26,27
ONTOGENY OF HEMATOPOIETIC STEM CELLS
The reconstitution of hematopoiesis by transplantation with cord blood indicates that hematopoietic stem cells are present at birth.28 However, the developmental origin of hematopoietic stem cells has not yet been defined. It was first postulated that hematopoietic stem cells originate independently in each hematopoietic site (yolk sac, liver, and bone marrow) of the embryo.29 However, experiments in the mammalian embryo indicate that the liver rudiment is seeded by exogenous hematopoietic cells.30,31 The marrow also is seeded by exogenously derived blood cells. Fetal liver provides a source of stem cells for myeloid and lymphoid reconstitution of fetal sheep and monkey transplant recipients.32 The immunological reconstitution of an immunodeficient human fetus with fetal liver-derived cells also indicates that hematopoietic stem cells exist in the fetal liver.13
Yolk sac stem cells were first thought to seed the liver and eventually the bone marrow.33 However, later experiments in avian and amphibian embryos indicated that the hematopoietic stem cells that seed the marrow arise within the body of the embryo proper rather than from the yolk sac.34,35 Investigations in the mouse embryo also suggest that prior to the fetal liver, the aorta-gonad-mesonephros (AGM) region of the embryo proper contains stem cells capable of engrafting myeloablated adult recipients.36 This correlates anatomically with the transient appearance of CD34-positive blood cells closely associated with the ventral wall of the aorta in several mammalian species, including the 5-week gestation human embryo.37,38 These studies suggest that the AGM-region-derived stem cells seed the liver and the marrow to provide lifelong hematopoiesis. The underlying relationship of primitive hematopoiesis in the yolk sac to definitive hematopoiesis in the fetal liver and the marrow is unclear.
SYNTHESIS OF FETAL HEMOGLOBINS
Human hemoglobin is a tetramer composed of two a-type and two b-type globin chains (Table 7-1). The a-globin gene cluster is located on chromosome 16 and contains the z gene 5′ to the pair of a-globin genes. The b-globin gene cluster is located on chromosome 11 and contains five globin genes oriented 5′ to 3′ as e-gA-gG-d-b.39 During embryogenesis the genes on both chromosomes are activated sequentially from the 5′ to the 3′ end. This globin “switching” is related not only to the relative positions of the globin genes within their respective chromosomal clusters, but also to interacting upstream “locus control regions.”40

TABLE 7-1 EMBRYONIC HEMOGLOBINS

Hb Gower 1 (z2e2), is the major hemoglobin in embryos less than 5 weeks of gestation (see Table 7-1).41 Hb Gower 2 (a2e2) has been found in embryos with a gestational age as young as 4 weeks and is absent in embryos older than 13 weeks.42 Hb Portland (z2g2) is found in young embryos but persists in infants with homozygous a thalassemia.11 Synthesis of the z and e chains decreases as that of a and g chains increases (Figure 7-1). The z to a globin switch precedes the e to g globin switch as the liver replaces the yolk sac as the main site of erythropoiesis.43,44

FIGURE 7-1 Changes in hemoglobin tetramers (a) and in globin subunits (b) during human development from embryo to early infancy. (Reproduced from HF Bunn and BG Forget, Hemoglobin: Molecular, Genetic and Clinical Aspects, Saunders, Philadelphia, 1986, with permission.)

Hb F (a2g2) is the major hemoglobin of fetal life45 (see Figure 7-1). Synthesis of Hb A can be demonstrated in fetuses as young as 9 weeks of gestation.46,47 In fetuses of 9 to 21 weeks of gestation, the amount of Hb A (a2b2) rises from 4 to 13 percent of the total hemoglobin.47 These levels of Hb A have enabled the antenatal diagnosis of b thalassemia using globin chain synthesis. After 34 to 36 weeks of gestation the percentage of Hb A rises, while that of Hb F decreases (see Figure 7-1). The mean synthesis of Hb F in term infants was 59.0 ± 10 percent (1 SD) of total hemoglobin synthesis as assessed by 14C-leucine uptake.48 The amount of Hb F in blood varies in term infants from 53 to 95 percent of total hemoglobin.49,50
The fetal hemoglobin concentration in blood decreases after birth by approximately 3 percent per week and is generally less than 2 to 3 percent of the total hemoglobin by 6 months of age. This rate of decrease in Hb F production is closely related to the gestational age of the infant and is not affected by the changes in environment and oxygen tension that occur at the time of birth.51 Hb A2 (a2d2) has not been detected in fetuses. Normal adult levels of Hb A2 are achieved by four months of age.52 Increased proportions of Hb F at birth have been reported in infants who are small for gestational age, who have experienced chronic intrauterine hypoxia, or who have trisomy 13.53,54,55 and 56 Decreased levels of Hb F at birth are found in trisomy 21.57 Persistence of the embryonic Hb Gower-1, Hb Gower-2, and Hb Portland has been described in some infants with developmental abnormalities, while persistently elevated levels of fetal hemoglobin have been observed in infants dying from the sudden infant death syndrome (SIDS).58
FETAL BLOOD
The fetal blood composition changes markedly during the second and third trimesters. The mean hemoglobin in fetuses progressively increases from 9.0 ± 2.8 g/dl at age 10 weeks to 16.5 ± 4.0 g/dl at 39 weeks.59 There is a concomitant decrease in the MCV of fetal red cells from a mean of 134 fl/cell at 18 weeks to 118 fl/cell at 30 weeks gestation.60 The total white blood cell count during the middle trimester is between 4 and 4.5 × 109/liter, with an 80 to 85 percent preponderance of lymphocytes and 5 to 10 percent neutrophils.60 The percentage of circulating nucleated red cells decreases from a mean of 12 percent at 18 weeks to 4 percent at 30 weeks.60 The platelet count remains greater than 150,000/µl from 15 weeks gestation to term.60,61
Large numbers of committed hematopoietic progenitors circulate in the fetal blood. Blood samples obtained by fetoscopy at 12 to 19 weeks of gestation reveal a mean of 20,450 BFU-E/ml and 12,490 CFU-GM/ml.62 This is in striking contrast to adult peripheral blood, which contains essentially no erythroid progenitors and 30 to 250 CFU-GM/ml.63 The cycling rate of 26 to 28 week gestation fetal hematopoietic progenitors is nearly maximal (70–80%) compared to the relative quiescence (0–5%) of adult marrow-derived progenitors.63
NEONATAL HEMATOPOIESIS
RED CELLS
NEONATAL ERYTHROPOIESIS
Hemoglobin, Hematocrit, and Indices The mean hemoglobin level in cord blood at term is 16.8 g/dl, with 95 percent of the values falling between 13.7 and 20.1 g/dl.64 This variation reflects perinatal events, particularly asphyxia,65 and also the amount of blood transferred from the placenta to the infant after delivery. Delay of cord clamping may increase the blood volume and red cell mass of the infant by as much as 55 percent.66,67 The mean total blood volume after birth is 86.3 ml/kg for the term infant and 89.4 ml/kg for the premature infant.68 The blood volume per kilogram decreases over the ensuing weeks, reaching a mean value of about 65 ml/kg by 3 to 4 months of age.
Normally the hemoglobin and hematocrit values rise in the first several hours after birth because of the movement of plasma from the intravascular to the extravascular space.69 A venous hemoglobin concentration of less than 14 g/dl in a term infant and/or a fall in hemoglobin or hematocrit level in the first day of life are abnormal. Normal red cell values from capillary blood samples are shown in Table 7-2 for term infants in the first 12 weeks of life.70 Capillary hematocrit values in newborns are higher than those in simultaneous venous samples, particularly during the first days of life, and the capillary/venous ratio is approximately 1.1:.71 This difference reflects circulatory factors and is greater in preterm and sick infants.

TABLE 7-2 RED CELL VALUES FOR TERM INFANTS DURING THE FIRST 12 WEEKS OF LIFE*

The red cells of the newborn are macrocytic, with a mean cell volume (MCV) in excess of 110 fl/cell. The MCV begins to fall after the first week, reaching adult values by the ninth week (see Table 7-2).70,72 The blood film from a newborn infant shows macrocytic normochromic cells, polychromasia, and a few nucleated red blood cells. Even in healthy infants there may be mild anisocytosis and poikilocytosis.73 Three to 5 percent of the red cells may be fragments, target cells, or distorted. By 3 to 5 days after birth, nucleated red blood cells are not found normally in the blood of term or premature infants, but they may be present in markedly elevated numbers in the presence of hemolysis or hypoxic stress.
There are significant numbers of circulating progenitor cells in cord blood.74,75,76 and 77 Cord blood BFU-E and CFU-E differentiate more rapidly than their adult counterparts.78 Furthermore, the proportion of cord blood hematopoietic progenitors in the mitotic cycle is approximately 50 percent, intermediate between the proportions found in fetal and adult progenitor cells.76
In several,79,80 but not all, studies81 premature infants at birth had lower hemoglobin levels, higher reticulocyte counts, and higher nucleated red cell counts than the term infants. The reticulocyte counts of premature infants are inversely proportional to their gestational age, with a mean of 8 percent reticulocytes evident at 32 weeks gestation and 4 to 5 percent at term.82 Infants who are small for their gestational ages have higher red cell counts, hematocrit levels, and hemoglobin concentrations compared to infants whose size is appropriate for their gestational age.80,83
Erythropoietin and Physiologic Anemia of the Newborn Erythropoietin is the primary regulator of erythropoiesis. While erythropoietin is present in cord blood, it falls to undetectable levels after birth in healthy infants.84 Subsequently, the reticulocyte count falls to less than one percent by the sixth day of life.85 The red cell, hemoglobin, and hematocrit values decrease only slightly during the first week but decline more rapidly in the following 5 to 8 weeks (see Table 7-2),70 producing the physiologic anemia of the newborn.86 The lowest hemoglobin values in the term infant occur at about 2 months of age.72 When the hemoglobin concentration falls below 11 g/dl, erythropoietic activity begins to increase. Erythropoietin can be measured after the 60th day of life,87 corresponding to the recovery from physiologic anemia. If there is sufficient stimulus, such as hemolytic anemia or cyanotic heart disease, the newborn infant is able to produce erythropoietin during the first several months of life.84
In the premature infant the fall in hemoglobin level is more pronounced. In one study of premature infants the mean hemoglobin level at 2 months was 9.4 g/dl, with a 95 percent range of 7.2 to 11.7 g/dl.88 In healthy premature infants erythropoietin becomes detectable when the hemoglobin level falls to about 12 g/dl. In infants with a lower percentage of Hb F (as from transfusion) and consequently better oxygen delivery, erythropoietin does not rise until the hemoglobin falls to about 9.5 g/dl.89 The mean values for iron-sufficient premature infants reached those of term infants by 4 months for red cell count, 5 months for hemoglobin level, and 6 months for mean corpuscular volume and mean corpuscular hemoglobin.88
Blood Viscosity The viscosity of blood increases logarithmically in relation to the hematocrit.90,91 Hyperviscosity has been found in 5 percent of infants in one series92 and in 18 percent of infants who are small for gestational age in another.93 Newborn infants with hematocrit values of greater than 65 to 70 percent may become symptomatic because of increased viscosity.94 Of 45 infants with documented hyperviscosity and a mean hematocrit greater than 65 percent, 17 (38%) had symptoms of irritability, hypotonia, tremors, or poor suck reflex.95 Partial plasma exchange transfusion reduced blood viscosity, improved cerebral blood flow, and relieved the symptoms. However, cerebral blood flow was normal in the asymptomatic infants with hyperviscosity, and there consequently was no benefit from exchange transfusion.95
Red Cell Antigens The blood group antigens on neonatal red cells differ from those of the older child and adult. The i antigen is expressed strongly while the I antigen and the A and B antigens are expressed only weakly on neonatal red cells. The i antigen is a straight-chain carbohydrate which is replaced by the branched-chain derivative, I antigen, as a result of the developmental acquisition of a glycosyltransferase.96 By one year of age the i antigen has become undetectable, and the ABH antigens increase to adult levels by age 3. The ABH, Kell, Duffy and Vel antigens can be detected on the cells of the fetus in the first trimester and are present at birth.97 The Lua and Lub antigens also are detectable on fetal red cells and are more weakly expressed at birth, increasing to adult levels by age 15.97 The Xg antigen is variably expressed in the fetus and is weaker on newborn than adult red cells. Moreover, particularly poor expression of Xg has been noted in newborns with trisomy 13, 18, and 21.97 The Lewis group (Lea/Leb) antigens are adsorbed on the red cell membrane and become detectable within 1 to 2 weeks after birth as the receptor sites develop. Anti-A and anti-B as isohemagglutinins develop during the first 6 months of life, reaching adult levels by 2 years of age.
Red Cell Life Span The life span of the red cells in the newborn infant is shorter than that of red cells in the adult. The average of several studies of mean half-life of newborn red cells labeled with chromium was 23.3 days in term infants and 16.6 days in premature infants. When corrected for the elution rate of chromium from newborn cells, the estimate of mean red cell survival in the newborn is 60 to 80 days.98 The reasons for this shortened survival are unclear, but the known susceptibility to oxidant injury of newborn red cells may be a contributing factor.
Iron and Transferrin The serum iron level in cord blood of the normal infant is elevated compared to maternal levels. The mean value is about 150 ± 40 µg/dl (1 SD).99 Infants on an iron-supplemented diet have a median serum iron level of 125 µg/dl at 1 month of age and of about 75 µg/dl at 6 months of age. The total iron- binding capacity rises throughout the first year of life. The median transferrin saturation falls from almost 65 percent at 0.5 months to 25 percent at 1 year, and saturations as low as 10 percent may be observed in the absence of iron deficiency.100 The mean serum ferritin levels in iron-sufficient infants are high at birth, 160 µg/l, rise further during the first month, and then fall to a mean of 30 µg/l by 1 year of age.101 The amount of stainable iron in the marrow at birth is small but increases in both term and premature infants during the first weeks of life. Stainable marrow iron begins to decrease after 2 months and is gone by 4 to 6 months in term infants and earlier in premature infants.102
RED CELL FUNCTIONS
Oxygen Delivery The oxygen affinity of cord blood is greater than that of maternal blood, since the affinity of Hb F for 2,3-bisphosphoglycerate (2,3-BPG) is less than that of Hb A.103 Levels of 2,3-BPG are lower in newborn red cells than in adult cells and even more decreased in the red cells of premature infants,104 and this low 2,3-BPG level further heightens the oxygen affinity of newborn red cells. Thus, the red cell oxygen equilibrium curve of the newborn is shifted to the left of that of the adult (Figure 7-2). The mean partial pressure of oxygen at which hemoglobin is 50 percent saturated with oxygen at 1 day of age in term infants is 19.4 ± 1.8 torr, as compared with the normal adult value of 27.0 ± 1.1 torr.105 This results in a decrease in the oxygen released at the tissue level, as shown in Figure 7-2. As the PO2 falls from 90 torr in arterial to 40 torr in the venous blood, 3.0 ml/dl of oxygen are released from newborn blood, while 4.5 ml/dl are released from adult, Hb A-containing blood. The shift to the left of the oxygen equilibrium curve is even more pronounced in the premature infant, requiring a larger fall in PO2 to release an equivalent amount of oxygen. After birth the oxygen equilibrium curve shifts gradually to the right, reaching the position of the adult curve by 6 months of age. The position of the curve in the premature infant correlates with gestational age rather than with postnatal age,105 and its shift to the adult position is more gradual.

FIGURE 7-2 The oxygen equilibrium curves are based on the assumption that the Hb concentration is 15 g/dl and that there is full O2 saturation of Hb at a PO2 of 100 torr. The O2 released is the difference in O2 content between a PO2 of 90 torr and the mixed venous PO2 of 40 torr. The O2 available is the difference in O2 content between a PO2 of 90 torr and a mixed venous PO2 of 20 torr. This is the maximum O2 available without evoking compensatory mechanisms such as increased cardiac output.

Metabolism Many differences have been found between the metabolism of the red cells of newborn infants and that of adults.106,107 Some of the differences may be explained by the younger mean cell age in the newborn, but others seem to be properties of the fetal cell. The glucose consumption in newborn cells is lower than that in adult cells.108 Elevated levels of glucose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase beyond those explainable by the young cell age have been found in neonatal cells.104,109 The level of phosphofructokinase is low in red cells of term and premature infants.104,109,110 The pentose phosphate shunt is active in red cells of term and premature infants,111 but there is glutathione instability and a heightened susceptibility to oxidant injury. Furthermore, there is relative instability of the 2,3-BPG concentration. Lower-than-adult activities have been found for several other red cell enzymes, including NADP-dependent methemoglobin reductase112 and glutathione peroxidase.113 The levels of ATP and ADP are higher in the red cells of term and preterm infants110 but may merely reflect the younger age of the erythrocyte population.114
Membrane The membrane of the newborn red cell also is different from that of the adult red cell. Ouabain-sensitive ATPase is decreased,115 and active potassium influx is significantly less in neonatal red cells.116 Newborn cells are more sensitive to osmotic hemolysis and to oxidant injury than are adult cells. Newborn red cell membranes have higher total lipid, phospholipid, and cholesterol per cell than adult red cells.117,118 The patterns of phospholipid and phospholipid fatty acid composition also differ from those in adult red cells. Red cells of newborns have the same pattern of membrane proteins on polyacrylamide gel electrophoresis119 and the same rate of mobility in an electric field120 as do red cells from adults. After trypsin treatment of newborn and adult cells, however, there is a difference in electrophoretic mobility, indicating that the surface trypsin-resistant proteins are different.120 The relationship of the metabolic and membrane alterations in neonatal red cells to their shorter life span is not clear.
WHITE CELLS
NEONATAL GRANULOPOIESIS
Colony-Stimulating Factors and Granulo-monopoiesis The absolute number of neutrophils in the blood of term and premature infants is usually greater than that found in older children (Table 7-3).121 The neutrophil count tends to be lower in the premature than in the term infant, and the proportion of myelocytes and band neutrophils is higher.122 Serum and urinary colony-stimulating activity are elevated during the period of neutrophilia.123,124 When granulopoiesis was studied in cord blood, blood, and marrow of infants, the macrophage colony-forming unit was predominant in spite of the clinical neutrophilia, and this pattern was not altered by different sources of colony-stimulating factors.125,126 The endogenous cytokines produced by mononuclear cells from cord or systemic venous blood support the growth of neutrophil colonies in assays using marrow from adults.125 However, there is diminished GM-CSF, G-CSF, and IL-3 production and diminished mRNA expression in stimulated newborn compared to adult mononuclear cells,127,128 and 129 which may limit the response to bacterial infection in the newborn. Smaller numbers of CFU-GM colonies were observed in the blood of sick infants, who have diminished endogenous production of CSF in culture.126 Dysregulation of neonatal granulopoiesis may impair the neonatal response to infection.130 The administration of stem cell factor with G-CSF to newborn rats reduces the mortality of experimental group B streptococcal infection, and this approach may be useful in human disease.131

TABLE 7-3 THE WHITE CELL COUNT AND THE DIFFERENTIAL COUNT DURING THE FIRST 2 WEEKS OF LIFE*

White Cell and Differential Counts The values for the white cell and differential counts during the first 2 weeks of life are given in Table 7-3. The absolute number of segmented neutrophils rises in both term and premature infants in the first 24 h of life.132 In term infants the mean value increases from 8 × 109/liter (8000/µl) to a peak of 13 × 109/liter (13,000/µl) and then falls to 4 × 109/liter (4000/µl) by 72 h of age, remaining at this level through the following 7 days. In the premature infant the mean values for neutrophils are 5 × 109/liter (5000/µl) at birth, 8 × 109/liter (8000/µl) at 12 h, and 4 × 109/liter (4000/µl) at 72 h. The mean count then falls gradually to 2.5 × 109/liter (2500/µl) by the 28th day of life. The level after the first 72 h is very stable for an individual infant, whether term or premature. Immature forms, including an occasional promyelocyte and blast cell, may be seen in the blood of healthy infants in the first few days of life and are more frequent in premature infants than in term infants.132 Segmented granulocytes are the predominant cells in the first few days of life. As their number decreases, the lymphocyte becomes the most numerous cell and remains so during the first 4 years of life. An absolute eosinophil count of greater than 0.7 × 109/liter (700/µl) was found in 76 percent of premature infants at 2 to 3 weeks of age. The onset of the eosinophilia coincided with the establishment of steady weight gain in the infants.133 It is increased by the use of total parenteral nutrition, endotracheal intubation, and blood transfusions.
PHAGOCYTE FUNCTIONS
Bacterial infections are a major cause of morbidity and mortality in the newborn period.134 The infections are frequently due to organisms of low virulence in normal children and adults, including Staphylococcus, Lancefield group B b-hemolytic streptococci, Pseudomonas, and other gram-negative bacilli. Cellular defense mechanisms and humoral immunity of the newborn differ from those found later in life, and these undoubtedly contribute to the unusual susceptibility to infection noted in the neonatal period.134
Opsonins and Complement Engulfment and destruction of bacteria by neutrophils depend on opsonic activity of the plasma and on chemotaxis, phagocytosis, and the bacteriocidal capacity of the leukocyte. The serum factors necessary for optimal phagocytosis (opsonins) include the immunoglobulins and complement components. In term infants, opsonic activity is normal for Staphylococcus aureus,135,136 but it is low for yeast137 and Escherichia coli.136 Diminished opsonic antibody has been associated with group B streptococcal infection and represents one risk factor for neonatal infection.138
In premature infants, opsonic activity is low for Staphylococcus aureus and Serratia marcescens135 but is normal for Pseudomonas aeruginosa.139 When serum concentrations of fibronectin and IgG subclasses C3 and C4 were measured at birth, 1 month, 3 months, and 6 months, early gestational age was correlated with lower initial levels.140 The decreased opsonic activity for some organisms in premature infants has been attributed to diminished IgG levels, since additional IgG will correct the opsonic defect both in vivo and in vitro.135 The added IgG improves bacterial opsonization by serum of premature infants in part because complement consumption and deposition of C3 on the bacterial surface is augmented.141,142
Complement components appear in fetal blood before 20 weeks of gestation and increase markedly during the third trimester. However, in many newborns both the classical and alternative complement pathways are decreased in activity and in levels of individual components.143 The mean level of C3, the first common component of the two pathways of complement activation, is about 65 percent of that in normal adults.144,145 and 146 There is no transplacental transfer of this protein, and levels in infants are lower than those in their mothers.144 Total hemolytic complement (CH50) and alternative pathway activity (PH50) in newborns are lower than in adults, as are mean levels of C1q, C2–C9, properdin, and factors B, I and H.145,146 and 147 In general, the mean levels in full-term infants are greater than 50 percent of those in normal adult controls and may be somewhat less in premature infants. There is considerable overlap, however, between levels in infants and in controls. A functional deficiency in the alternative pathway has been detected in infants.148
Fibronectin mediates more efficient interactions between phagocytes and infectious agents. Fibronectin, a 450 kD glycoprotein found in plasma and in the intercellular matrix, promotes the attachment of staphylococci to neutrophils149 and enhances opsonic activity of antibodies against group B streptococci.150 Since both these bacteria are common pathogens for neonates, the deficiency in fibronectin observed in neonates151 may further compromise opsonic capacity and hence bactericidal activity in the neonate.
The administration of intravenous IgG may be useful in the treatment or prophylaxis of infection in preterm infants based on the reduced placental transfer of maternal antibody and the restricted endogenous synthesis of IgG.152 IgG administered to septic neonates appears to enhance serum opsonic capacity as well as to increase the quantity of circulating neutrophils.153 Added IgG heightens phagocytosis of granulocytes from premature neonates,154 and intravenous IgG has been reported to effectively treat infected premature neonates, but these reports involved small numbers of subjects.155,156 The clinical efficacy of IgG prophylaxis against neonatal pathogens is not firmly established.157,158 New IgG preparations with consistent, adequate levels of antibodies directed against neonatal pathogens can be achieved by selection of sera with high levels of functional antibodies,159 or potentially by the addition of monoclonal antibodies, and these may prove more clearly effective.
Chemotaxis Chemotactic function of leukocytes is low in neonates, while random motility is normal.160,161 and 162 Neonatal serum does not generate as much chemotactic factor as does adult serum, even after the addition of purified C3. The defect in chemotaxis may be related to decreased granulocyte deformability and impaired capping of cell surface receptors.163 The role of observed cAMP and membrane potential alterations in the defective chemotaxis is not clear.163
The densities of the C3bi receptor (CD11b/CD18) and of the low-affinity receptor for immunoglobulin, FcRIII (CD16), are decreased on neutrophils of premature infants, whereas term infants’ cells show a lesser impairment.164,165,166 and 167 The deficient up-regulation of C3bi has been correlated with decreased adherence and chemotaxis by neonatal neutrophils.168 Low FcRIII was associated with impaired chemotaxis of neonatal neutrophils,169 although decreased FcRIII might also be responsible for subtle defects in adherence and subsequent phagocytosis of opsonized159 and unopsonized170 organisms by neutrophils.
Phagocytic and Bactericidal Activity Phagocytosis of bacteria and latex granules by neutrophils from premature and term infants is normal.135,136,137,138 and 139,172,173 Bactericidal activity varies according to the conditions of testing and the clinical status of the neonates. The intracellular killing of Staphylococcus aureus and Serratia marcescens in cells from most term and low-birth-weight infants is normal,135,174 as is that of Escherichia coli in term infants.136 Similar studies have shown defective bactericidal activity against S. aureus in some infants in the first 12 h of life,172 P. aeruginosa in cells from premature infants,139 and Candida albicans in granulocytes from term and premature infants.175 With bacteria/neutrophil ratios of 1:1, newborn cells kill S. aureus and E. coli as effectively as controls; however, at the higher ratio of 100:1 killing and oxidative response as measured by chemiluminescence are markedly depressed, although phagocytosis is normal.173 Depressed activity also has been found in cells from newborns who have had clinical stress, either from infection or other disorders, shown both as decreased chemiluminescence and impaired bactericidal activity against S. aureus, E. coli, and group B streptococci.176,177 and 178 The decreased granulocyte function shown in these studies also is found in liquid culture, where neutrophils from newborns do not survive as long as those from adults, perhaps because of decreased resistance to autoxidation.179 Although superoxide dismutase levels are normal and superoxide production is normal or increased in neutrophils from newborns, glutathione peroxidase and catalase levels are decreased.180,181 The relationship of these in vitro cellular defects to bacterial infections in the newborn is still not clear.
Monocytes from newborn infants have normal nitroblue tetrazolium (NBT) reduction,182 normal antibody-dependent cellular cytotoxicity,183 and normal in vitro killing of S. aureus and E. coli.184 However, they are slower than monocytes from adults in phagocytosis of polystyrene spheres,185 and they have reduced ATP production.186 Furthermore, chemotaxis to serum-derived factors is decreased, as is monocyte appearance in skin windows.187 These functional aspects may contribute to the observed susceptibility of newborns to a variety of infectious agents.
Cytokine Effects on Neonatal Phagocytic Function There is a complex interaction between cytokines produced by lymphocytes and macrophages, and the activation status of neutrophils during infection. There is decreased production of g-interferon by neonatal leukocytes.188,189 and 190 g-interferon causes the up-regulation of the C3bi receptor and induces the surface expression of the high-affinity immunoglobulin receptor FcRI (CD64)191 on neutrophils. C3bi is required for adherence and efficient chemotaxis by neutrophils. Complement-mediated phagocytosis and oxidative metabolism also are impaired by low levels of this receptor. FcRI mediates oxidative responses as well, and appears on neutrophils of adults during infection. The diminished production of G-CSF and GM-CSF by neonatal mononuclear cells127,128 and 129 may not only limit progenitor colony growth but also impair neonatal neutrophil functions, including chemotaxis, superoxide production, and C3bi expression, which are enhanced by these factors.192,193 Tumor necrosis factor alpha (TNF-a) and interleukin 4 (IL-4), cytokines which modulate neutrophil functions, also may be produced at lower levels in neonates.194 Interleukin 8 (IL-8), a cytokine that enhances neutrophil functions, has not been adequately studied in neonates.
PLATELETS
NEONATAL THROMBOPOIESIS
The platelet counts in term and preterm infants are between 150 and 400 × 109/liter (150,000 to 400,000/µl), comparable to adult values.195 and 196 Thrombocytopenia of less than 100 × 109/liter (100,000/ µl) may occur in high-risk infants with respiratory distress or sepsis,197 small-for-date infants,198 and newborns with trisomy syndromes.199 Even normal newborns are unable to regulate thrombopoiesis and myelopoiesis in a totally effective manner.200 Although committed megakaryocyte progenitors (CFU-Meg) are increased in the marrow and cord blood of newborns, they are less able to produce adequate numbers of platelets when severely stressed. Thrombopoiesis-stimulating activity appears lower in cord serum than in adult serum,201 and reduced levels of G-CSF, GM-CSF, IL-3, and IL-11 may play a role in the impaired response.202 IL-11 and IL-3 act synergistically to enhance mouse CFU-Meg, and the role of these growth factors and others, such as TPO, IL-6, and Steel factor are currently being explored.
PLATELET FUNCTIONS
Bleeding Time The expected inverse relationship between the platelet count and bleeding time has been described in term and preterm newborns.203 However, the bleeding time often is longer than would be predicted by the platelet count because of sepsis or respiratory distress resulting in impaired platelet function, aggravating the effects of thrombocytopenia.
The bleeding time reflects platelet function and capillary integrity as well as the platelet count and traditionally has been used to assess these parameters. However, there are technical difficulties in applying a technique for measuring bleeding time to neonates or preterm infants because of the need for venous occlusion of the forearm, where the test normally is performed, and for a minimal incision to avoid scarring of the skin. Bleeding times were measured using an automatic device to minimize trauma in normal neonates, with venous occlusion of 20 torr for infants less than 1000 g, 25 torr for those 1000 to 2000 g, and 30 torr for those over 2000 g. In 82 observations, 97 percent of the measurements were below 3.5 min, which was suggested as the upper limit for normal in these infants.204 A similar upper limit (200 s) for the bleeding time of normal infants has been obtained using an automated device and vertical incisions.205 Generally, newborn infants have shorter bleeding times than those of children and adults, and this may reflect their higher hematocrit, increased concentration of von Willebrand factor, and higher proportion of high molecular weight multimers of von Willebrand factor.206 Children have longer bleeding times than either adults or newborns,207 and the upper limit measured with an automated pediatric device may be as high as 13 min before age 10, compared to an upper limit of 7 min in adults measured with the same device.207
The bleeding times in newborns may be prolonged for a variety of reasons, including neonatal infection and respiratory distress syndrome, which do not necessarily result in thrombocytopenia.208 The use of indomethacin for treatment of patent ductus arteriosus in preterm infants has been questioned because this agent interferes with prostaglandin metabolism and the production of thromboxane A2, an important initiator of platelet aggregation. Although bleeding times are prolonged from a normal of 3.5 min to approximately 9 min in indomethacin-treated patients,209 indomethacin did not result in an increase in periventricular or intraventricular hemorrhage in preterm infants treated for patent ductus arteriosus.
Platelet Aggregation and Metabolism A variety of differences have been described in the platelet function of neonates. These include decreased ADP release, platelet factor 3 activity, platelet adhesiveness, and platelet aggregation in response to ADP, epinephrine, collagen, or thrombin.210,211 These defects result from intrinsic differences in neonatal compared to adult platelets.212 Paradoxically, these insufficiencies have little effect on the bleeding time of neonates. The in vitro findings do not appear related to a significant defect in prostaglandin synthesis or to storage pool deficiency of adenine nucleotides.210 Further, electron micrographs of neonatal platelets do not differ from those of platelets from normal adults.213 This leaves unexplained the in vitro observations in neonatal platelets, which may be related to platelet membrane immaturity. These in vitro abnormalities may aggravate the impairment in platelet function and the predisposition to bleeding which results from neonatal diseases, particularly respiratory distress syndrome and sepsis.
Aspirin ingestion by mothers also results in abnormalities in platelet aggregation in response to collagen.214,215 However, aspirin has been studied extensively in patients with preeclampsia, and there is no significant bleeding in the fetus or newborn.216,217
Newborn infants commonly have petechiae, particularly on the head, neck, and shoulders after vertex deliveries. They are presumably due to trauma associated with passage through the birth canal and disappear within a few days. Petechiae usually are not present in infants delivered by cesarean section.
Platelet Antigens and Glycoproteins The glycoprotein complex GPIIb/IIIa represents about 15 percent of platelet surface protein and exhibits two allelic forms, PlA1 and PlA2.218 The PlA1 antigen can be identified on fetal platelets by 16 weeks gestation.210 PlA1 antigen is observed in a higher percentage of fetuses between 18 and 26 weeks than in adults. Approximately 2 percent of the population in the United States of European descent is homozygous for PlA2 and hence PlA1 negative. The complete expression of the PlA1 antigen during early gestation likely permits early sensitization in women who are PlA1 negative even during their first pregnancy.219 The membrane glycoprotein GPIb, as well as the GPIIb/IIIa complex, is expressed by 18 weeks of gestation.219 The gene for GPIIb/IIIa has been cloned, and the difference between PlA1 and PlA2 is a leucine 33/ proline 33 amino acid polymorphism in glycoprotein IIIA.218 Prenatal diagnosis of the glycoprotein genotype using DNA from amniocytes and the polymerase chain reaction can establish the potential for neonatal alloimmune thrombocytopenia220 as well as the diagnosis of Glanzmann’s thrombasthenia. Rarely, other fetal platelet antigens such as PlE2, DUZOa, Koa and Baka have caused maternal sensitization and neonatal alloimmune thrombocytopenia.221 The gestational ages for expression of these antigens have not been defined but are sufficiently early to permit sensitization.
NEONATAL LYMPHOPOIESIS
T-LYMPHOCYTE FUNCTIONS—CELLULAR IMMUNITY
The absolute number of lymphocytes in the newborn is equivalent to that in older children (Table 7-4), with lower values in premature infants at birth. Thymus-derived cells (T cells) develop early in gestation.222 The various lymphocyte subsets in newborns are shown in Table 7-4.223 The absolute number of CD3+ and CD4+ (helper/ inducer phenotype) T-cell subsets in blood of newborns is significantly higher than in adults.13,224 This is due to an increased total lymphocyte count in neonates (and older children) compared to adults.225 The percentages of major lymphoid subsets (CD2, CD3, CD4, CD8, CD19, CD16) are not markedly different in neonates, children, and adults when measured by flow cytometry methods.226 There is a trend to increased CD4 and decreased CD8 lymphocytes in newborns and children, resulting in an increased CD4/CD8 ratio.227,228 In spite of this, T-cell suppressor activity may be increased in newborns.229 Most responses of the cellular immunity system, such as antigen recognition and binding, antibody-dependent cytotoxicity and graft-versus-host reactivity are present in the newborn,229 although some are decreased in comparison with adults.230 The in vitro response to phytohemagglutinin of cord blood lymphocytes is increased,231,232 but the response of the newborn to 2,4-dinitrofluorobenzene, a potent inducer of delayed hypersensitivity, is not as consistent as that seen in older children.233 Impaired T-cell production of g interferon and other lymphokines may be related to immature macrophage rather than T-lymphocyte function, since intercellular cooperation is a requisite for these processes.234 Further, cord blood T-lymphocytes form a functional IL-2 receptor complex and have normal IL-2 receptors, but they do not up-regulate g interferon in response to IL2.235

TABLE 7-4 LYMPHOCYTE SUBSETS IN NEONATAL (CORD) BLOOD

B-LYMPHOCYTE FUNCTIONS— HUMORAL IMMUNITY
Humoral (B-cell) immunity also develops early in gestation,222 but it is not fully active until after birth. In the newborn, about 15 percent of lymphocytes have immunoglobulin on their surface, with all Ig isotypes represented.236 A percentage of these cells are CD5+ B cells (B-1 cells), which produce polyreactive autoantibodies whose function is yet unclear.237 The proportion of CD5+ B cells is markedly higher in the fetus compared to adults. The percentages of B cells expressing specific immunoglobulin isotypes are not related to the plasma levels of those isotypes. Variation in antibody response to specific antigens relates to the interaction of macrophages, T cells, and B cells; B lymphocytes are well represented in newborns.238
Fetal lymphocytes synthesize little immunoglobulin, presumably because of the sheltered environment in utero. Animals kept germ-free after birth have few plasma cells and markedly decreased production of immunoglobulins.239 IgG levels of term infants are similar to maternal levels because of transplacental transfer.240 IgM, IgD, and IgE do not cross the placenta,240,241 and the levels of these immunoglobulins and of IgA are low or not detectable at birth. Breast feeding provides some transfer of antibodies, particularly secretory IgA, lysozyme, and lactoferrin. Large numbers of lymphocytes and monocytes (106 cells/ml) are found in colostrum and milk during the first two months postpartum.242 These may provide local gastrointestinal protection against infection,243 and there is some evidence for absorption of immunoglobulin and transfer of tuberculin sensitivity to the infant.
Although the newborn infant can produce specific IgG antibody,244 only small amounts of IgG are usually produced by the fetus. IgG levels in premature infants are reduced in relation to gestational age because of the low placental transport early in pregnancy.245,246 and 247 The ability of the fetus to produce IgM and IgA with appropriate stimuli is indicated by the presence of these antibodies in many newborn infants who have had prenatal infections248 and by the presence of IgM isohemagglutinins in more than one-half of term newborn infants.249 In human newborns and in fetal animals the IgM response is predominant, and the appearance of IgG after exposure to specific antigens is delayed. These differences from the adult may relate to functional immaturity of B and T lymphocytes,250,251 and 252 to increased activity of suppressor T cells239,250 and perhaps to altered macrophage function.253
Newborns also may have relative splenic hypofunction, suggested by the large number of “pocked” red cells seen in the blood films of neonates, particularly premature infants. These “pocks” represent residual intraerythrocyte inclusions, which remain because of monocyte and macrophage hypofunction.254,255
COAGULATION IN THE NEONATE
PLASMA COAGULATION FACTORS
When the term newborn is compared to older children and adults, several differences in the coagulation and fibrinolytic systems have been described.256,257,258,259,260 and 261 A comprehensive evaluation of the developmental changes in the levels of clotting factors and coagulation tests in preterm and term infants has been published.262,263 The term newborn has reduced mean plasma levels (<60% of adult levels) of factors II, IX, X, XI, XII, prekallikrein, and high molecular weight kininogen (Table 7-5). In contrast, the plasma concentration of factor VIII is similar and von Willebrand factor is increased compared to older children and adults. In spite of the lower levels of factors, the functional tests (prothrombin and partial thromboplastin times) are only slightly prolonged compared to adult normal values (see Table 7-5). Although different coagulation factors show different postnatal patterns of maturation, near-adult values are achieved for most components by 6 months of life.259

TABLE 7-5 REFERENCE VALUES FOR COAGULATION TESTS IN PRETERM AND FULL-TERM INFANTS*

Factor II (prothrombin), VII, IX, and X require vitamin K for the final gamma glutamyl carboxylation step in their synthesis.264 These factors decrease during the first 3 to 4 days after birth. This fall may be lessened by administration of vitamin K,265 effectively preventing classical, early-occurring (first few days of life) hemorrhagic disease of the newborn. Inactive prothrombin molecules have been found in the plasma of some newborns, but they disappear after administration of vitamin K.266 Early-occurring hemorrhagic disease is most often associated with maternal administration of medications such as phenytoin (Dilantin)267 and warfarin268 which reduce the vitamin K-dependent factors. In rare cases no contributing factor is found.
A hemorrhagic diathesis also may occur later, 2 to 12 weeks after birth, due to lack of vitamin K and is called late hemorrhagic disease of the newborn, or acquired prothrombin complex deficiency.269,270 The etiology of the vitamin K lack is unclear but may result from poor dietary intake, particularly related to breast feeding, alterations in liver function with cholestasis and decreased vitamin K absorption, or a toxic or infectious impairment of hepatic utilization.269 Unfortunately, intracranial hemorrhage frequently is the presenting event in this condition. This problem can be prevented by parenteral or oral vitamin K, but the preferred route of administration remains controversial.271 The parenteral route may result rarely in neuromuscular complications,272 and an association of intramuscular vitamin K prophylaxis and cancer in infancy was suggested but not substantiated. Oral administration, however, may be less reliable and require repeated doses.269 The most current recommendation of the Scientific and Standardization Subcommittee on Perinatal Haemostasis suggests that present practice should not be changed at this time.270 Many institutions in the United States administer 1 mg vitamin K1 intramuscularly at birth with effective prophylaxis. A new mixed micellar vitamin K1 preparation is particularly well absorbed273 and may permit prophylaxis with a single oral dose.
The values for coagulation factors in healthy 30- to 36-week- gestation premature infants are shown in Table 7-5. More prominent decreases in factors IX, XI, and XII are noted, which tend to prolong the partial thromboplastin time. The values for coagulation factors in 28- to 31-week-gestation infants also are shown in Table 7-5. All of the coagulation factors are lower at earlier gestational ages.
There are no significant differences in mean prothrombin time determinations between 30- to 36-week-premature and full-term infants who have not received vitamin K.274 Premature infants given vitamin K have a longer mean prothrombin time than term infants similarly treated. In some small infants there is no improvement in prothrombin time or levels of prothrombin and factors VII and X after the intramuscular administration of vitamin K.265,275 These results suggest a greater degree of “immaturity” of the liver in the small infants.
BLEEDING AND THROMBOSIS
Significant bleeding occurs more often in low-birth-weight infants than in term newborn infants. Increased capillary fragility is frequently found in premature infants in the first 2 days after birth and is not associated with thrombocytopenia.265 Bleeding under the scalp or in other superficial areas may be due to trauma coupled with increased capillary fragility. The more serious disorders of periventricular-intraventricular hemorrhage and pulmonary hemorrhage probably are not primarily due to coagulation disorders, although such disorders may increase the bleeding.276 Hypoxia seems to affect the clotting status of low birth weight infants.277 Many infants with markedly abnormal prothrombin times have had hypoxia during delivery or shortly thereafter.274 Cardiovascular collapse seen with episodes of cardiac arrest or with profound shock may cause disseminated intravascular coagulation and generalized bleeding. In many sick premature infants, a combination of shock, sepsis, liver immaturity, hypoxia, and other factors may contribute to the pathogenesis of coagulation abnormalities.
Arterial and venous thromboses are relatively frequent in newborns compared to other age groups, but greater than 90 percent of arterial and greater than 80 percent of venous clots are related to catheters. Spontaneous thromboses are much less common, and most involve the renal veins or rarely the pulmonary vasculature.278 Relative hypercoagulability in the newborn could result from a difference in the vascular endothelium, activation of the coagulation cascade, diminished coagulation inhibitor activity, or a defect in fibrinolysis. Inhibitors of coagulation include antithrombin, heparin cofactor II, protein C, and protein S.263,279 The levels of proteins C and S, which are vitamin K-dependent, as well as antithrombin and heparin cofactor II, are low in the newborn; they are in a range associated with thrombotic episodes in adults with inherited deficiencies.279 In addition, the presence of factor V Leiden may occur in as many as 6 percent of newborns.280 This produces resistance to the action of protein C and may heighten the susceptibility to thrombosis. Further, hyperprothrombinemia secondary to the 20210A allele prothrombin gene may affect 1 percent of the population281 and has been associated with heightened venous thrombosis.282 The combined deficiency of these anticoagulant proteins may further intensify the thrombotic risk. However, the precise role of these inhibitors of coagulation in newborn hypercoagulability is uncertain, since a proportionate decrease in vitamin K dependent procoagulant factors (II, VII, IX, X) also is present, and an additional inhibitor, a2-macroglobulin, is increased. The values for plasma inhibitors of coagulation in premature and term infants are shown in Table 7-6.

TABLE 7-6 REFERENCE VALUES FOR INHIBITORS OF COAGULATION IN PRETERM AND FULL-TERM INFANTS*

HEMATOLOGIC EFFECTS OF MATERNAL DRUGS ON THE FETUS AND NEWBORN
HEMOSTATIC EFFECTS
A number of maternally administered pharmacologic agents have been implicated in hematologic abnormalities of the fetus or newborn (Table 7-7). Maternal aspirin ingestion results in impaired platelet aggregation but does not foster neonatal bleeding. Other agents taken by the mother, including diazoxide and thiazides, may be associated with neonatal thrombocytopenia.283,284 and 285

TABLE 7-7 HEMATOLOGIC EFFECTS OF MATERNAL DRUGS ON THE FETUS AND NEWBORN

The newborn’s plasma coagulation factors may be depressed by maternal warfarin ingestion.268 This drug is best avoided during pregnancy, as it is teratogenic (first trimester) and may cause growth retardation of the fetus as well as bleeding.268 In contrast, heparin does not cross the placenta, and maternal treatment with heparin appears to be safe for the fetus.286
Dilantin and/or phenobarbital also may reduce the newborn’s vitamin-K dependent factors, possibly by microsomal enzyme induction, which enhances their degradation.267 Furthermore, phenytoin (Dilantin) may depress the platelet count as a result of prenatal exposure287 and cause teratogenic effects, e.g., the fetal hydantoin syndrome.288 The decision to use this agent during pregnancy should reflect an assessment of the need for this specific drug, and also the risk of maternal seizures to the fetus and mother versus the potential side effects of treatment. Newborns of mothers taking rifampin and isoniazid also may have depressed vitamin K-dependent factors.289
BILIRUBIN/KERNICTERUS
Nitrofurantoin and nalidixic acid may cause oxidant injury to the red cell membrane and hemoglobin.290,291 If there is glucose-6-phosphate dehydrogenase deficiency or if reduced glutathione is diminished, as in newborn red cells, these drugs have the potential to induce hemolysis and heighten neonatal hyperbilirubinemia. Although this problem has not been documented by transplacental transfer of nitrofurantoin or nalidixic acid, hemolysis has occurred in glucose-6-phosphate dehydrogenase–deficient infants who acquired the drugs from breast milk.291,292 Alternatively, sulfonamides may cause displacement of bilirubin bound to albumin and heighten the risk of kernicterus.293 Salicylates, phenylbutazone, and naproxen may have a similar effect at very high plasma concentrations.293
Ideally, all these medications should be avoided during pregnancy unless their indication outweighs the potential risk to the fetus and newborn.
CHAPTER REFERENCES

1.
Bloom W, Bartelmez GW: Hematopoiesis in young human embryos. Am J Anat 67:21, 1940.

2.
Gilmour JR: Normal hemopoiesis in intrauterine and neonatal life. J Pathol 52:25, 1942.

3.
Zon L: Developmental biology of hematopoiesis. Blood 86:2876, 1995.

4.
Shivdasani RA, Mayer EL, Orkin SH: Absence of blood formation in mice lacking T-cell leukemia oncoprotein tal-1/SCL. Nature 373:432, 1995.

5.
Warren AJ, Colledge WH, Carlton MBL, Evans M, Smith AJH, Rabbitts TH: The oncogenic cysteine-rich LIM domain protein is essential for erythroid development. Cell 78:45, 1994.

6.
Fujiwara Y, Browne CP, Cuniff K, Goff SC, Orkin SH: Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1. Proc Natl Acad Sci 93:12355, 1996.

7.
Peschle C, Migliaccio AR, Migliaccio G, Ptrini M, Calandrini M, Russo G, et al: Embryonic to fetal Hb switch in humans: studies on erythroid bursts generated by embryonic progenitors from yolk sac and liver. Proc Natl Acad Sci USA 81:2416, 1984.

8.
Migliaccio G, Migliaccio AR, Petti S, et al: Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac—liver transition. J Clin Invest 78:51, 1986.

9.
Huyhn A, Dommergues M, Izac B, Croisille L, Katz A, Vainchenker W, Coulombel L: Characterization of hematopoietic progenitors from human yolk sacs and embryos. Blood 86:4474, 1995.

10.
Dommergues M, Aubeny E, Dumez Y, Durandy A, Coulombel L: Hematopoiesis in the human yolk sac: quantitation of erythroid and granulopoietic progenitors between 3.5 and 8 weeks of development. Bone Marrow Transplant 9:23, 1992.

11.
Keleman E, Calvo W, Fliedner TM: Atlas of Human Hemopoietic Development, Springer-Verlag, Berlin, 1979.

12.
Lin C-S, Lim S-K, D’Agati V, Constantini F: Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis. Genes Dev 10:154, 1996.

13.
Neubauer H, Cumano A, Muller M, Wu H, Huffstadt U, Pfeffer K: Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis. Cell 93:397, 1998.

14.
Valtieri M, Gabbianelli M, Pelosi E, Bassano E, Petti S, Russo G, et al: Erythropoietin alone induces erythroid burst formation by human embryonic but not adult BFU-E in unicellular serum-free culture. Blood 74:460, 1989.

15.
Emerson SG, Shanti T, Ferrara JL, Greenstein JL: Developmental regulation of erythropoiesis by hematopoietic growth factors: analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver. Blood 74:49, 1989.

16.
Dame C, Fahnenstich H, Feitag P, Hofmann D, Abdul-Nour T, Bartmann P, Fandrey J: Erythropoietin mRNA expression in human fetal and neonatal tissue. Blood 92:3218, 1998.

17.
Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner MC, Miller TA, et al: A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell 65:677, 1991.

18.
Nuez B, Michalovich D, Bygrave A, Ploemacher R, Grosveld F: Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature 375:316, 1995.

19.
Hann IM, Bodger MP, Hoffbrand AV: Development of pluripotent hematopoietic progenitor cells in the human fetus. Blood 62:118, 1983.

20.
Porcellini A, Manna A, Manna M, et al. Ontogeny of granulocyte- macrophage progenitor cells in the human fetus. Int J Cell Clon 1:92, 1983.

21.
Nicola NA, Metcalf D: Specificity of action of colony-stimulating factors in the differentiation of granulocytes and macrophages. CIBA Found Symp 118:7, 1986.

22.
Ohls RK, Li Y, Abdel-Mageed A, Buchanan G Jr., Mandell L, Christensen RD. Neutrophil pool sizes and granulocyte colony-stimulating factor production in human mid-trimester fetuses. Pediatric Res 37(6):806, 1995.

23.
Slayton WB, Juul SE, Calhoun DA, Li Y, Braylan RC, Christensen RD: Hematopoiesis in the liver and marrow of human fetuses at 5 to 16 weeks postconception: quantitative assessment of macrophage and neutrophil populations. Pediatric Res 43:774, 1998.

24.
Charbord P, Tavian M., Humeau L, Peault B: Early ontogeny of the human marrow from long bones: an immunohistochemical study of hematopoiesis and its microenvironment. Blood 87:4109, 1996.

25.
Gupta S, Pahwa R, O’Reilly R, et al: Ontogeny of lymphocyte subpopulation in human fetal liver. Proc Natl Acad Sci USA 73:919, 1976.

26.
Rainaut M, Pagniez M, Hercend T, Daffos F, Forestier F: Characterization of mononuclear cell subpopulations in normal fetal peripheral blood. Hum Immunol 18:331, 1987.

27.
Hann IM, Gibson BES, Letsky EA: Fetal and Neonatal Haematology, Baillaire Tindale, Philadelphia, 1991.

28.
Cairo MS, Wagner JE: Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. J Am Soc Hematol 90:4665, 1997.

29.
Maximow AA: Relation of blood cells to connective tissues and endothelium. Physiol Rev IV(4):532, 1924.

30.
Houssaint E: Differentiation of the mouse hepatic primordium. II. Extrinsic origin of the haemopoietic cell line. Cell Differ 10:243, 1981.

31.
Cudennec CA, Johnson GR: Presence of multipotential hemopoietic cells in teratocarcinoma cultures. J Embryol Exp Morphol 61:51, 1981.

32.
Gupta S, Pahwa R, O’Reilly R, et al: Ontogeny of lymphocyte subpopulation in human fetal liver. Proc Natl Acad Sci USA 73:919, 1976.

33.
Moore MAS, Owen JJT: Stem-cell migration in developing myeloid and lymphoid systems. Lancet i:658, 1967.

34.
Dieterlen-Lievre F: On the origin of hematopoietic stem cells in the avian embryo: an experimental approach. J Embryol Exp Morphol 33:607, 1975.

35.
Carpenter KL, Turpen JB: Experimental studies on hemopoiesis in the pronephros of Rana pipiens. Differentiation 14:167, 1979.

36.
Muller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E: Development of hematopoietic stem cell activity in the mouse embryo. Immunity 1:291, 1994.

37.
Smith RA, Glomski CA: “Hemogenic endothelium” of the embryonic aorta: does it exist? Dev Comp Immunol 6:359, 1982.

38.
Tavian M, Coulombel L, Luton D, San Clemente H, Dieterlen-Lievre F, Peault B: Aorta-associated CD-34+ hematopoietic cells in the early human embryo. Blood 87:67, 1996.

39.
Proudfoot NJ, Shander MH, Manley JL, Gefter ML, Maniatis T: Structure and in vitro transcription of human globin genes. Science 209:1329, 1980.

40.
Grosveld F, van Assendelft GB, Greaves DR, Kolias B: Position independent, high-level expression of the human b globin gene in transgenic mice. Cell 51:975, 1987.

41.
Hecht F, Motulsky AG, Lemire RJ, et al: Predominance of hemoglobin Gower 1 in early human embryonic development. Science 152:91, 1966.

42.
Huehns ER, Dance N, Beaven GH, et al: Human embryonic hemoglobins. Cold Spring Harbor Symp Quant Biol 29:327, 1964.

43.
Gale RE, Clegg JB, Huehns ER: Human embryonic haemoglobins Gower 1 and Gower 2. Nature 280:162, 1979.

44.
Peschle C, Mavilio F, Care A, Migliaccio G, Migliaccio AR, Salvo G, et al: Haemoglobin switching in human embryos: asynchrony of the z to a and e to g globin switches in primitive and definitive erythropoietic lineage. Nature 313:235, 1985.

45.
Pataryas HA, Stomatoyannopoulos G: Hemoglobins in human fetuses: evidence of adult hemoglobin production after the 11th gestational week. Blood 39:688, 1972.

46.
Thomas ED, Lochte HL Jr, Greenough WB III, et al: In vitro synthesis of foetal and adult haemoglobin by foetal haematopoietic tissues. Nature 185:396, 1960.

47.
Kazazian HH, Woodhead AP: Hemoglobin A synthesis in the developing fetus. N Engl J Med 289:58, 1973.

48.
Bard H: The effect of placental insufficiency on fetal and adult hemoglobin synthesis. Am J Obstet Gynecol 120:67, 1974.

49.
Kirschbaum T: Fetal hemoglobin content of cord blood determined by column chromatography. Am J Obstet Gynecol 84:1375, 1962.

50.
Armstrong D, Schroeder WA, Fenninger W: A comparison of the percentage of fetal hemoglobin in human umbilical cord blood as determined by chromatography and by alkali denaturation. Blood 22:554, 1963.

51.
Bard H: Postnatal fetal and adult hemoglobin synthesis in early preterm newborn infants. J Clin Invest 52:1789, 1973.

52.
Metaxotou-Mavromati AD, Antonopoulou HK, Laskari SA, Tsiarta HK, Ladis VA, Kattamis CA: Developmental changes in hemoglobin F levels during the first two years of life in normal and heterozygous b-thalassemia infants. Pediatrics 69:734, 1982.

53.
Bard H, Makowski EL, Meschia G, et al: The relative rates of synthesis of hemoglobins A and F in red cells of newborn infants. Pediatrics 45:766, 1970.

54.
Bromberg YN, Abrahamov A, Salzberger M: The effect of maternal anoxemia on the foetal haemoglobin of the newborn. J Obstet Gynaecol Br Commonw 63:875, 1956.

55.
Huehns ER, Hecht F, Keil JV, et al: Developmental hemoglobin anomalies in a chromosomal triplication. Proc Natl Acad Sci USA 51:89, 1964.

56.
Lee CSN, Boyer SH, Bowen P, et al: The D1 trisomy syndrome: three subjects with unequally advancing development. Johns Hopkins Med J 118:374, 1966.

57.
Wilson MG, Schroeder WA, Graves DA: Postnatal change of hemoglobins F and A2 in infants with Down’s syndrome (G trisomy). Pediatrics 42:349, 1968.

58.
Giulian GG, Gilbert EF, Moss RL: Elevated fetal hemoglobin levels in sudden infant death syndrome. N Engl J Med 316:1122, 1987.

59.
Brown MS: Fetal and neonatal erythropoieses, in Developmental and Neonatal Hematology, edited by JA Stockman, III and C Pochedly, pp 39. Raven Press, New York, 1988.

60.
Forestier F, Daffos F, Galacteros F, et al: Haematological values of 163 normal fetuses between 18 and 30 weeks of gestation. Paediat Res 20:342, 1986.

61.
Millar DS, Davis LR, et al. Normal blood cell values in the early mid- trimester fetus. Prenat Diagn, 5:367, 1985.

62.
Linch DC, Knott LJ, et al. Studies of circulating hemopoietic progenitor cells in human fetal blood. Blood 59:976, 1982.

63.
Christensen RD. Hematopoiesis in the fetus and neonate. Pediatr Res 26:531, 1989.

64.
Marks J, Gairdner D, Roscoe JD: Blood formation in infancy. III. Cord blood. Arch Dis Child 30:117, 1955.

65.
Linderkamp O, Versmold HT, Messow-Zahn K, et al: The effect of intrapartum and intra-uterine asphyxia on placental transfusion in premature and full-term infants. Eur J Pediatr 127:91, 1978.

66.
Yao AC, Hirvensalo M, Lind J: Placental transfusion rate and uterine contraction. Lancet 1:380, 1968.

67.
Usher R, Shepard M, et al: The blood volume of the newborn and placental transfusion. Acta Paediatr 52:497, 1963.

68.
Bratteby LE: Studies on erythro-kinetics in infancy. XI. The change in circulating red cell volume during the first five months of life. Acta Paediatr Scand 57:215, 1968.

69.
McCue CM, Garner FB, Hurt WG, et al: Placental transfusion. J Pediatr 72:15, 1968.

70.
Matoth Y, Zaizor R, Varsano I: Postnatal changes in some red cell parameters. Acta Paediatr Scand 60:317, 1971.

71.
Linderkamp O, Versmold HT, Strohhacker I, et al: Capillary-venous hematocrit differences in newborn infants. Eur J Pediatr 127:9, 1977.

72.
Saarinen UM, Simmes MA: Developmental changes in red blood cell counts and indices of infants after exclusion of iron deficiency by laboratory criteria and continuous iron supplementation. J Pediatr 92:412, 1978.

73.
Zipursky A, Brown E, Palko J, et al: The erythrocyte differential count in newborn infants. Am J Pediatr Hematol Oncol 5:45, 1983.

74.
Shannon KM, Naylor GS, Torkildson JC, et al: Circulating erythroid progenitors in the anemia of prematurity. N Engl J Med 317:728, 1987.

75.
Linch DC, Knott LJ, Rodeck CH, Huehns ER: Studies of circulating hemopoietic progenitor cells in human fetal blood. Blood 59:976, 1983.

76.
Christensen RD: Circulating pluripotent hematopoietic progenitor cells in neonates. J Pediatr 11:622, 1987.

77.
Clapp DW, Baley JE, Gerson SL: Gestational age dependent changes in circulating hematopoietic stem cells in newborn infants. J Lab Clin Med 113:422, 1989.

78.
Holbrook SR, Christensen RD, Rothstein G: Erythroid colonies derived from fetal blood display different growth patterns from those derived from adult marrow. Pediatr Res 24:605, 1988.

79.
Burman D, Morris AF: Cord hemoglobin in low birth weight infants. Arch Dis Child 49:382, 1974.

80.
Meberg A: Haemoglobin concentrations and erythropoietin levels in appropriate and small for gestational age infants. Scand J Haematol 24:162, 1980.

81.
Zaizov R, Matoth Y: Red cell values on the first postnatal day during the last 16 weeks of gestation. Am J Hematol 1:275, 1976.

82.
Lockridge S, Pass R, Cassidy G: Reticulocyte counts in intrauterine growth retardation. Pediatrics 47:919, 1971.

83.
Humbert JR, Abelson H, Hathaway WE, et al: Polycythemia in small for gestational age infants. J Pediatr 75:1812, 1969.

84.
Halvorsen S, Finne PH: Erythropoietin production in the human fetus and newborn. Ann NY Acad Sci 149:576, 1968.

85.
Seip M: The reticulocyte level and the erythrocyte production judged from reticulocyte studies in newborn infants during the first week of life. Acta Paediatr Scand 44:355, 1955.

86.
Stockman JA III, Oski FA: Physiological anaemia of infancy and the anaemia of prematurity. Clin Haematol 7:3, 1978.

87.
Mann DL, Sites ML, Donati RM, et al: Erythropoietic stimulating activity during the first ninety days of life. Proc Soc Exp Biol Med 118:212, 1965.

88.
Lundstrom U, Simmes MA: Red blood cell values in low-birth-weight infants: Ages at which values become equivalent to those of term infants. J Pediatr 96:1040, 1980.

89.
Stockman JA III, Garcia JF, Oski FA: The anemia of prematurity: factors governing the erythropoietin response. N Engl J Med 296:647, 1977.

90.
MackIntosh TF, Walker CHM: Blood viscosity in the newborn. Arch Dis Child 48:547, 1973.

91.
Bergqvist G: Viscosity of the blood in the newborn infant. Acta Paediatr Scand 63:858, 1974.

92.
Wirth FH, Goldberg WR, Lubchenco L: Neonatal hyperviscosity. I. Incidence. Pediatrics 63:833, 1979.

93.
Hakanson DO, Oh W: Hyperviscosity in the small-for-gestational age infant. Biol Neonate 37:190, 1980.

94.
Ramamurthy RS, Berlanga M. Postnatal alteration in hematocrit and viscosity in normal and polycythemic infants. J Pediatr 110:929, 1987.

95.
Bada HS, Korones SB, Pourcyrous M, et al: Asymptomatic syndrome of polycythemic hyperviscosity: Effect of partial plasma exchange transfusion. J Pediatr 120:579, 1992.

96.
Bierhuizen MFA, Mattei MG, Fukuda M: Expression of the developmental I antigen by a cloned human cDNA encoding a member of a b-1,6-N-acetylglucosaminyltransferase gene family. Gene Dev 7:468, 1993.

97.
Race RR, Sanger R: Blood Groups in Man, 6th ed. Blackwell Scientific Publications, London, 1975.

98.
Pearson HA: Life-span of the fetal red blood cell. J Pediatr 70:166, 1967.

99.
Weipple G, Pantlitschko M, Bauer P, et al: Normal values and distribution of serum iron in cord blood. Clin Chim Acta 44:147, 1973.

100.
Saarinen UM, Siimes MA: Developmental changes in serum iron, total iron-binding capacity, and transferrin saturation in infancy. J Pediatr 91:875, 1977.

101.
Saarinen UM, Siimes MA: Serum ferritin in assessment of iron nutrition in healthy infants. Acta Paediatr Scand 67:745, 1978.

102.
Seip M, Halvorsen S: Erythrocyte production and iron stores in premature infants during the first months of life. The anemia of prematurity—etiology, pathogenesis, iron requirement. Acta Paediatr Scand 45:600, 1956.

103.
Bauer C, Ludwig I, Ludwig M: Different effects of 2,3-diphosphoglycerate and adenosine triphosphate on oxygen affinity of adult and fetal hemoglobin. Life Sci 7:1339, 1968.

104.
Oski FA: Red cell metabolism in the newborn infant. V. Glycolytic intermediates and glycolytic enzymes. Pediatrics 44:84, 1969.

105.
Oski FA, Delivoria-Papadopoulos M: The red cell, 2, 3-diphosphoglycerate, and tissue oxygen release. J Pediatr 77:941, 1970.

106.
Zipursky A: The erythrocytes of the newborn infant. Semin Hematol 2:167, 1965.

107.
Oski FA, Komazawa M: Metabolism of the erythrocytes of the newborn infant. Semin Hematol 12:209, 1975.

108.
Oski FA, Smith CA: Red cell metabolism in the premature infant. III. Apparent inappropriate glucose consumption for cell age. Pediatrics 41:473, 1968.

109.
Konrad PN, Valentine WN, Paglia DE: Enzymatic activities and glutathione content of erythrocytes in the newborn: comparison with red cells of older normal subjects and those with comparable reticulocytosis. Acta Haematol 48:193, 1972.

110.
Gross RT, Schroeder EAR, Brounstein SA: Energy metabolism in the erythrocytes of premature infants compared to full term newborn infants and adults. Blood 21:755, 1963.

111.
Oski FA: Red cell metabolism in the premature infant. II. The pentose phosphate pathway. Pediatrics 39:689, 1967.

112.
Ross JD: Deficient activity of DPNH-dependent methemoglobin diaphorase in cord blood erythrocytes. Blood 21:51, 1963.

113.
Gross RT, Bracci R, Rudolph N, et al: Hydrogen peroxide toxicity and detoxification in erythrocytes of newborn infants. Blood 29:481, 1967.

114.
Travis SF, Kumar SP, Delivoria-Papadopoulos M: Red cell metabolic alterations in postnatal life in term infants: glycolytic intermediates and adenosine triphosphate. Pediatr Res 15:34, 1981.

115.
Whaun JM, Oski FA: Red cell stromal adenosine triphosphatase (ATPase) of newborn infants. Pediatr Res 3:105, 1969.

116.
Blum SF, Oski FA: Red cell metabolism in the newborn infant. IV. Transmembrane potassium flux. Pediatrics 43:396, 1969.

117.
Crowley J, Ways P, Jones JW: Human fetal erythrocyte and plasma lipids. J Clin Invest 44:989, 1965.

118.
Neerhout RC: Erythrocyte lipids in the neonate. Pediatr Res 2:172, 1968.

119.
Shapiro DL, Pasqualini P: Erythrocyte membrane proteins of premature and full-term infants. Pediatr Res 12:176, 1978.

120.
Kosztolanyi G, Jobst K: Electrokinetic analysis of the fetal erythrocyte membrane after trypsin digestion. Pediatr Res 14:138, 1980.

121.
Altman PL, Dittmer, DS: Blood and Other Body Fluids. Federation of American Societies for Experimental Biology, Washington, DC, 1961.

122.
Coulombel L, Dehan M, Tchernia G, Hill C, et al: The number of polymorphonuclear leukocytes in relation to gestational age in the newborn. Acta Paediatr Scand 68:709, 1979.

123.
Barak Y, Blachar Y, Levin S: Neonatal neutrophilia: Possible role of a humoral granulopoietic factor. Pediatr Res 14:1026, 1980.

124.
Laver J, Duncan E, Abboud M, et al: High levels of granulocyte and granulocyte-macrophage colony-stimulating factors in cord blood of normal full-term neonates. J Pediatr 116:627, 1990.

125.
Ijima H, Suda T, Miura Y: Predominance of macrophage-colony formation in human cord blood. Exp Hematol 10:234, 1982.

126.
Prindull G, Ben-Ishay Z, Gabriel M, et al: A comparison of spontaneous and CSF added CFU-MG colony formation in healthy, sick and hypotrophic pre-term infants. Blut 45:167, 1982.

127.
Cairo MS, Suen Y, Knoppel E, Van De Ven C, Nguyen A, Sender L: Decreased stimulated GM-CSF production and GM-CSF gene expression but normal numbers of GM-CSF receptors in human term newborns compared with adults. Pediatr Res 30:362, 1991.

128.
English BK, Hammond WP, Lewis DB, Brown CB, Wilson CB: Decreased granulocyte-macrophage colony-stimulating factor production by human neonatal blood mononuclear cells and T cells. Pediatr Res 31:211, 1992.

129.
Cairo MS, Suen Y, Knoppel E, et al: Decreased G-CSF and IL-3 production and gene expression from mononuclear cells of newborn infants. Pediatr Res 31:574, 1992.

130.
Rosenthal J, Cairo MS: The role of cytokines in modulating neonatal myelopoiesis and host defense. Cytokin Mol Ther 1:165, 1995.

131.
Cairo MS, Plunkett JM, Nguyen A, Van De Ven C: Effect of stem cell factor with and without granulocyte colony-stimulating factor on neonatal hematopoiesis: in vivo induction of newborn myelopoiesis and reduction of mortality during experimental group B streptococcal sepsis. Blood 80:96, 1992.

132.
Xanthou M: Leucocyte blood picture in healthy full-term and premature babies during neonatal period. Arch Dis Child 45:242, 1970.

133.
Gibson EL, Vaucher Y, Corrigan JJ Jr: Eosinophilia in premature infants. Relationship to weight gain. J Pediatr 95:99, 1979.

134.
Siegel JD, McCracken GH Jr: Sepsis neonatorum. N Engl J Med 304:642, 1981.

135.
Forman ML, Stiehm ER: Impaired opsonic activity but normal phagocytosis in low-birth-weight infants. N Engl J Med 281:926, 1969.

136.
Dossett JH, Williams RC Jr, Quie PG: Studies on interaction of bacteria, serum factors and polymorphonuclear leukocytes in mothers and newborns. Pediatrics 44:49, 1969.

137.
Miller ME: Phagocytosis in the newborn infant: humoral and cellular factors. J Pediatr 74:255, 1969.

138.
Hill HR, Shigeoka AO, Pincus S, Christensen RD: Intravenous IgG in combination with other modalities in the treatment of neonatal infection. Pediatr Inf Dis 5:180, 1986.

139.
Cocchi P, Marianelli L: Phagocytosis and intracellular killing of Pseudomonas aeruginosa in premature infants. Helv Paediatr Acta 22:110, 1967.

140.
Drossou V, Kanakoudi F, Diamanti E, et al: Concentrations of main serum opsonins in early infancy. Arch Dis of Childhood (Fetal and Neonatal Edition) 72:F172, 1995.

141.
Yang KD, Bathras JM, Shigeoka AO, James J, Pincus SH, Hill HR: Mechanisms of bacterial opsonization by immune globulin intravenous correlation of complement consumption with opsonic activity and protective efficacy. J Infect Dis 159:701, 1989.

142.
Shaio MF, Yang KD, Bohnsack JF, Hill HR: Effect of immune globulin intravenous on opsonization of bacteria by classic and alternative complement pathways in premature serum. Pediatr Res 25:634, 1989.

143.
Hill H: Host defenses in the neonate: prospects for enhancement. Semin Perinatol 9:2, 1985.

144.
Propp RP, Alper CA: C3 synthesis in the human fetus and lack of transplacental passage. Science 162:672, 1968.

145.
Johnston RB Jr, Altenburger KM, Atkinson AW Jr, et al: Complement in the newborn infant. Pediatrics 64:781, 1979.

146.
Strunk RC, Fenton LJ, Gaines JA: Alternative pathway of complement activation in full term and premature infants. Pediatr Res 13:641, 1979.

147.
Davis CA, Vallota EH, Forristal J: Serum complement levels in infancy: age related changes. Pediatr Res 13:1043, 1979.

148.
Mills EL, Bjorksten B, Quie PG: Deficient alternative complement pathway activity in newborn sera. Pediatr Res 13:1341, 1979.

149.
Proctor RA, Prendergast E, Mosher DF: Fibronectin mediates attachment of Staphylococcus aureus to human neutrophils. Blood 59:681, 1982.

150.
Hill HR, Shigeoka AO, Augustine NH, Pritchard D, Lundblad JL, Schwartz RS: Fibronectin enhances the opsonic and protective activity of monoclonal and polyclonal antibody against group B streptococci. J Exp Med 159:1618, 1984.

151.
Harris MC, Levitt J, Douglas SD, Gerdes JS, Polin RA: Effect of fibronectin on adherence of neutrophils from newborn infants. J Clin Microbiol 21:243, 1985.

152.
Hill HR, Shigeoka AO, Gonzales LA, Christensen RD: Intravenous immune globulin use in newborns. J Allergy Clin Immunol 84:617, 1989.

153.
Christensen RD, Brown MS, Hall DC, Lassiter HA, Hill HR: Effect on neutrophil kinetics and serum opsonic capacity of intravenous administration of immune globulin to neonates with clinical signs of early-onset sepsis. J Pediatr 118:606, 1991.

154.
Fujiwara T, Taniuchi S, Hattori K, Kobayashi T, Kinoshita Y, Kobayashi Y: Effect of immunoglobulin therapy on phagocytosis by polymorphonuclear leucocytes in whole blood of neonates. Clin Exp Immunol 107:435, 1997.

155.
Weisman LE, Stoll BJ, Kueser TJ, et al: Intravenous immune globulin therapy for early-onset sepsis in premature neonates. J Pediatr 121:434, 1992.

156.
Schreiber JR, Berger M: Intravenous immune globulin therapy for sepsis in premature neonates. J Pediatr 121:401, 1992.

157.
Baker CJ, Melish ME, Hall RT, et al: Intravenous immune globulin for the prevention of nosocomial infection in low-birth-weight infants. N Engl J Med 327:213, 1992.

158.
Fanaroff A, Wright E, Korones S, Wright L: A controlled trial of prophylactic intravenous immunoglobulin to reduce nosocomial infections in VLBW infants. Pediatr Res 31:202A, 1992.

159.
Fischer GW, Weisman LE, Hemming VG: Directed immune globulin for the prevention or treatment of neonatal group B streptococcal infections: A review. Clin Immunol Immunopathol 62:S92, 1992.

160.
Miller ME: Chemotactic function in the neonate. Humoral and cellular aspects. Pediatr Res 5:487, 1971.

161.
Klei RB, Fischer TJ, Gard SE, et al: Decreased mononuclear and polymorphonuclear chemotaxis in human newborns, infants, and young children. Pediatrics 60:467, 1977.

162.
Tono-oka T, Nakayama M, Uehara H, et al: Characteristics of impaired chemotactic function in cord blood leukocytes. Pediatr Res 13:148, 1979.

163.
Hill HR, Augustine NH, Newton JA, Shigeoka AO, Morris E, Sacchi F: Correction of a developmental defect in neutrophil activation and movement. Am J Pathol 128:307, 1987.

164.
Bruce MC, Baley JE, Medvik KA, Berger M: Impaired surface membrane expression of C3bi but not C3b receptors on neonatal neutrophils. Pediatr Res 21:306, 1987.

165.
Anderson DC, Freeman KLB, Heerdt B, Hughes BJ, Jack RM, Smith CW: Abnormal stimulated adherence of neonatal granulocytes: impaired induction of surface MAC-1 by chemotactic factors or secretagogues. Blood 70:740, 1987.

166.
Smith JB, Campbell DE, Ludomirsky A, et al: Expression of the complement receptors CR1 and CR3 and the type III Pc-gamma receptor on neutrophils from newborn infants and from fetuses with Rh disease. Pediatr Res 28:120, 1990.

167.
Carr R, Davies JM: Abnormal PcRIII expression by neutrophils from very preterm neonates. Blood 76:607, 1990.

168.
Anderson DC, Rothlein R, Marlin SD, Krater SS, Smith CW: Impaired transendothelial migration by neonatal neutrophils: abnormalities of Mac-1(CD11b/CD18)-dependent adherence reactions. Blood 76:2613, 1990.

169.
Masuda K, Kinoshita Y, Kobayashi Y: Heterogeneity of Fc expression in chemotaxis and adherence of neonatal neutrophils. Pediatr Res 25:6, 1989.

170.
Tosi MF, Berger M: Functional differences between the 40 kDa and 50 kDa IgG Fc receptors on human neutrophils revealed by elastase treatment and antireceptor antibodies. J Immunol 141:2097, 1988.

171.
Salmon JE, Kapur S, Kimberly RP: Opsonin-independent ligation of Fc-gamma receptors. J Exp Med 166:1798, 1987.

172.
Coen R, Grush O, Kander E: Studies of bactericidal activity and metabolism of the leukocyte in full-term neonates. J Pediatr 78:400, 1969.

173.
Mills EL, Thompson T, Bjorksten B, et al: The chemiluminescence response and bactericidal activity of polymorphonuclear neutrophils from newborns and their mothers. Pediatrics 63:429, 1979.

174.
Park BH, Holmes B, Good RA: Metabolic activities in leukocytes of newborn infants. J Pediatr 76:237, 1970.

175.
Xanthou M, Valassi-Adam E, Kintronidou E, et al: Phagocytosis and killing ability of Candida albicans by blood leucocytes of healthy term and preterm babies. Arch Dis Child 50:72, 1975.

176.
Wright WC Jr, Ank BJ, Herbert J, et al: Decreased bactericidal activity of leukocytes of stressed newborn infants. Pediatrics 56:569, 1975.

177.
Shigeoka AO, Santos JI, Hill HR: Functional analysis of neutrophil granulocytes from healthy, infected, and stressed neonates. J Pediatr 95:454, 1979.

178.
Shigeoka AO, Charette RP, Wyman ML, et al: Defective oxidative metabolic responses of neutrophils from stressed neonates. J Pediatr 98:392, 1981.

179.
Strauss RG, Snyder EL: Neutrophils from human infants exhibit decreased viability. Pediatr Res 15:794, 1981.

180.
Strauss RG, Snyder EL, Wallace PO, et al: Oxygen-detoxifying enzymes in neutrophils of infants and their mothers. J Lab Clin Med 95:897, 1980.

181.
Yamazaki M, Matsuoka T, Yasui K, Komiyama A, Akabane T: Increased production of superoxide anion by neonatal polymorphonuclear leukocytes stimulated with a chemotactic peptide. Amer J Hematol 27:169, 1988.

182.
Kretschmer RR, Papierniak CK, Stewardson-Krieger P, et al: Quantitative nitrobluetetrazolium reduction by normal newborn monocytes. J Pediatr 91:306, 1977.

183.
Milgrom H, Shore SL: Assessment of monocyte function in the normal newborn infant by antibody-dependent cellular cytotoxicity. J Pediatr 91:612, 1977.

184.
Orlowski JP, Sieger L, Anthony BF: Bactericidal capacity of monocytes of newborn infants. J Pediatr 89:797, 1976.

185.
Schuit KE, Powell DA: Phagocytic dysfunction in monocytes of normal newborn infants. Pediatrics 65:501, 1980.

186.
Das M, Henderson T, Feig SA: Neonatal mononuclear cell metabolism: Further evidence for diminished monocyte function in the neonate. Pediatr Res 13:632, 1979.

187.
Mills EL: Mononuclear phagocytes in the newborn: their relation to the state of relative immunodeficiency. Am J Pediatr Hematol 5:189, 1983.

188.
Bryson YJ, Winter HS, Gard SE, Fisher TJ, Stiehm ERL: Deficiency of immune interferon production by leukocytes of normal newborns. Cell Immunol 55:191, 1987.

189.
Frenkel L, Bryson YJ: Ontogeny of phytohemagglutinin-induced gamma interferon by leukocytes of healthy infants and children: evidence for decreased production in infants younger than 2 months of age. J Pediatr 111:97, 1987.

190.
Wilson CB, Westall J, Johnston L, Lewis DB, Dower SK, Alper AR: Decreased production of interferon-gamma by human neonatal cells. J Clin Invest 77:860, 1986.

191.
Perussia B, Dayton ET, Lazarus R, Fenning V, Trinchieri G: Immune interferon induces the receptor for monomeric IgG on human monocytic and myeloid cells. J Exp Med 158:1092, 1983.

192.
Cairo MS: Review of G-CSF and GM-CSF effects on neonatal neutrophil kinetics. Am J Pediatr Hematol Oncol 11:238, 1989.

193.
Cairo MS, VandeVen C, Toy C, Suen Y, Mauss D, Sender L: GM- CSF primes and modulates neonatal PMN motility: up-regulation of C3bi (Mol) expression with alteration in PMN adherence and aggregation. Am J Pediatr Hematol Oncol 13:249, 1991.

194.
Sautois B, Fillet G and Beguin Y. Comparative cytokine production by in vitro stimulated mononucleated cells from cord blood and adult blood. Exp Hematol 25:103, 1997.

195.
Fogel BJ, Arais D, Kung F: Platelet counts in healthy premature infants. J Pediatr 73:108, 1968.

196.
Sell EJ, Corrigan JJ: Platelet counts, fibrinogen concentrations and factor V and factor VIII levels in healthy infants according to gestational age. J Pediatr 82:1028, 1973.

197.
Mehta P, Vasa R, Neumann L, Karpatkin M: Thrombocytopenia in the high-risk infant. J Pediatr 97:791, 1980.

198.
Meberg A, Halvorsen S, Orstavik I: Transitory thrombocytopenia in small-for-dates infants, possibly related to maternal smoking. Lancet 2:303, 1977.

199.
Thuring W, Tonz O: Neonatale Thrombozytenwere be: Kindern mit Down-Syndrom und anderen autosomalen Trisomien. Helv Paediatr Acta 34:545, 1979.

200.
Cairo, MS: The regulation of hematopoietic growth factor production from cord mononuclear cells and its effect on newborn rat hematopoiesis. J Hematother 2:217, 1993.

201.
Sirota L, Bessler H, Weissman Z, Dulitzky F, Djaldetti M: Thrombopoietic activity in preterm newborns and infants. Arch Dis Childhood 61:585, 1986.

202.
Suen Y, Chang M, Lee SM, Buzby JS, Cairo MS: Regulation of interleukin-11 protein and mRNA expression in neonatal and adult fibroblasts and endothelial cells. Blood 84:4125, 1994.

203.
Feusner JH: Normal and abnormal bleeding times in neonates and young children utilizing a fully standardized template technic. Am Soc Clin Pathol 74:73, 1980.

204.
Rennie JM, Gibson T, Cooke RWI: Micromethod for bleeding time in the newborn. Arch Dis Child 60:51, 1985.

205.
Andrew M, Paes B, Bowker J, Vegh P: Evaluation of an automated bleeding time device in the newborn. Am J Hematol 35:275, 1990.

206.
Weinstein MJ, Blanchard R, Moake JL, Vosburgh E, Moise K: Fetal and neonatal von Willebrand factor (vWF) is unusually large and similar to the vWF in patients with thrombotic thrombocytopenic purpura. Br J Haematol 72:68, 1989.

207.
Andrew M, Vegh P, Johnston M, Bowker J, Ofosu F, Mitchell L: Maturation of the hemostatic system during childhood. Blood 80:1998, 1992.

208.
Andrew M, Castle V, Saigal S, et al: Clinical impact of neonatal thrombocytopenia. J Pediatr 110:457, 1987.

209.
Corazza MS, Davis RF, Merritt TA, et al: Prolonged bleeding time in preterm infants receiving indomethacin for patent ductus arteriosus. J Pediatr 105:292, 1984.

210.
Stuart MJ: Platelet function in the neonate. Am J Pediatr Hematol Oncol 1:227, 1979.

211.
Israels SJ, Daniels M, McMillan EM: Deficient collagen-induced activation in the newborn platelet. Pediatr Res 27:337, 1990.

212.
Rajasekhar D, Kestin AS, Bednarek FJ, Ellis PA, Barnard MR, Michelson AD: Neonatal platelets are less reactive than adult platelets to physiological agonists in whole blood. Thromb Haemost 72:957, 1994.

213.
Ts’ao C, Green D, Schultz K: Function and ultrastructure of platelets of neonates; enhanced ristocetin aggregation of neonatal platelets. Br J Haematol 32:225, 1976.

214.
Blieyer WA, Breckenridge RT: Studies on the detection of adverse drug reactions in the newborn. II. The effects of prenatal aspirin on newborn hemostasis. JAMA 213:2049, 1970.

215.
Corby DG, Schulman I: The effects of antenatal drug administration on aggregation of platelets of newborn infants. J Pediatr 79:307, 1971.

216.
Hauth JC, Goldenberg RL, Parker CR Jr, Cutter GR, Cliver SP: Low- dose aspirin: lack of association with an increase in abruptio placentae or perinatal mortality. Obstet Gynecol 85:1055, 1995.

217.
Sibai BM, Caritis SN, Thom E, Shaw K, McNellis D: Low-dose aspirin in nulliparous women: safety of continuous epidural block and correlation between bleeding time and maternal-neonatal bleeding complications. National Institute of Child Health and Human Developmental Maternal-Fetal Medicine Network. Am J Obstet Gynecol 172:1553, 1995.

218.
Newman PJ, Derbes RS, Aster RH: The human platelet alloantigens, PIA1 and PIA2, are associated with a leucine 33/proline 33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing. J Clin Invest 83:1778, 1989.

219.
Gruel Y, Boizard B, Daffos F, Forestier F, Caen J, Wautier JL: Determination of platelet antigens and glycoproteins in the human fetus. Blood 68:488, 1986.

220.
McFarland JG, Aster RH, Bussel JB, Gianopoulos JG, Derbes RS, Newman PJ: Prenatal diagnosis of neonatal alloimmune thrombocytopenia using allele-specific oligonucleotide probes. Blood 78:2276, 1991.

221.
Shulman NR, Jordan JV Jr: Platelet immunology, in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 2nd ed, edited by RW Colman, J Hirsh, VJ Marder, EW Salzman, pp 476–483. JB Lippincott, Philadelphia, 1987.

222.
Pabst HF: Ontogeny of the immune response as a basis of childhood diseases. J Pediatr 97:519, 1980.

223.
Erkeller-Yuksel FM, Deneys V, Yuksel B, et al: Age related changes in human blood lymphocyte subpopulations. J Pediatr 120:216, 1992.

224.
De Waele M, Foulon W, Renmans W, et al: Hematologic values and lymphocyte subsets in fetal blood. Am J Clin Pathol 89:742, 1988.

225.
Hicks MJ, Jones JF, Minnich LL, Weigle KA, Thies AC, Layton JM: Age-related changes in T- and B-lymphocyte subpopulations in the peripheral blood. Arch Pathol Lab Med 107:518, 1983.

226.
Kotylo PA, Baenzinger JC, Yoder MC, Engle WA, Bolinger CD: Rapid analysis of lymphocyte subsets in cord blood. Am J Clin Pathol 93:263, 1990.

227.
Slukvin II, Chernishov VP: Two-color flow cytometric analysis of natural killer and cytotoxic T-lymphocyte subsets in peripheral blood of normal human neonates. Biol Neonate 61(3):156–61, 1992.

228.
Neubert R, Delgado I, Abraham K, Schuster C, Helge H: Evaluation of the age-dependent development of lymphocyte surface receptors in children. Life Sci 62:1099, 1998.

229.
Miller ME: Immune-inflammatory response in the human neonate. Am J Pediatr Hematol 3:199, 1981.

230.
Stiehm ER, Winter HS, Bryson YF: Cellular (T cell) immunity in the human newborn. Pediatrics 64:814, 1979.

231.
Carr MC, Stites DP, Fudenberg HH: Cellular immune aspects of the human fetal-maternal relationship. I. In vitro response of cord blood lymphocytes to phytohemagglutinin. Cell Immunol 5:21, 1972.

232.
Papiernick M: Comparison of human foetal with child blood lymphocytic kinetics. Biol Neonate 19:163, 1971.

233.
Uhr JW, Dancis J, Newmann CG: Delayed-type hypersensitivity in premature neonatal humans. Nature 187:1130, 1960.

234.
Blaese RM, Poplack DG, Muchmore AV: The mononuclear phagocyte system: Role in expression of immunocompetence in neonatal and adult life. Pediatrics 64(suppl):829, 1979.

235.
Von Freeden U, Zessack N, Van Valen F, Burdach S: Defective interferon gamma production in neonatal T cells is independent of interleukin-2 receptor binding. Pediatr Res 30:270, 1991.

236.
Sterm CMM: Changes in lymphocytes subpopulations in the blood of healthy and sick newborn infants. Pediatr Res 13:792, 1979.

237.
Raveche ES: Possible immunoregulatory role for CD5+ B cells. Clin Immuno Immunopath 56:135, 1990.

238.
Lawton AR, Cooper MD: B cell ontogeny: immunoglobulin genes and their expression. Pediatrics 64:750, 1979.

239.
Gustafsson BE, Laurell CB: Gamma globulin production in germ free rats after bacterial contamination. J Exp Med 110:675, 1959.

240.
Gitlin D: The differentiation and maturation of specific immune mechanisms. Acta Pediatr Scand 172(suppl):60, 1967.

241.
Stiehm ER: Fetal defense mechanisms. Am J Dis Child 129:438, 1975.

242.
Goldman AS, Garza C, Nichols BL, Goldblum RM: Immunological factors in human milk during the first year of lactation. J Pediatr 100:563, 1982.

243.
Goldman AS, Ham Pong AJ, Goldblum RM: Host defenses: development and maternal contributions. Adv Pediatr 32:71, 1985.

244.
Rothberg RM: Immunoglobulin and specific antibody synthesis during the first weeks of life of premature infants. J Pediatr 75:391, 1969.

245.
Harworth JC, Norris M, Dilling L: A study of the immunoglobulins in premature infants. Arch Dis Child 40:243, 1965.

246.
Thom H, McKay E, Gray DWG: Protein concentrations in the umbilical cord plasma of premature and mature infants. Clin Sci 33:433, 1967.

247.
Yeung CY, Hoffs JR: Serum g-G-globulin levels in normal, premature, postmature, and “small-for-dates” newborn babies. Lancet 1:1167, 1968.

248.
Sever JH: Immunological responses to perinatal responses to perinatal infections. J Pediatr 75:1111, 1969.

249.
Thomaidis T, Agathopoulos A, Matsaniotis N: Natural isohemagglutinin production by the fetus. J Pediatr 74:39, 1969.

250.
Morito T, Bankhurst AD, Williams RC Jr: Studies of human cord blood and adult lymphocyte interactions with in vitro immunoglobulin production. J Clin Invest 64:990, 1979.

251.
Miyagawa Y, Sugita K, Komiyama A, et al: Delayed in vitro immunoglobulin production by cord lymphocytes. Pediatrics 65:497, 1980.

252.
Ferguson AC, Cheung SC: Modulation of immunoglobulin M and G synthesis by monocytes and T lymphocytes in the newborn infant. J Pediatr 98:385, 1981.

253.
Blaese RM, Poplack DG, Muchmore AV: The mononuclear phagocyte system: role in expression of immunocompetence in neonatal and adult life. Pediatrics 64:829, 1977.

254.
Holroyde CP, Oski FA, Gardner FH: The “pocked” erythrocyte. N Engl J Med 281:516, 1969.

255.
Freedman RM, Johnston D, Mahoney MJ, et al: Development of splenic reticuloendothelial function in neonates. J Pediatr 96:466, 1980.

256.
Gross SJ, Stuart MJ: Hemostasis in the premature infant. Clin Perinatol 4:259, 1977.

257.
Barnard DR, Hathaway WE: Neonatal thrombosis. Am J Pediatr Hematol Oncol 1:235, 1979.

258.
Bleyer WA, Hakami N, Shepard TH: The development of hemostasis in the human fetus and newborn infant. J Pediatr 79:838, 1971.

259.
Andrew M, Paes B, Milner B, et al: Development of the human coagulation system in the full-term infant. Blood 70:165, 1987.

260.
Andrew M, Paes B, Milner R, et al: Development of the human coagulation system in the healthy premature infant. Blood 72:1651, 1988.

261.
Corrigan JJ Jr: Neonatal thrombosis and the thrombolytic system: pathophysiology and therapy. Am J Pediatr Hematol Oncol 10:83, 1988.

262.
Andrew M, Paes B, Johnston M: Development of the hemostatic system in the neonate and young infant. Am J Pediatr Hematol Oncol 12:95, 1990.

263.
Andrew M: The relevance of developmental hemostasis to hemorrhagic disorders of newborns. Semin Perinatol 21:70, 1997.

264.
Furie B, Furie BC: Molecular basis of gamma-carboxylation. Role of the propeptide in the vitamin K-dependent proteins. Ann NY Acad Sci 614:1, 1991.

265.
Aballi AJ, deLamerens S: Coagulation changes in the neonatal period and in early infancy. Pediatr Clin North Am 9:785, 1962.

266.
Muntean W, Petek W, Rosanelli K, et al: Immunologic studies of prothrombin in newborns. Pediatr Res 13:1262, 1979.

267.
Lane PA, Hathaway WE: Vitamin K in infancy. J Pediatr 106:351, 1985.

268.
Stevenson RE, Burton OM,, Ferlauto GJ, et al: Hazards of oral anticoagulants during pregnancy. JAMA 243:1549, 1980.

269.
Shearer MJ: Annotation: Vitamin K and vitamin K-dependent proteins. Br J Haematol 75:156, 1990.

270.
von Kries R, Hanawa Y: Neonatal vitamin K prophylaxis. Report of Scientific and Standardization Subcommittee on Perinatal Haemostasis. Thromb Haemost 69:293, 1993.

271.
Sutor AH, Gobel U, Kries RV, Kunzer W, Landbeck G: Vitamin K prophylaxis in the newborn. Blut 60:275, 1990.

272.
Hathaway WE, Isarangkura PB, Mahasandana C, et al: Comparison of oral and parenteral vitamin K prophylaxis for prevention of late hemorrhagic disease of the newborn. J Pediatr 119:461, 1991.

273.
Amadee-Manesme O, Labert WE, Alagille D, De Leenheer AP: Pharmacokinetics and safety of a new solution of vitamin K1 (20) in children with cholestasis. J Pediatr Gastroenterol Nutr 14:160, 1996.

274.
Aballi AJ: The action of vitamin K in the neonatal period. South Med J 58:48, 1965.

275.
Gray OP, Ackerman A, Fraser AJ: Intracranial haemorrhage and clotting in low birth weight infants. Lancet 1:543, 1968.

276.
Volpe JJ: Neonatal intraventricular hemorrhage. N Engl J Med 304:886, 1981.

277.
Appleyard WJ, Cottom DG: Effect of asphyxia on thrombotest values in low birthweight infants. Arch Dis Child 45:705, 1970.

278.
Schmidt B, Zipursky A: Thrombotic disease in newborn infants. Clin Perinatol 2:461, 1984.

279.
Rodgers GM, Shuman MA: Congenital thrombotic disorders. Am J Hematol 21:419, 1986.

280.
Sifontes MT, Nuss R, Hunger SP, Jacobson LJ, Waters J, Manco-Johnson MJ: Correlation between the functional assay for activated protein C resistance and factor V Leiden in the neonate. Pediatr Res 42:776, 1997.

281.
Leroyer C, et al: Prevalence of 20210 A allele of the prothrombin gene in venous thromboembolism patients. Thromb Haemost 80:49, 1998.

282.
Poort SR, Rosendaal FR, Reitsma PH, Bertina RM: A common genetic variation in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 88:3698, 1996.

283.
Miller RK, Kellogg CR, Saltzman RA: Reproductive and perinatal toxicology, in Handbook of Toxicology, edited by TJ Haley, WO Berndt, pp 195–309. Hemisphere Publishing, Washington, DC, 1987.

284.
Gray MJ: Use and abuse of thiazides in pregnancy. Clin Obstet Gynecol 11:568, 1968.

285.
Leikin SL: Thiazide and neonatal thrombocytopenia. N Engl J Med 271:161, 1964.

286.
Ginsberg JS, Kowalchuk G, Hirsh J, Brill-Edwards P, Burrows R: Heparin therapy during pregnancy. Arch Intern Med 149:2233, 1989.

287.
Page TE, Hoyme HE, Markarian M, et al: Neonatal hemorrhage secondary to thrombocytopenia: an occasional effect of prenatal hydantoin exposure. Birth Defects 18:47, 1982.

288.
Hanson JW, Buehler BA: Fetal hydantoin syndrome: current status. J Pediatr 101:816, 1982.

289.
Eggermont E, Logghe N, van de Casseye W, et al: Haemorrhagic disease of the newborn in the offspring of rifampin and isoniazid treated mothers. Acta Pediatr Belg 29:87, 1976.

290.
Powell RD, DeGowin RL, Alving AS, et al: Nitrofurantoin-induced hemolysis. J Lab Clin Med 62:1002, 1963.

291.
Belton EM, Jones RV: Haemolytic anaemia due to nalidixic acid. Lancet 2:691, 1965.

292.
Varsano I, Fischl J, Tikvah P, et al: The excretion of orally ingested nitrofurantoin in human milk. J Pediatr 82:886, 1973.

293.
Brodersen R: Prevention of kernicterus, based on recent progress in bilirubin chemistry. Acta Paediatr 66:625, 1977.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

 

FREE downloads:

i will be here

shot caller

side by side

CHAPTER 6 THE INFLAMMATORY RESPONSE

CHAPTER 6 THE INFLAMMATORY RESPONSE
Williams Hematology

CHAPTER 6 THE INFLAMMATORY RESPONSE

JEFFREY S. WARREN
PETER A. WARD

History
General Characteristics of Inflammation
Acute Inflammation

Hemodynamic Changes

Leukocyte Recruitment

Regulation of the Inflammatory Response
Chronic Inflammation and Repair
Chapter References

The inflammatory response is characterized by a series of events that encompass a rapid and relatively short-lived increase in local blood flow, an increase in microvascular permeability, and the sequential recruitment of different types of leukocytes. Superimposed upon the inflammatory response is a series of reparative processes (e.g., parenchymal regeneration, angiogenesis, production of extracellular matrix material, and scar formation). Early hemodynamic changes at a site of an inflammation establish conditions that enable marginated leukocytes to engage in low-affinity selectin-mediated rolling interactions with endothelial cells. In response to locally produced soluble and cell surface mediators, endothelial cells and rolling leukocytes become activated and sequentially express several sets of complementary adhesion molecules which include b2 integrins, members of the selectin family, and members of the immunoglobulin superfamily. Leukocyte and endothelial cell adhesion molecules mediate the high-affinity adhesive interactions necessary for leukocyte emigration from the vascular space and across specific chemotactic gradients. Analogous, temporally regulated soluble mediators and cellular adhesion molecules also orchestrate the monocyte- and lymphocyte-rich chronic inflammatory response. This basic paradigm is modulated by a large number of surface-active and soluble inflammatory mediators which include vasoactive amines and lipids, reactive oxygen and nitrogen intermediates, cytokines, chemokines, and many plasma proteins (e.g., complement system, kinins, and coagulation cascade).

Acronyms and abbreviations that appear in this chapter include: cGMP, cyclic guanosine 3,5-monophosphate; EDRF, endothelium-derived relaxing factor; ELAM-1, endothelial cell leukocyte adhesion molecule-1; GMP-140, granule membrane protein-140; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid; ICAM-1, intercellular adhesion molecule-1; ICAM-2, intercellular adhesion molecule-2; ICAM-3, intercellular adhesion molecule-3; IFN-g, interferon-g; IL-1, interleukin-1; IL-6, interleukin-6; IL-8, interleukin-8; IL-b, interleukin-b; g-IP-10, g-interferon-inducible protein; LAM-1, leukocyte adhesion molecule-1; LTB4, leukotriene B4; LTC4, leukotriene C4; LTD4, leukotriene D4; LDE4, leukotriene E4; LFA-1, lymphocyte function-associated antigen-1; MCP-1, monocyte chemoattractant protein-1; MGSA (or GROa), melanocyte growth-stimulatory activity; MIP-1a, macrophage inflammatory protein-1a; MIP-1b, macrophage inflammatory protein-1b; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NAP-2, neutrophil-activating peptide; NOS, nitric oxide synthase; PAF, platelet-activating factor; PECAM-1, platelet endothelial cell adhesion molecule; PF4, platelet factor 4; RANTES, regulated upon activation, normal T cell expressed and presumably secreted; RGD; TNF-a, tumor necrosis factor-a; VCAM-1, vascular cell adhesion molecule-1; VLA-4, very late antigen 4.

HISTORY
The sentinel clinical features of acute inflammation, rubor, calor, tumor, and dolor, have been recognized for at least five thousand years. Dr. John Hunter, the renowned late eighteenth century Scottish surgeon, observed that the inflammatory response is not a disease per se but rather a nonspecific and salutary response to a variety of insults. Through his microscopic examinations of transparent vital membrane preparations, Julius Cohnheim concluded that the inflammatory response is fundamentally a vascular phenomenon. Leukocytic phagocytosis was discovered late in the nineteenth century by Eli Metchnikoff and his colleagues. Morphologic studies, using both live animals and fixed histologic preparations, transformed our understanding of inflammation and led to the currently held concepts of inflammation-associated hemodynamic alterations, “acute” inflammation, and “chronic” inflammation.1 It has been during the past thirty to forty years that the modern techniques of biochemistry (e.g., protein and lipid purification and the measurements of reactive oxygen and nitrogen species), tissue culture, monoclonal antibody production, recombinant DNA technology, and the genetic manipulation of isolated cells and whole animals, have enabled a more detailed understanding of the cellular and molecular mechanisms which characterize the inflammatory response. These studies, in concert with “experiments of nature” such as chronic granulomatous disease (see Chap. 67 and Chap. 72) and the leukocyte adhesion deficiency disorders (see Chap. 72), have provided for the formulation of complex, yet elegant, models of acute and chronic inflammation and the development of incisive therapeutic approaches that promise to exploit this knowledge. A vast array of human diseases is marked by either defects in the development of the inflammatory response or the deleterious effects of the inflammatory response itself.
GENERAL CHARACTERISTICS OF INFLAMMATION
While necessarily contrived, it remains useful to consider inflammation as an acute or chronic process. “Acute” inflammation lasts from minutes to several days and is characterized by local hemodynamic and microvascular changes and leukocyte accumulation.2 The acute inflammatory response is consistently marked by microvascular leakage and the accumulation of neutrophils. The four cardinal signs of inflammation, alluded to above, can be accounted for within the physiologic terms of acute inflammation.
In contrast, the chronic inflammatory response lasts much longer and is more varied in its effects.2 Cellular infiltrates are composed primarily of lymphocytes and monocytes but there are many variations in the cellular composition, anatomic distribution, and tempo of development of chronic inflammatory lesions. The chronic inflammatory response is also marked by the proliferation of resident fibroblasts and the growth of new capillaries. Chronic inflammatory processes are classified according to these variations. For example, granulomatous inflammation is a chronic process marked by nodular aggregates of mononuclear phagocytes that have become “transformed” into so-called epithelioid histiocytes because of their similar appearance to epithelial cells. In many cases there are accompanying multinucleate giant cells. Granulomas may be distributed along blood vessels (e.g., angiocentric), along airways (e.g., bronchocentric), or randomly throughout the interstitium or parenchyma of an organ. Some chronic inflammatory processes are marked by the appearance of plasma cells or eosinophils.
Superimposed upon the acute and chronic inflammatory response is repair.2 Repair may entail the regeneration of parenchymal cells damaged as the result of an insult per se or damaged secondary to the inflammatory response to the insult. Repair is characterized by the growth of new capillaries (angiogenesis) and the activation of fibroblasts which produce extracellular matrix molecules (e.g., scar tissue). In some circumstances an acute inflammatory response is self-contained and nonprogressive. In other situations the response progresses to a chronic process which can persist for years (e.g., tuberculous granulomas).
This chapter will first address acute inflammation, which encompasses localized changes in blood flow, alterations in microvascular permeability, and neutrophil exudation. There has been a rapid advance in understanding of the processes of endothelial cell activation, leukocyte-endothelial cell rolling and adhesive interactions, leukocyte emigration, and leukocyte activation. The second section of this chapter will introduce the vast array of soluble and surface-active mediators that orchestrate both acute and chronic inflammatory responses. These mediators include substances that range from short-lived reactive oxygen and nitrogen intermediates to entire regulatory systems (e.g., complement system and coagulation cascade). Finally, a brief overview of chronic inflammation and tissue repair will be provided. The goal of this chapter is to provide a framework for understanding the basic processes of inflammation while gaining an appreciation for the highly complex and integrated nature of the regulated inflammatory response.
ACUTE INFLAMMATION
HEMODYNAMIC CHANGES
The hemodynamic changes that occur early in the acute phase of inflammation include arteriolar vasodilatation and localized increases in microvascular permeability. In many but not all circumstances arteriolar vasodilatation follows a rapidly developing (within seconds) and brief (seconds) period of vasoconstriction.3 Arteriolar vasodilation results in increased blood flow, thus explaining the familiar redness and warmth which characterize a site of acute inflammation. The increase in blood flow, coupled with increases in microvascular permeability, results in hemoconcentration and increased local viscosity. These localized hemodynamic changes are critical to subsequent leukocyte emigration because selectin-mediated low-affinity rolling leukocyte-endothelial adhesive interactions can efficiently occur only under such conditions of low shear force (see below). Experimental studies using in vitro flow chambers and live animals indicate that selectin-mediated leukocyte-endothelial rolling adhesive interactions cannot occur in the face of the shear forces exerted by normal blood flow. Increased microvascular permeability leads to the exudation of protein-rich plasma which is a fundamental characteristic of acute inflammation. Microvascular leakage occurs through a variety of mechanisms that include venular endothelial cell contraction, which is accompanied by widening of intercellular junctions; so-called endothelial cell retraction, which is not well understood but involves cytosketetal changes; leukocyte-mediated endothelial cell injury; direct endothelial injury; and leakage via new capillaries which do not yet possess fully “closed” intercellular junctions.4,5 Increases in rate of transcytosis in which plasma constituents cross endothelial cells in vesicles or vacuoles have a role in neoplastic blood vessels and may play a role in inflammation.6 Alterations in local blood flow occur at the level of arterioles which are regulated largely by the autonomic nervous system, vasoactive peptides, and eicosanoids. A variety of soluble mediators [e.g., histamine, leukotrienes, complement components C3a and C5a, interleukin-1, tumor necrosis factor-a (TNF-a), and interferon-g (IFN-g)] can induce increases in microvascular permeability through several of the above-mentioned mechanisms.
LEUKOCYTE RECRUITMENT
The orchestrated recruitment of leukocytes into a site of inflammation is a fundamental characteristic of the inflammatory response.6 The importance of white blood cells in host defense is highlighted in patients with genetic defects in white blood cell function [e.g., absence respiratory burst in patients with chronic granulomatous disease (Chap. 67) and diminished leukocyte emigration in patients with leukocyte adhesion deficiency (Chap. 72)]. Leukocytes are critical because of their central role in the phagocytosis and containment or killing of microbes and in the digestion of necrotic tissue debris. Leukocyte-derived products such as proteolytic enzymes and reactive oxygen intermediates contribute to tissue injury.
Leukocyte Adhesion and Transmigration
When vascular stasis occurs as the result of the hemodynamic changes of early acute inflammation, leukocytes are pushed from the central axial column of blood cells to a position along the endothelial surface. This process, called margination, occurs under conditions of slow blood flow.2 Individual leukocytes adhere transiently and weakly to the endothelial surface. Studies using vital membrane preparations and flow chamber studies using endothelial cell monolayers and suspensions of purified leukocytes have revealed that cells roll along the endothelial surface.6,7,8,9,10,11,12 and 13 Rolling neutrophil-endothelial adhesive interactions occur early (minutes) after the initiation of an acute inflammatory response and can, depending upon the time within the evolution of an inflammatory response, involve neutrophils, lymphocytes, monocytes, basophils, or eosinophils. The leukocyte-endothelial cell rolling adhesive interaction is a specific and necessary step that precedes tight adhesion and emigration.10 Studies indicate that early rolling adhesive interactions are mediated largely by selectins.11 In turn, the cell surface expression of selectins (and other intercellular adhesion molecules, see below) is regulated by a number of locally produced proinflammatory mediators.6,8,9,10,11,12 and 13
Selectins contain a C-terminal cytoplasmic tail, a lipophilic transmembrane domain, a series of complement regulatory domains, an epidermal growth factor–like domain, and an extracellular N-terminal carbohydrate-binding region which is homologous to mammalian lectins (Table 6-1).9,10,11,12 and 13 P-selectin (previously known as GMP-140) is expressed by endothelial cells and platelets, E-selectin (formerly ELAM-1) by endothelial cells, and L-selectin (also known as LAM-1) by most white blood cells. P-selectin is stored in endothelial intracytoplasmic granules called Weibel-Palade bodies.14,15 When endothelial cells are exposed to histamine, thrombin, or platelet-activating factor, P-selectin is rapidly (minutes) translocated to the endothelial surface where it engages marginated leukocytes via carbohydrate moieties that contain sialic acid residues (e.g., sialyl LewisX glycoprotein).9,10,11,12 and 13 This transient, low-affinity binding interaction which can withstand only the low-flow shear force conditions found in stasis, accounts in part for the early rolling leukocyte-endothelial cell adhesive interactions (Fig. 6-1). Exposure of endothelial cells to TNF-a or IL-b results in protein synthesis–dependent expression of E-selectin, a response that occurs within 1–2 hours and peaks at 4–6 hours.16,17 As in the case of P-selectin–mediated leukocyte adhesion, E-selectin–mediated adhesion occurs via a series of sialylated and fucosylated carbohydrate moieties related to the sialyl LewisX (SLeX) and sialyl LewisA (SLeA) blood group antigens on leukocytes (Table 6-1).13,18 L-selectin is constitutively expressed by leukocytes, participates in white blood cell-endothelial cell adhesive interactions via mucin-like glycoproteins (e.g., CD34, GlyCAM), and is shed when the leukocyte is activated (Table 6-1).9,19 It is believed that L-selectin shedding facilitates leukocyte emigration by allowing the white blood cell to detach from the endothelium. Low-affinity rolling adhesive interactions set the stage for b-integrin and immunoglobulin superfamily-mediated high-affinity adhesive interactions and leukocyte transmigration.10

TABLE 6-1 ADHESION MOLECULES IN INFLAMMATION

FIGURE 6-1 Leukocyte-endothelial adhesive interactions. Early in the acute inflammatory response, marginated leukocytes engage in transient, low-affinity, selectin-mediated adhesive interactions with endothelial cells. As the response evolves, activated leukocytes and endothelial cells engage in high-affinity b2-integrin and immunoglobulin superfamily-mediated adhesive interactions. A variety of chemotactic factors can provide the motive force for leukocyte emigration. [Modified and redrawn from multiple references (7,8,9,10 and 11)].

Relatively weak selectin-mediated and high-affinity adhesive interactions are not temporally or mechanistically discrete. For example, TNF-a and IL-b induce both E-selectin, which is not expressed by quiescent cells, and increases in endothelial expression of ICAM-1 and VCAM-1, which are constitutively expressed in low concentrations and are involved in the recruitment of all types of leukocytes in the case of ICAM-1, and chronic inflammatory leukocytes (lymphocytes, monocytes, eosinophils, and basophils) in the case of VCAM-1.6,8,9,10,11 and 12,20 Intercellular adhesion molecule-1 binds to b2 (leukocyte) integrins (e.g., CD11a/CD18, CD11b/CD18) and VCAM-1 binds to b1 integrins (e.g., VLA-4/a4b1) (Table 6-1).21 It is believed that activated endothelial cells secrete such mediators as platelet-activating factor and IL-8 which in turn activate overlying leukocytes.6 Leukocyte CD11a/CD18 (LFA-1) undergoes a conformational change by which there is an increase in its binding affinity for endothelial ICAM-1. The b2 integrins are heterodimeric structures which contain varied alpha chains (CD11a, CD11b, CD11c) and a common beta chain (CD18).21 The role of CD11c/CD18 is less clearcut than those of CD11a/CD18 and CD11b/CD18. Intercellular adhesion molecules (ICAM-1, ICAM-2, ICAM-3) are found on a variety of cell types aside from endothelial cells.22,23 and 24 CD11a/CD18 interacts with both ICAM-1 and ICAM-2 while CD11b/CD18 binds to ICAM-2 and the complement activation product, iC3b (see below). The role of ICAM-3 in leukocyte-endothelial adhesion is less well established. b1 integrins, notably VLA-4, are found primarily on chronic inflammatory leukocytes (e.g., lymphocytes, monocytes, basophils, and eosinophils) and mediate leukocyte binding via VCAM-1.25,26 and 27 b1-integrin–mediated adhesive interactions occur via RGD amino acid sequences within VCAM-1 as well as other molecules (e.g., fibronectin). b2 integrin-ICAM and b1-VCAM-1–mediated adhesive interactions occur later (hours-days) in the inflammatory response than do selectin-mediated interactions. Studies indicate that additional adhesive interactions are also involved in leukocyte transmigration [e.g., CD31 or PECAM-1 (platelet endothelial cell adhesion molecule)].28 The functional importance of the various complementary leukocyte-endothelial adhesive interactions has been clarified by in vitro leukocyte-endothelial binding studies and in vivo studies that have employed neutralizing antibodies directed against adhesion molecules, pharmacologic antagonists of adhesion molecules, and knockout mice.29,30 and 31 The functional importance of leukocyte integrins (CD11a/CD18, CD11b/CD18, CD11c/CD18) has also been highlighted by clinical and experimental observations in patients with leukocyte adhesion deficiencies (see Chap. 72).32
LEUKOCYTE CHEMOTAXIS AND ACTIVATION
Leukocytes that are tightly bound to endothelium emigrate from the vascular space into the interstitium by extending pseudopods between intercellular junctions (Fig. 6-1). Secreted specific granule proteases play a role in the passage or “invasion” of leukocytes through subendothelial extracellular matrix material (e.g., basement membrane). Leukocyte emigration and movement through the interstitium is facilitated by binding interactions between leukocyte integrins and complementary sites on extracellular matrix molecules (e.g., fibronectin).33 A wide variety of soluble mediators can provide the motive force (chemotaxis) for this process.34 Chemotactic factors for neurophils include peptides derived from bacteria (e.g., N-formyl-methionyl peptides), complement-derived peptides (e.g., C5a, see below), chemotactic lipids [e.g., leukotriene B4 (LTB4) and others, see below], and locally produced cytokines (e.g., TNF-a and IL-1b) and chemokines (e.g., IL-8, see below). Chemotactic factors vary with respect to their specificity for different types of leukocytes. For example, C5a and N-formyl peptides both induce neutrophil and monocyte chemotaxis, IL-8 induces neutrophil chemotaxis, and monocyte chemoattractant protein-1 (MCP-1) induces chemotactic responses in monocytes and a specific subset of memory T lymphocytes. Each of these chemotactic factors activates “target” cells by engaging specific, cell surface receptors which in turn are linked to the contractile cell motility apparatus (e.g., microfilament proteins such as myosin and actin, and actin-regulating proteins such as gelsolin, filamen, profilin, and calmodulin) via complex signal-transduction pathways.33,34 In addition to chemotaxis, soluble and cell surface mediators induce leukocyte activation which is manifested by a wide array of changes in cellular function (e.g., adhesion molecule upregulation and increased adhesion molecule binding avidity (e.g., CD11a/CD18), selectin shedding (e.g., L-selectin), lysosome degranulation, and initiation of the respiratory burst). There have been great advances in understanding of the biochemical pathways involved in chemotaxis and cell activation. While there are many nuances in the signal-transduction pathways involved in these processes, several themes have emerged. Cell surface receptors are activated by specific ligands (e.g., C5a, LTB4, IL-8, etc.) and receptor activation is transduced via specific G proteins and membrane-associated phospholipases which in turn leads to mobilization of intracellular calcium, influx of extracellular calcium, and protein phosphorylation. Genetic defects in the regulation of many of these processes have been described and are detailed elsewhere throughout this text.
The principal result of neutrophil and monocyte recruitment are to provide 1) high concentrations of activated leukocytes that can release lytic substances and reactive oxygen and nitrogen intermediates needed to destroy foreign invaders, and 2) a vehicle to contain foreign particulates through phagocytosis. The products and functions of activated inflammatory cells are at once salutary because they contain and destroy invaders and deleterious because they cause tissue damage.
Leukocyte activation, especially that of neutrophils and mononuclear phagocytes, induced either by soluble mediators or by the process of phagocytosis, results in the secretion of many lysosomal substances (e.g., myeloperoxidase by neutrophils), the generation of reactive oxygen and nitrogen intermediates (e.g.,
, H2O2, NO), the generation of arachidonate metabolites (e.g., leukotrienes and prostaglandins), and the production of other mediators (see below).35,36 In some circumstances these materials are released into phagolysosomes where they contribute to the destruction of engulfed microbes while in other circumstances they are secreted into the extracellular milieu where they may amplify the inflammatory response and cause tissue damage.
Phagocytosis involves three distinct steps: recognition and attachment, engulfment, and degradation (killing) of the ingested material.37,38 Phagocytosis is enhanced greatly when particles (e.g., bacteria) are coated with opsonins which in turn function as ligands for leukocyte surface receptors. The major opsonins include the Fc domain of IgG and IgM immunoglobulins and the complement-derived fragments, C3b and iC3b, which covalently link to the surfaces of particles and large molecules. There are a variety of Fc receptors (FcgRI, FcgRII. FcgRIII, etc.) and complement receptors (e.g., CR1, CR2, CR3) which specifically engage their respective opsonins when the latter coat foreign particulates. As noted in Table 6-1, some enhanced phagocytic reactions occur independently of opsonins (e.g., CR3, the b2 integrin Mac-1, binds lipopolysaccharide directly). Engulfment is triggered as the result of engagement of FcgR and is enhanced by the concurrent engagement of complement receptors. In some circumstances, engulfment is enhanced by the simultaneous binding of the leukocyte to specific extracellular matrix molecules (e.g., fibronectin) or soluble cytokines. Engulfment results in the formation of phagosomes which fuse with lysosomes to form phagolysosomes in which the foreign particle is degraded. Numerous mechanisms for killing and/or degradation of microbes have been elucidated (Table 6-2). Although these mechanisms are classified as either oxygen-dependent or oxygen-independent, both types of processes may be involved in the destruction of a given microorganism, and a given microorganism may vary greatly in its susceptibility to various mechanisms of destruction.35,39

TABLE 6-2 KILLING AND DEGRADATION OF MICROORGANISMS IN PHAGOCYTES

REGULATION OF THE INFLAMMATORY RESPONSE
The foregoing sections provide a conceptual framework for the inflammatory response, specifically, the hemodynamic alterations, mechanisms of specific leukocyte-endothelial adhesive interactions, chemotaxis, and leukocyte activation and phagocytosis. The many steps that constitute this paradigm are regulated by a variety of soluble mediators that are produced by endothelial cells and leukocytes at a site of inflammation, by other resident cells (e.g., tissue macrophages, fibroblasts, mast cells), and as by-products of blood-borne proteins (e.g., complement system, coagulation cascade). These inflammatory mediator systems are summarized in Table 6-3.

TABLE 6-3 INFLAMMATORY MEDIATOR SYSTEMS

REACTIVE OXYGEN INTERMEDIATES
Since the early 1970s it has been recognized that activated phagocytes exhibit a transient but marked increase in oxygen consumption and the generation of reduced oxygen metabolites.35 Although small quantities of reactive oxygen intermediates are produced as by-products of a variety of biochemical pathways, the chief source is the leukocyte membrane-associated NADPH oxidase, an enzyme complex that is defective in patients with chronic granulomatous disease (see Chap. 67). Reactive oxygen intermediates include superoxide anion (
), hydrogen peroxide (H2O2), hydroxyl radical (H
), and singlet oxygen (1O2). These reduced oxygen products play a major role in intraphagolysosomal killing of microorganisms and when released extracellularly are directly or indirectly responsible for a variety of inflammatory processes including endothelial cell lysis, extracellular matrix degradation, activation of latent proteolytic enzymes (collagenase, gelatinase), inactivation of antiproteases, interaction with toxic metabolites of L-arginine, and generation of chemotactic factors from arachidonic acid and the complement component, C5. In addition to their role in endothelial cytotoxicity, reactive oxygen intermediates have been shown to be cytotoxic for fibroblasts, erythrocytes, tumor cells, and various parenchymal cells. The biochemical mechanisms implicated include lipid peroxidation, the formation of carbonyl moieties and nitrosylation products, intracellular enzyme inactivation, protein oxidation, and oxidant-mediated DNA damage. Studies indicate that reactive oxygen intermediates (e.g.,
) can also undergo reactions with reactive nitrogen intermediates (e.g., NO, see below) to generate toxic NO derivatives.
REACTIVE NITROGEN INTERMEDIATES
Described in 1980 as endothelium-derived relaxing factor (EDRF), NO is the soluble, short-acting biosynthetic product of L-arginine, O2, NADPH, and nitric oxide synthase (NOS).40,41 As suggested by its original name, NO mediates vascular smooth muscle relaxation. NO binds to the heme moiety of guanylyl cyclase to trigger the generation of intracytoplasmic cGMP and, through the activation of a series of kinases, induces smooth muscle relaxation and vasodilatation. At least three different forms of NOS have been characterized. Nitric oxide can be produced either constitutively or induced in a wide variety of cell types (e.g., endothelial cells, neurons, macrophages). In addition to its activity as a vasodilator, NO plays important roles in the inhibition of smooth muscle proliferation and in inflammation. For instance, NO can react with reactive oxygen intermediates to form both reactive oxygen and nitrogen species (e.g., NO +
® N
+ H
), it can inhibit DNA synthesis, it can directly kill microbes and tumor cells, and it can inactivate cytosolic glutathione and a number of sulfhydryl enzymes. In vivo studies have confirmed that inhibition of NO synthesis with antagonistic L-arginine analogs can reduce tissue injury in models of inflammation.42,43
LYSOSOMAL GRANULE CONSTITUENTS
The activation of neutrophils, monocytes, and macrophages results in the release, either through exocytosis or as the result of cell death, of a wide variety of proinflammatory mediators that have important roles in the inflammatory response.44 Neutrophils contain two major types of granules (see Chap. 64, Chap. 65, and Chap. 67). Large, primary (azurophilic) granules contain lysozyme, a variety of cationic proteins, myeloperoxidase, defensins, phospholipase, acid hydrolases, and neutral proteases (e.g., proteinase 3, collagenases, elastase). Smaller, secondary (specific) granules contain lysozyme, lactoferrin, type IV collagenase, alkaline phosphatase, membrane-associated NADPH oxidase, and the b2 integrins. Acid proteases function most efficiently within phagolysosomes where the pH is low, while neutral proteases can function efficiently within extracellular inflammatory exudates. Lysosomal granule constituents contribute to the inflammatory response and tissue injury through a wide array of mechanisms (e.g., degradation of extracellular matrix, proteolytic generation of chemotactic peptide, catalysis of reactive oxygen metabolite generation).
CYTOKINES AND CHEMOKINES
Cytokines are relatively small (5–20kD) proteins that modulate the function of other cell types. A large number of cytokines and chemokines have been identified and characterized in recent years.45,46 and 47 In addition to their important roles in regulating various aspects of the immune response, many cytokines participate in inflammatory processes. Among the most thoroughly characterized cytokines are IL-1 and TNF-a. Interleukin 1 and TNF-a are structurally dissimilar but share many biologic activities and function as autocrine, paracrine, and endocrine mediators (Table 6-4).

TABLE 6-4 INTERLEUKIN-1 AND TUMOR NECROSIS FACTOR IN INFLAMMATION

IL-1 is a 17 kD protein that exhibits a wide variety of biological activities. Initially termed “endogenous pyrogen” due to its ability to induce temperature elevation and the acute-phase response, IL-1 is now known to be relevant to acute inflammation because of its ability to induce cytokine production in monocytes, macrophages, fibroblasts, and endothelial cells (TNF-a, IL-1, and IL-6). Interleukin-1 can also induce NOS. As noted in the section that describes endothelial-leukocyte adhesive interactions, IL-1 can activate endothelial cells, resulting in the expression of adhesion molecules.
Tumor necrosis factor-a (TNF-a), or cachectin, is also a 17 kD protein. Like IL-1, TNF-a can induce cytokine production in a variety of cells. TNF-a can induce neutrophil activation and the expression of adhesion molecules on endothelial cells. In contrast to IL-1, TNF-a possesses potent cytotoxic activities for certain types of cells. Both IL-1 and TNF-a are produced in response to endotoxemia and both can initiate a systemic shock-like response.
Chemokines, or “intercrines,” are cytokines which exhibit prominent chemotactic activities.48,49,50 and 51 The two major subfamilies include the alpha, or “-C-X-C-,” chemokines and the beta, or “C-C,” chemokines. “C-X-C” chemokines are so-designated because the first two N-terminal cystine residues are separated by a single amino acid. Alpha chemokines, of which IL-8 is the prototype, consistently exhibit neutrophil chemotactic activity, while the beta, or “-C-C,” chemokines, of which monocyte chemoattractant protein-1 (MCP-1) is the prototype, exhibit monocyte chemotactic activity (Table 6-5). Chemokines activate leukocytes through membrane receptors (serpentines) which contain seven transmembrane domains and are linked to cytosolic surface G proteins.52

TABLE 6-5 CHEMOKINES

INFLAMMATORY LIPIDS
Arachidonic acid is a 20-carbon polyunsaturated fatty acid (5, 8, 11, 14-eicosatetraenoic acid) derived either from dietary sources or by conversion from linoleic acid. Arachidonic acid is maintained in cell membranes as an esterified phospholipid. The two families of inflammatory mediators derived from arachidonic acid are generated via the cyclooxygenase and lipoxygenase pathways (resulting in the appearance of prostaglandins and leukotrienes, respectively).53 Cell activation or mechanical stress can result in the release of arachidonic acid. Activation of the cyclooxygenase family of phospholipases results in prostaglandins synthesis. Members of this group of mediators exhibit several proinflammatory activities which include vasodilatation, vasoconstriction, increases in permeability, and platelet activation (aggregation). Activation of the lipoxygenase pathway results in the synthesis of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is a potent chemoattractant of neutrophils and is further modified to yield a series of leukotrienes. Leukotriene B4 (LTB4) induces neutrophil chemotaxis, aggregation, degranulation, and adherence, while LTC4, LTD4, and LTE4 trigger smooth muscle constriction and increases in vascular permeability. Members of both of these families of lipid-derived mediators have been detected in inflammatory exudates. Nonsteroidal anti-inflammatory agents and aspirin, which inhibit cyclooxygenase, emphasize the importance of these mediators in the development of an acute inflammatory response.
Platelet-activating factor (PAF) is a potent proinflammatory lipid produced by a variety of cell types including neutrophils, monocytes, endothelial cells, and IgE-sensitized basophils.54 Derived from the cell membrane constituent, choline phosphoglyceride, PAF is an acetyl glycerol ether phosphocholine which is synthesized following the activation of phospholipase A2. PAF triggers platelet aggregation and degranulation, increases vascular permeability, and promotes leukocyte accumulation and activation. In vivo studies using specific PAF antagonists have suggested a role for PAF in ischemia-reperfusion of the heart and gut and in immune complex–mediated injury in the skin, lung, and kidney.54 In addition, a PAF-like lipid has been measured in the blood of patients with angioedema and cold urticaria.
KININS
The kinin system is activated by contact activation of clotting factor XII (Hageman factor) (see Chap. 112).55 Activation of the kinin system results in the generation of the vasoactive nine amino acid peptide, bradykinin. Bradykinin possesses several activities, including the capacity to increase vascular permeability, to induce smooth muscle contraction, to trigger vasodilation, and to cause pain.55 Activated Hageman factor (factor XIIa), also known as the prekallikrein activator, converts plasma prekallikrein to kallikrein. Kallikrein cleaves high-molecular-weight kininogen to produce bradykinin. Models of septic shock have revealed decreases in plasma kininogen that parallel decreases in peripheral arterial resistance.55 The presence of plasma kininases precludes the routine measurement of bradykinin by functional or immunochemical approaches.
VASOACTIVE AMINES
Histamine and serotonin (5-hydroxytryptamine) are low molecular weight vasoactive amines. Histamine is contained in mast cell and basophil granules while platelets are the chief source of serotonin.56 Localized release of histamine results in wheal formation due to increases in vascular permeability. Histamine induces the formation of reversible openings in endothelial tight junctions, triggers the formation of prostacyclin in endothelium, and induces NO release from the endothelium. In addition, histamine, like thrombin, can induce the rapid upregulation of endothelial P-selectin.57 Serotonin, which acts through receptors on vascular smooth muscle cells, is responsible for vasoconstriction, whereas interaction with endothelial receptors results in vasodilation (via release of NO) and increased permeability. Release of histamine and serotonin from mast cells and platelets can be triggered by IgE-mediated type I hypersensitivity reactions, directly by C3a or C5a, and directly by neutrophil granule-derived cationic proteins.
COMPLEMENT
The complement system, including its soluble and cell membrane–associated regulators, consists of nearly two dozen plasma proteins that give rise to mediators of chemotaxis, increased vascular permeability, opsonic activity, phagocytic activation, and cytolysis.58 In a manner analogous to coagulation, the complement system is activated through a cascade of proteolytic cleavage reactions. There are two convergent pathways (Figure 6-2). The first of these, the classical pathway, is initiated by complement-fixing immune complexes (IgG and IgM), while the second, the alternative pathway, is triggered by a variety of substances that include IgA aggregates, endotoxin, cobra venom factor, and the polysaccharide components of some bacterial and fungal cell walls. The classical pathway is initiated by the fixation of C1 (C1qr2s2) by the Fc portion of surface-bound IgG or IgM immunoglobulins. Activated C1 (C1qr2s2) cleaves C2 and C4 which leads to the formation of “classical pathway” C3 convertase, C4b2a. Activation of the alternative pathway results in the formation of an “alternative pathway” C3 convertase following direct cleavage of C3 and subsequent interactions of C3b with factors B and D in the presence of Mg2+. The resulting complex, C3bBb is stabilized by properdin, leading to a stable C3 convertase, C3bBbP. C3 convertases generated via either pathway can cleave C3 to form C3a and C3b. C3b can bind to either the classical or alternative pathway C3 convertase to form a C5 convertase, which cleaves C5 into C5a and C5b. C5a is released into the fluid phase, like C3a, whereas C5b combines first with C6 and then C7 to form C5b-7 which in turn binds with C8 and multiple C9 molecules to form C5b-9, the membrane attack complex. In addition to the cell-activating and cytolytic activities of C5b-9, individual complement cleavage products and complexes have a wide variety of specific and potent proinflammatory activities.58 These various functions, combined with the rapid amplification in numbers of complement-derived mediators, emphasize the vital role of complement in acute inflammation. The most important activation products of complement appear to be the major chemotactic factor, C5a, and the anaphylatoxins (C3a, C4a, C5a), of which C3a is the most abundant. C5b-9 appears to be a major cytotoxic product, provided that this complex is assembled on the surface of a susceptible cell (e.g., bacterium). A series of soluble and cell membrane–associated complement proteins play important roles in the regulation of the complement cascade.58

FIGURE 6-2 The complement system. The complement system consists of a series of soluble and surface-associated mediators which are functionally organized into the classical and alternative pathways. The classical and alternative pathways of complement converge and lead to the production of the pore-forming membrane attack complex. The classical pathway is most often activated by IgG- and IgM-containing immune complexes while the alternative pathway can be activated by a variety of particulates. In both cases, complex multicomponent enzyme complexes called C3 and C5 convertases are formed. A variety of proinflammatory peptide fragments (e.g., C3a, C5a) are generated as a result of complement activation.

COAGULATION SYSTEM
The coagulation system, its disorders, and the clinical management of its disorders, are reviewed in detail in Chap. 112, Chap. 113, Chap. 114, Chap. 115, Chap. 116, Chap. 117, Chap. 118, Chap. 119, Chap. 120, Chap. 121, Chap. 122, Chap. 123, Chap. 124, Chap. 125, Chap. 126, Chap. 127, Chap. 128, Chap. 129, Chap. 130, Chap. 131, Chap. 132, Chap. 133 and Chap. 134. Activation of the clotting cascade results in the generation of fibrinopeptides which increase vascular permeability and are chemotactic for leukocytes. Thrombin has been shown to induce endothelial expression of P-selectin, resulting in increased neutrophil adhesion.59 In addition, plasmin is responsible for the activation of Hageman factor, which then can activate the kinin system, and can cleave C3 into its active components; it can also generate fibrin-split products. The induction of procoagulant activity in endothelial cells exposed to TNF-a and IL-1 further links the coagulation system to the inflammatory response.60
CHRONIC INFLAMMATION AND REPAIR
The chronic inflammatory response and repair processes are, like the acute inflammatory response, highly regulated. Chronic inflammation is characterized by the recruitment of lymphocytes, monocytes, and plasma cells as well as by the proliferation of new capillaries (angiogenesis) and increases in the deposition of extracellular matrix molecules.1,2 and 3 The recruitment of this wide variety of cell types is achieved by a complex interaction among cytokines, chemokines, and indigenous cells. Great advances in understanding of angiogenesis and extracellular matrix molecule metabolism have been made in recent years. The characteristics of individual chronic inflammatory responses are dependent upon the location of the injury and the type of injurious agent. For instance, lymphocyte- and monocyte-binding interactions with endothelial cells are mediated by selectins, b1 and b2 integrins, and both ICAM and VCAM-1. Bacterium-derived chemotactic peptides play a role in monocyte recruitment, and members of the b chemokine subfamily induce monocyte and lymphocyte recruitment. These several factors have been observed to play a key role in the development of some models of chronic inflammation (Table 6-5).
The proliferation of fibroblasts and the induction of angiogenesis that accompany chronic inflammation are mediated by a variety of cytokines and growth factors derived from platelets, macrophages, and lymphocytes. For instance, fibroblast chemotaxis has been observed in response to a variety of mediators including TNF-a, C5a, collagen fragments, and growth factors (e.g., transforming growth factor-b, platelet-derived growth factor, epidermal growth factor, and basic fibroblast growth factor).1,2 and 3 Chronic inflammatory responses can persist for lengthy periods of time and are less stereotypic than acute responses.
CHAPTER REFERENCES

1.
Weissman G: Inflammation: historical perspectives, in Inflammation: Basic Principles and Clinical Correlates, 2d ed, edited by JJ Gallin et al, pp. 5–13. New York, Raven Press, 1992.

2.
Acute and chronic inflammation, in Robbins’ Pathologic Basis of Disease, 6th ed, edited by RS Cotran, V Kumar, T Collins, pp. 50–88. W.B. Saunders, Philadelphia, 1999.

3.
Majno G: The capillary then and now: an overview of capillary pathology. Mod Pathol 5:9, 1992.

4.
Feng D, et al: Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine and serotonin. J Exp Med 183:1981, 1996.

5.
Brett J, et al: Tumor necrosis factor/cachectin increases permeability of endothelial cell monolayers by a mechanism involving regulatory G proteins. J Exp Med 169:1977, 1989.

6.
Lampugnani MG, Dejana E: Interendothelial junctions: structure, signaling and functional roles. Curr Opin Cell Biol 9:674, 1997.

7.
Cotran RS, Mayadas TN: Endothelial adhesion molecules in health and disease. Pathol Biol 46:164, 1998.

8.
Springer TA: Traffic signals for lymphocyte circulation and leukocyte migration: the multistep paradigm. Cell 76:301, 1994.

9.
Lasky LA: Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science 258:964, 1992.

10.
McEver RP: Perspectives series: cell adhesion in vascular biology: role in PSGL-1 binding to selectins in leukocyte recruitment. J Clin Invest 100:485, 1997.

11.
Bevilacqua MP, Nelson RM: Selectins. J Clin Invest 91:379, 1993.

12.
Ley K, Gaehtgens P, Fennie C, et al: Lectin-like cell adhesion molecule 1 mediates leukocyte rolling in mesenteric venules in vivo. Blood 77:2553, 1991.

13.
Polley MJ, Phillips ML, Wayner E, et al: CD62 and endothelial cell-leukocyte adhesion molecule-1 (ELAM-1) recognizes the same carbohydrate ligand, sialyl LewisX. Proc Natl Acad Sci USA 88:6224, 1991.

14.
Lorant DE, et al: Inflammatory roles of P-selectin. J Clin Invest 92:559, 1993.

15.
Geng JG, Bevilacqua MP, Moore KL, et al: Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature 343:757, 1990.

16.
Bevilacqua MP, Stengelin S, Gimbrone MAJ, Seed B: Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 243:1160, 1989.

17.
Cotran RS, Briscoe DM: Endothelial cells in inflammation, in Textbook of Rheumatology, 5th ed, edited by W. Kelley et al, pp. 183–198. WB Saunders, Philadelphia, 1997.

18.
Lowe JB, Stoolman LM, Nair RP, et al: ELAM-1 dependent cell adhesion to vascular endothelium determined by a transfected human fucosyl-transferase cDNA. Cell 63:475, 1990.

19.
Picker LJ, Warnock RA, Burns AR, et al: The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140. Cell 66:921, 1991.

20.
Smith CW, Marlin SD, Rothlein R, et al: Role of ICAM-1 in the adherence of human neutrophils to human endothelial cells in vitro, in Structure and Function of Molecules Involved in Leukocyte Adhesion, edited by TA Springer, DC Anderson, R Rothlein, AS Rosenthal, p 170. Springer-Verlag, New York, 1989.

21.
Arnout MA: Structure and function of the leukocyte integrins (CD18/CD11). Blood 75:1037, 1990.

22.
Bevilacqua MP, Pober JS, Mendrick DL, et al: Identification of an inducible endothelial leukocyte adhesion molecule. Proc Natl Acad Sci USA 84:9238, 1987.

23.
Larson RS, Springer TA: Structure and function of leukocyte integrins. Immunol Rev 114:181, 1990.

24.
Christensen PJ, Kim S, Simon RH, et al: Differentiation-related expression of ICAM-1 by rat alveolar epithelial cells. Am J Respir Cell Mol Biol 8:9, 1993.

25.
Hemler ME: VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol 8:365, 1990.

26.
Wayner EA, Garcia-Pardo A, Humphries MJ, et al: Identification and characterization of the T-lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin. J Cell Biol 109:1321, 1989.

27.
Osborn L, Hession C, Tizard R, et al: Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 59:1203, 1989.

28.
Liao F, et al: Soluble domain 1 of platelet-endothelial cell adhesion molecule (PECAM) is sufficient to block transendothelial migration in vitro and in vivo. J Exp Med 185:1349, 1997.

29.
Mulligan MS, Varani J, Warren JS, et al: Roles of b2 integrins of rat neutrophils in complement- and oxygen radical-mediated acute inflammatory injury. J Immunol 148:1847, 1992.

30.
Arfors KE, Lundberg C, Lindom L, et al: A monoclonal antibody to the membrane glycoprotein complex CD18 inhibits polymorphonuclear leukocyte accumulation and plasma leakage in vivo. Blood 69:338, 1987.

31.
Doerschuk CM, Winn RK, Coxson HO, Harlan JM: CD18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits. J Immunol 144:2327, 1990.

32.
Arnaout AM: A leukocyte adhesion molecule deficiency. Immunol Rev 114:145, 1990.

33.
Stossel TP: On the crawling of animal cells. Science 260:1045, 1993.

34.
Foxman EF, et al: Multistep navigation and the combinatorial control of leukocyte chemotaxis. J Cell Biol 139:1349–1360, 1997.

35.
Klebanoff SJ: Oxygen metabolites from phagocytes, in Inflammation: Basic Principles and Clinical Correlates, 2d ed, edited by JJ Gallin et al, pp. 541–589. Raven Press, New York, 1992.

36.
Serhan CN, et al: Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J 10:1147–11158, 1996.

37.
Henson PM, et al: Phagocytosis, in Inflammation: Basic Principles and Clinical Correlates, 2d ed, edited by JJ Gallin et al, pp. 511–541. Raven Press, New York, 1992.

38.
Gallin JJ (eds.): Disorders of phagocytic cells, in Inflammation: Basic Principles and Clinical Correlates, 2d ed, edited by JJ Gallin et al, pp. 859–875. Raven Press, New York, 1992.

39.
Martin E, Gantz T, Lehrer RI: Defensins and other endogenous peptide antibiotics of vertebrates. J Leukoc Biol 58:128–136, 1995.

40.
Furchgott RF and Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373, 1980.

41.
MacMicking JD, et al: Nitric oxide and macrophage function. Annu Rev Immunol 15:323, 1997.

42.
Mulligan MS, Warren JS, Smith CW, et al: Lung injury after deposition of IgA immune complexes: requirements for CD18 and L-arginine. J Immunol 148:3086, 1992.

43.
Mulligan MS, Moncada S, Ward PA: Protective effects of inhibitors of nitric oxide synthase in immune complex-induced vasculitis. Br J Pharmacol 107:1159, 1992.

44.
Venge P, et al: Neutrophils and eosinophils, in Textbook of Rheumatology, 5th ed, edited by W. Kelley et al, pp. 146–160. Philadelphia, WB Saunders, 1997.

45.
Gauldie J, Baumann H: Cytokines and acute-phase protein expression, in Cytokines in Inflammation, edited by ES Kimball, pp. 275–298. CRC Press, Boca Raton, FL, 1992.

46.
Beutler B: TNF, immunity and inflammatory disease: lessons of the past decade. J Invest Med 43:227–235, 1995.

47.
Dinarello CA: Biologic basis for interleukin-1 in disease. Blood 87:2095–2147, 1996.

48.
Taub DD, Oppenheim JJ: Review of the chemokines. The Third International Meeting of Chemotactic Cytokines. Cytokine 5:175, 1993.

49.
Adams DH, Lloyd AR: Chemokines: leukocyte recruitment and activation cytokines. Lancet 349:490–495, 1997.

50.
Schall TJ: Biology of RANTES/SIS cytokine family. Cytokine 3:165, 1991.

51.
Wolpe SD, Cerami A: Macrophage inflammatory proteins 1 and 2: members of a novel superfamily of cytokines. FASEB J 3:2565, 1989.

52.
Kelvin DJ, et al: Chemokines and serpentines. J Leukocyte Biol 54:605, 1993.

53.
Zurier RB: Prostaglandins, leukotrienes, and related compounds. In Kelley WN, et al (eds.): Textbook of Rheumatology, 4th ed. pp. 201–212. W. B. Saunders Co., Philadelphia, 1992.

54.
Zimmerman G, et al: A fluid phase and cell-associated mediator of inflammation. In Gallin JJ, et al (eds.): Inflammation: Basic Principles and Clinical Correlates, 2nd ed. pp. 149–176. Raven Press, New York, 1992.

55.
Carmeliet P, Collen D: Molecular genetics of the fibrinolytic and coagulation systems in haemostasis, thrombogenesis, restenosis and atherosclerosis. Curr Opin Lipidol 8:118–125, 1997.

56.
Busse W: Histamine mediator and modulator in inflammation, in Chemical Messengers of the Inflammatory Process, edited by JC Houck, p 1. Elsevier, Amsterdam, 1979.

57.
Bonfanti R, Furie BC, Furie B, Wagner DD: PADGEM (GMP-140) is a component of Weibel-Palade bodies of human endothelial cells. Blood 73:1109, 1989.

58.
Asghar SS, Pasch MC: Complement as a promiscuous signal transduction device. Lab Invest 78:1203, 1998.

59.
McEver RP, Beckstead JH, Moore KL, et al: GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies. J Clin Invest 84:92, 1989.

60.
Argiles JM, et al: Journey from cachexia to obesity by TNF. FASEB J 11:743–751, 1997.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

CHAPTER 5 THE LYMPHOID TISSUES

CHAPTER 5 THE LYMPHOID TISSUES
Williams Hematology

CHAPTER 5 THE LYMPHOID TISSUES

THOMAS J. KIPPS

The Thymus

Thymic Anatomy

Thymic Structure

Thymic Immune Function
The Spleen

Splenic Anatomy

Splenic Structure

Splenic Function
Lymph Nodes

Lymph Node Anatomy

Lymph Node Structure

Lymph Node Function
Peripheral Lymphoid Tissues

Mucosa-Associated Lymphoid Tissues
Chapter References

The lymphoid tissues can be divided into primary and secondary lymphoid organs. Primary lymphoid tissues are sites where lymphocytes develop from progenitor cells into functional and mature lymphocytes. The major primary lymphoid tissue is the marrow, the site where all lymphocyte progenitor cells reside and initially differentiate. This organ is discussed in Chap. 4. The other primary lymphoid tissue is the thymus, the site where progenitor cells from the marrow differentiate into mature thymus-derived (T) cells. Secondary lymphoid tissues are sites where lymphocytes interact with each other and nonlymphoid cells to generate immune responses to antigens. These include the spleen, lymph nodes, and mucosa-associated lymphoid tissues (MALT). The structure of these tissues provides insight into how the immune system discriminates between self-antigens and foreign antigens and develops the capacity to orchestrate a variety of specific and nonspecific defenses against invading pathogens.

Acronyms and abbreviations that appear in this chapter include: CT, computed tomography; MALT, mucosa-associated lymphoid tissues; MHC, major histocompatibility complex; PALS, periarteriolar lymphoid sheath; T, thymus-derived; TCR, T-cell receptor.

THE THYMUS
The thymus is the site for development of thymic-dependent lymphocytes, or T cells. It is a primary lymphoid organ in that it is a major site of lymphopoiesis (lymphocyte development). In this organ, developing T cells, called thymocytes, differentiate from lymphoid stem cells derived from the marrow into functional, mature T cells. It is here that T cells acquire their repertoire of specific antigen receptors to cope with the antigenic challenges received throughout one’s life span. Once they have completed their maturation, the T cells leave the thymus and circulate in the blood and through secondary lymphoid tissues.
THYMIC ANATOMY
The thymus is located in the superior mediastinum, overlying, in order, the left brachiocephalic (or innominate) vein, the innominate artery, the left common carotid artery, and the trachea. It overlaps the upper limit of the pericardial sac below and extends into the neck beneath the upper anterior ribs. It receives its blood supply from the internal thoracic arteries. Venous blood from the thymus drains into the brachiocephalic and internal thoracic veins, which communicate above with the inferior thyroid veins.
Arising from the third and fourth brachial pouches as an epithelial organ populated by lymphoid cells, the thymus develops at about the eighth week of gestation. The thymus increases in size through fetal and postnatal life and remains ample into puberty,1 when it weighs about 40 g. Thereafter, the size progressively decreases with aging as a consequence of thymic involution.2,3
The volume of the thymus can be estimated by sonography. In one study of 149 healthy term infants within 1 week of birth, there was a significant correlation between the estimated thymic volume and the weight of the infant.4 However, no correlation was apparent between the estimated thymic volume and the infant’s sex, length, or gestational age. Also, there was no apparent correlation between estimated volume and the proportions of CD4+ T cells or CD8+ T cells found in the blood. The estimated thymic volume of healthy infants increases from birth to 4 and 8 months of age and then decreases.1 Most of the individual variation at 4 and 10 months of age appears to correlate with breast-feeding status, body size, and, to a lesser extent, illness. Breast-fed infants at 4 months of age have significantly larger estimated thymic volumes than do age-matched formula-fed infants with similar thymic volumes at birth.5
THYMIC STRUCTURE
A longitudinal fissure divides the thymus into two asymmetrical lobes, a larger right and a smaller left, that are derived from the right and left brachial pouches, respectively. These two developmentally separate parts of the thymus are easily separated from each other by blunt dissection.
Each lobe of the thymus is divided into multiple lobules by fibrous septa. Each lobule consists of an outer cortex and an inner medulla. The cortex contains dense collections of thymocytes that appear as lymphocytes of slightly variable size with scattered, rare mitoses. The lighter-staining medulla is more sparsely populated with cells. It contains loosely arranged mature thymocytes and characteristic tightly packed whorls of squamous-appearing epithelial cells, called Hassall’s corpuscles. These appear to be remnants of degenerating cells and are rich in high-molecular-weight cytokeratins.
The thymus contains several other important cell types in addition to thymocytes. There are three types of specialized epithelial cells within the thymus: the medullary epithelial cells, which are organized into clusters; the cortical epithelial cells, which form an epithelial network; and the epithelial cells of the outer cortex.6 The epithelial cells in the cortex and medulla often have a stellate shape, display desmosomal connections to one another, and may function as nurse cells to developing thymocytes. In addition, the thymus contains marrow-derived antigen-presenting cells, primarily interdigitating dendritic cells and macrophages, particularly at the corticomedullary junction.
After puberty, thymic involution begins within the cortex. This region may disappear completely with aging, while medullary remnants persist throughout life. Corticosteroids also may induce atrophy of the cortex secondary to glucocorticoid-induced apoptosis of cortical thymocytes.7 This also may be seen in conditions that are associated with increases in circulating steroids, for example, pregnancy or stress.8,9
THYMIC IMMUNE FUNCTION
Prothymocytes originate in the marrow and migrate to the thymus, where they mature into T cells (see Chap. 82 and Chap. 84). Maturation of T cells is accompanied by the sequential acquisition by thymocytes of the various T-cell markers (Fig. 5-1). Terminal deoxynucleotidyl transferase is found in prothymocytes and immature thymocytes but is absent in mature T cells.

FIGURE 5-1 Structure of the thymus. The top half of the figure provides a cross section of a thymic lobule, indicating the outer cortex (left), inner medulla (center), and periphery (far right). The marked arrows indicate various structures and cell types. As thymocytes mature, they migrate from the cortex toward the medullary region and acquire phenotypic features that are outlined at the bottom of the figure, as described in the text (see Chap. 82).

Pre–T cells enter the cortex via small blood vessels and are double negative for CD4 and CD8 antigens. One of the earliest identifiable T-cell membrane antigens is CD2. As the thymocytes proliferate and differentiate in the cortex, they acquire CD4 and CD8 antigens. They subsequently acquire the CD3 antigen and the T-cell receptor for antigen as they migrate toward the medulla.
Positive and negative selection of maturing T cells takes place in the thymus. “Double-positive” (CD4+ and CD8+) thymocytes undergo an initial positive selection step that is mediated exclusively by thymic cortical epithelium.10 Thymocytes that have T-cell receptors (TCR) capable of interacting with the major histocompatibility complex (MHC) molecules expressed by thymic cortical epithelial cells will undergo expansion, while thymocytes with defective TCR will undergo apoptosis.11,12 As these positively selected cells migrate toward the medulla, they experience negative selection. Those thymocytes that have TCR that react too vigorously with the MHC molecules of the medullary epithelium and marrow-derived cells will undergo apoptosis.12,13 and 14 Most of the developing thymocytes are destroyed. In this way, only those T cells that have the right level of low affinity for self-MHC molecules will reach the final maturation stages and be allowed to exit the thymus.
The selected thymocytes enter the thymic medulla, where they further mature and differentiate to become “single positive” for either CD4 or CD8 and acquire the capacity for future helper and cytolytic functions, respectively. Here they also may interact with scattered B cells during their final stages of thymic education (see Chap. 15, Chap. 82, and Chap. 84). A small percentage of the lymphocytes produced in the thymus finally exit the medulla via efferent lymphatics as mature, naive T cells.
THE SPLEEN
The spleen is a secondary lymphoid organ. Secondary lymphoid tissues provide an environment in which the cells of the immune system can interact with antigen and with one another to develop an immune response to antigen. The spleen is a major site of immune response to blood-borne antigens. In addition, the splenic red pulp contains macrophages that are responsible for clearing the blood of unwanted foreign substances and senescent erythrocytes, even in the absence of specific immunity. Thus, it acts as a filter for the blood.
SPLENIC ANATOMY
The spleen is located within the peritoneum in the left upper quadrant of the abdomen between the fundus of the stomach and the diaphragm. It receives its blood supply from the systemic circulation via the splenic artery, which branches off the celiac trunk, and the left gastroepiploic artery.15 The blood returning from the spleen drains into the portal circulation via the splenic vein. Therefore, the spleen can become congested with blood and increase in size when there is portal hypertension.
About 10 percent of individuals have one or more accessory spleens. Accessory spleens are usually 1 cm in diameter and resemble lymph nodes. However, they usually are covered with peritoneum, as is the spleen itself. Accessory spleens typically lie along the course of the splenic artery or its gastroepiploic branch, but they may be elsewhere.16 The commonest location is near the hilus of the spleen, but approximately one in six accessory spleens can be found embedded in the tail of the pancreas, where they may be occasionally mistaken for a pancreatic mass lesion.17,18,19,20 and 21
The average weight of the spleen in the adult human is 135 g, ranging from 100 to 250 g. However, when emptied of blood it weighs only about 80 g. On autopsy of 539 subjects with normal spleens, there was a positive correlation between the spleen weight and both the degree of acute splenic congestion and the subject’s height and weight but not with the subject’s sex or age.22
The splenic volume can be estimated by computed tomography (CT) of the abdomen.23,24 In one study, the splenic volume was calculated from the linear and the maximal cross-sectional area measurements of the spleen, using the following formula: splenic volume = 30 cm3 + 0.58 × the product of the measured width, length, and thickness of the spleen.23 Using this formula, the mean value of the calculated splenic volume for 47 normal subjects was 214.6 cm3, with a range from 107.2 to 314.5 cm3. The calculated splenic volume did not appear to vary significantly with the subject’s age, gender, height, weight, body mass index, or the diameter of the first lumbar vertebra, the latter being considered representative of body habitus on CT.
The splenic volume also can be estimated by sonography. In one study of 32 normal spleens from adult corpses, the ultrasound measurements of maximal height, width, and breadth of the spleen were compared with the actual volume displaced by the excised organ.25 The mean actual splenic volume was approximately 148 cm3 (± 81 cm3 SD), whereas mean splenic volume estimated from ultrasound was 284 cm3 (± 168 cm3 SD). Despite the differences between the actual and estimated volumes, these investigators did find a roughly linear correlation between actual splenic volume and the estimated splenic volume measured by ultrasound. However, there may be operator-to-operator variation in measurement of the estimated splenic volume, making the use of sonography in longitudinal studies technically demanding.
SPLENIC STRUCTURE
The spleen has an “open” circulation, which lacks endothelial continuity from artery to vein.26 When isolated spleens are perfused in washout studies, erythrocytes that appear in the splenic vein appear to be flushed out from three compartments. The red cells that are flushed out first come from a compartment that presumably is formed by the splenic vessels. The erythrocytes that are flushed out next come from a second compartment, where they presumably are loosely held within the filtration beds. The erythrocytes that are flushed out last presumably were adherent to cells of the filtration beds. Although 90 percent of the blood flow passes through the splenic vessels, only about 10 percent of the total splenic red cells are found within this first compartment. The second compartment is perfused by 9 percent of the total inflow yet contains 70 percent of the splenic red cells. The last compartment is perfused by only 1 percent of the inflow but contains 20 percent of the splenic red cells.
These compartments reflect the anatomy of the spleen and its stroma. The stroma is composed of branched, fibroblast-like cells called reticular cells. These cells produce slender collagen fibers, the reticular fibers, which are rich in type IV collagen. The reticular cells and fibers form a meshwork, or reticulum, which filters the blood. Three major types of filtration beds can be distinguished by their structure and content: the white pulp, the marginal zone, and the red pulp.
WHITE PULP
The white pulp contains the lymphocytes and other mononuclear cells that surround the arterioles branching off the splenic artery. After the splenic artery pierces the splenic capsule at the hilum, it divides into progressively smaller branches. Each branch is called a central artery because it runs through the central longitudinal axis of a distinctive filtration bed that surrounds each central artery (Fig. 5-2). This is composed of a cuff of lymphocytes called the periarteriolar lymphoid sheath (or PALS). The PALS is contained within a protective and supporting fibrous trabecula and is composed mostly of T lymphocytes, about two-thirds of which are CD4+ T cells. Attached to the PALS are lymphoid follicles, some of which contain pale kernels of activated lymphocytes interspersed with large, pale macrophages and dendritic cells called germinal centers.27 On gross inspection of the surface of a freshly cut spleen, these appear as white dots referred to as Malpighian corpuscles. These corpuscles predominantly contain a germinal center and have the same anatomic features and functions as secondary follicles in the lymph node (Fig. 5-3).

FIGURE 5-2 Structure of the spleen. A branch of the splenic artery enters the pulp and becomes a central artery. Surrounding the central artery is a PALS. At the circumference of the PALS is the marginal zone, which generally separates the white pulp of the PALS from the red pulp. Follicles of B cells with occasional germinal centers (Malpighian corpuscles) are located at the outer margins of the PALS for the depicted central artery and the PALS of central arteries that are in a different plane from that of the figure.

FIGURE 5-3 Structure of the lymph node. The lymph enters via afferent lymphatic channels and exits via the efferent lymphatic channel. The large arrows indicate the direction of the lymphatic flow into and out of the lymph node. The legend shows the symbols used for the T-cell zone (x) and the B-cell zone (shaded) of each follicle. The follicle in the lower left part of the node contains a primary follicle lacking a germinal center. The follicle immediately above this follicle contains a germinal center. Thus, the entire follicle delineated by the dashed lines is a secondary follicle. The cortex, paracortical area, and medulla are also shown.

Branches coming off the central artery deliver disproportionate amounts of plasma and lymphocytes to the rims of the PALS. These branches tend to run at acute angles, leading to a selective loss of plasma from the blood, a phenomenon referred to as skimming. After becoming relatively depleted of plasma, the arterioles then carry high-hematocrit blood into the filtration beds of the red pulp and marginal zone. As a result, the red pulp and marginal zone beds contain relatively high concentrations of red cells.
THE MARGINAL ZONE
The marginal zone surrounds the PALS and follicles. It is composed of reticulum, which forms a finely meshed filtration bed, serving as a vestibule for much of the blood that flows through the spleen. The marginal zone surrounds the white pulp and merges insensibly into the red pulp. It contains more lymphocytes than the red pulp. These are primarily memory B cells and CD4+ T cells.28,29 and 30 However, like the red pulp, the marginal zone may become congested and clear imperfect and senescent red cells and parasites.
THE RED PULP
The red pulp of the spleen is composed of a reticular meshwork, called the splenic cords of Billroth, and splenic sinuses. This region predominantly contains erythrocytes but has large numbers of macrophages and dendritic cells. There are relatively few lymphocytes and plasma cells in this area.
As the central arteries branch and decrease in size, the PALS also branches and decreases in diameter to but a few cells surrounding the arteriole. The small arteriole finally emerges from its sheath and then terminates in either the marginal zone or the red pulp. Here these vessels are suspended and anchored by adventitial reticular cells in the periarterial beds. They often terminate abruptly as arteriolar capillaries or as vessels with a trumpetlike flare with widened slits called interendothelial slits. The blood flows through these slits into filtration beds composed of large-meshed loculi that open to one another.
The blood in the red pulp and marginal zone drains into venous sinuses that form anastomosing, blind-ending vessels. These venous sinuses actually are specialized postcapillary venules. The endothelial cells are shaped as tapered rods that are stiffened by basal, longitudinal, intermediate cytoskeletal filaments and contractile filaments of actin and myosin. These intracellular contractile filaments can shorten the vein, causing the endothelium to buckle and form interendothelial gaps, favoring transmural passage.
The endothelial cells are attached to a basement membrane. While this appears to be fashioned of fibers, the basement membrane actually is an extracellular membranous wall with large, regular defects that expose considerable basal endothelial surface. This includes the interendothelial slits, through which blood may flow from the filtration bed and into the vein. Ordinarily the interendothelial slits are narrow or even closed unless forced apart by cells in transmural transit or by endothelial contraction.
Splenic arterioles terminate at varied distances from the walls of venous vessels. Blood flowing from arterioles that terminate at the venous vessel wall may flow directly into the splenic vein. However, blood flowing from arterioles that terminate at a distance from a vein must traffic through the spleen. In so doing, the blood either may pass quickly through a nonsinusal venous aperture or slowly through sinusal interendothelial slits and the fibroblast stroma.
The fibroblast stroma contains reticular cells and myofibroblast cells, also called barrier cells.31 The latter may fuse with each other to form a syncytial membrane that connects the arterial terminals with venous interendothelial slits or apertures. Like other myofibroblasts, these cells contain actin and myosin and may contract, thereby approximating splenic arterial and venous vessels with one another. Thus, the fibroblast stroma may affect the relative proportion of blood that flows through the sinusal interendothelial slits and the stroma itself.
SPLENIC FUNCTION
RED CELL CLEARANCE
Mixed within the stroma of the red pulp and marginal zone are monocytes and macrophages. As the blood passes through the stroma, monocytes may be held on the stroma, where the microenvironment is conducive to their maturation into macrophages and large, dendritic, lysosome-rich phagocytes. These cells may assist the reticular cells in mechanical filtration. More important, these cells have phagocytic activity that allows them to ingest imperfect erythrocytes, store platelets, and remove infectious agents, such as Plasmodia, from the circulation. In addition, these cells have nonphagocytic functions, such as the presentation of antigens to T cells or the elaboration of certain cytokines.
Collectively, the anatomy of the spleen allows the marginal zone and red pulp to cull defective erythrocytes. As the blood passes slowly through the sinusal interendothelial slits and the fibroblast stroma, the erythrocytes must undergo alterations in shape to squeeze through the mechanical barrier generated by this filtration compartment. Normal red cells that are supple may pass through readily because the interendothelial slits can open to about 0.5 µm. However, blood cells containing large, rigid inclusions, such as plasmodium-containing erythrocytes, are delayed or sequestered. Moreover, splenic macrophages residing within these filtration beds can sequester erythrocytes that are coated with antibody.
When these filtration beds sequester imperfect red cells, the blood pools inside the spleen, causing stasis and congestion. This stimulates sphincterlike contraction of the distal vein, resulting in proximal plasma transudation that produces a viscous luminal mass of high-hematocrit blood. During episodes of enhanced red cell sequestration, as occur during malarial crises or hemolytic episodes in sickle cell disease, the splenic volume and weight may increase ten- to twenty-fold.32,33 Although the white pulp may enlarge, particularly in germinal centers, the marginal zones and red pulp become greatly widened with pooled erythrocytes and macrophages in this setting.
SPLENIC IMMUNE FUNCTION
The spleen and its responses to antigens are similar to those of lymph nodes, the major difference being that the spleen is the major site of immune responses to blood-borne antigens, while lymph nodes are involved in responses to antigens in the lymph. Antigens and lymphocytes enter the spleen through the vascular sinusoids, since the spleen lacks high endothelial venules. Upon entry, the lymphocytes home to the white pulp. T cells migrate to the PALS and B cells to the lymphoid nodules. T cells and B cells migrate within these compartments for about 5 and 7 h, respectively. In the absence of an immune response, these cells migrate through a reticulum arranged around the circumference of the central artery.
Upon immune activation in response to antigen, the lymphocytes may remain in the spleen to sustain a primary or secondary immune response. Activation of B cells is initiated in the marginal zones that are adjacent to CD4+ T cells in the PALS. Activated B cells then migrate into germinal centers or into the red pulp.34 Lymphoid nodules appear and expand by recruiting lymphocytes from the blood and the peripheral zone of the follicles, termed the mantle zone. These cells then proliferate and differentiate in the center of a lymphoid nodule, forming a germinal center.35 In their path from the marginal zone to the follicles, B cells pass the PALS, where they remain in contact with T lymphocytes for a few hours, allowing ample time for T-B cell interaction in response to antigens. If they are not recruited in an immune response to antigen, both T and B lymphocytes exit the spleen via deep efferent lymphatics, not the splenic veins.
These efferent lymphatics are not distinguished as separate structures within the PALS, being quite thin-walled and often packed with efferent lymphocytes. However, they are important in moving nonreactive lymphocytes out of the spleen and in producing high-hematocrit pulp blood. After leaving the spleen, the efferent lymphocytes become the afferent lymphatics of the perisplenic mesenteric lymph nodes or empty into the thoracic duct. This duct empties into the left subclavian vein, thus returning the lymphocytes to the venous circulation.
LYMPH NODES
The lymphoid nodes are secondary lymphoid tissues. They form part of a network that filters antigens from the interstitial tissue fluid and lymph during its passage from the periphery to the thoracic duct. Thus, the lymph nodes are the primary sites of immune response to tissue antigens.
LYMPH NODE ANATOMY
The lymph nodes are round or kidney-shaped clusters of mononuclear cells that normally are less than 1 cm in diameter. A collagenous capsule surrounds a typical lymph node and has an indentation called the hilus where blood vessels enter and leave.
Lymph nodes typically are present at the branches of the lymphatic vessels and form part of the extensive network of lymphatic channels that extends throughout the body. Several afferent lymphatic channels that drain lymph from regional tissues into the lymph node perforate the capsule of each lymph node. The lymph draining from the node leaves through one efferent lymphatic vessel at the hilus. The lymph from the node, in turn, empties into efferent lymphatic vessels that eventually drain into larger lymphatic channels leading eventually to the thoracic duct. The thoracic duct in turns drains into the left subclavian vein, thus returning lymph into the systemic circulation.
Clusters of lymph nodes are placed strategically in areas that drain various superficial and deep regions of the body, such as the neck, axillae, groin, mediastinum, and abdominal cavity. The lymph nodes that receive lymph that drains from the skin, termed somatic nodes, are superficial. The lymph nodes that receive their lymph from the mucosal surface of the respiratory, digestive, or genitourinary tract, termed visceral nodes, are usually deep within body cavities.
LYMPH NODE STRUCTURE
Beneath the collagenous capsule is the subcapsular sinus, into which the afferent lymphatic channels drain (see Fig. 5-3). This sinus is lined with phagocytic cells. Fibrous trabeculae radiate from the medulla adjacent to the hilus of the node to the subcapsular sinus, thus breaking the node into several follicles, called cortical follicles. These trabeculae, together with the capsule and a network of reticulin fibers, support the various cellular components of the node and serve as the scaffolding for lymphatic spaces, namely, the subcapsular and cortical sinuses. These lymphatic spaces are continuous with medullary sinuses and the solitary efferent lymphatic channel exiting the hilus.
Each cortical follicle contains dense collections of small, mature, recirculating lymphocytes. These consist of a B-cell area (cortex), a T-cell area (paracortex), and a central medulla with cellular cords that contain T cells, B cells, plasma cells, and macrophages. Some follicles contain lightly staining areas of 1- to 2-mm in diameter, called germinal centers. Germinal centers are the specialized sites for the generation of memory B cells and antibody affinity maturation via the process of immunoglobulin variable-region somatic hypermutation.36,37 Follicles without germinal centers are called primary follicles, and those with germinal centers are called secondary follicles. Primary lymphoid follicles contain nodules that consist predominantly of small, mature, recirculating B lymphocytes.
Within 1 week after antigenic stimulation, secondary follicles develop a germinal center, which contains proliferating B cells and macrophages.27,38 The small, nonreactive B cells are apparently forced to the periphery of the follicle, where they form a dense follicular mantle. The B cells within the germinal center, on the other hand, are highly activated, typically forming blasts that have abundant cytoplasm and round, cleaved, or convoluted shapes. Follicular dendritic cells also are found within the germinal centers. These cells can trap and retain antigens for months, possibly in the form of immune complexes.39 The germinal centers of the secondary follicle may gradually regress after the antigenic stimulus is eliminated.
Surrounding the lymphoid follicles of the superficial cortex are sheets of lymphocytes that extend to the deep cortex, the so-called paracortex, that blend into medullary cords of cells. The paracortical zones are formed mostly of T cells. The ratio of T cells to B cells in these zones is about 3:1. The medulla, however, contains scattered B cells, dendritic cells, macrophages, and, during an immune response, plasma cells. The superficial cortex and medulla of the lymph nodes are the thymic-independent areas, while the deep cortex is particularly enriched with T cells, forming an area that sometimes is referred to as the thymic-dependent area. The major T-cell population found within the lymph node consists of CD4+ T cells. The scattering of CD4+ T cells in the follicles, and in more prominent numbers in the interfollicular zones, reveals the proximity of CD4+ T and B cells important for T-B cooperation during proliferation and maturation of antigen-stimulated B cells.40
Lymphocytes primarily enter lymphatic tissues from the blood by migrating across the tall, active endothelium of specialized post-capillary venules called high endothelial venules.41 Cellular adhesion molecules and various chemokines are responsible for the pattern of lymphocyte trafficking and determine the microanatomy of the lymphoid tissues.42
LYMPH NODE FUNCTION
The lymph node is the site where different types of lymphocytes, macrophages, and dendritic cells can interact with one another to generate an immune response to antigens carried within the lymph. As the lymph passes across the nodes from afferent to efferent lymphatic vessels, particulate antigens are removed by the phagocytic cells and transported into the lymphoid tissue of the lymph node. Abnormal cells within the lymph, such as neoplastic cells, also can be trapped within the lymph node.
Within the lymph node, antigen is presented to T cells as processed peptides by MHC molecules of antigen-presenting cells (see Chap. 84). Various T-cell subsets comprise a network of interactive cells. CD4+ and CD8+ cell-mediated contacts, as well as T cell–derived soluble factors, induce and regulate the immune response (see Chap. 15). T-cell recognition is mediated by the TCR for antigen (see Chap. 84). Which T cells are activated is determined by the specificity of the TCRs (see Chap. 86), the structure of MHC molecules, and the nature of antigen-presenting cells, including the dendritic reticular cells, macrophages, and B cells.
However, along with TCR recognition of processed antigen presented in the MHC of the antigen-presenting cell, adequate T-cell activation requires second signals, or costimulation, delivered through accessory molecules, such as CD28 on T cells (see Chap. 84).43 Without these second signals, the T cells may become anergic, or specifically nonresponsive to antigen stimulation. This specific suppression is thought to play an important regulatory role in the maintenance of self-tolerance.44,45
T-cell recognition of specific antigen may induce release of soluble factors, such as the interleukins, that can activate T cells, B cells, and/or monocytes.46,47,48 and 49 Also, the activated T cells express surface molecules, such as CD40 ligand, that also can activate B cells, dendritic cells, or macrophages.50,51
The T-dependent immune response includes the formation of early germinal centers within days after antigen exposure. There is a mixture of B cells and activated CD4+ T cells in the lymphoid follicles. T-B cooperation involves the accessory B-cell antigen CD40 and the CD40 ligand expressed on activated T cells (see Chap. 15). Activated B cells become blasts and comprise the largest numbers of cells in the early germinal center.27 Subsequently, B-cell blasts give rise to smaller B cells, the centrocytes. B cells undergo affinity maturation within the germinal center. During this process, the genes encoding the surface immunoglobulin of B cells undergo high rates of mutation, called somatic hypermutation.35,52 B cells, including the centrocytes, that express immunoglobulin with little or no affinity for antigen undergo apoptosis.53 The resulting cellular debris is tingible, or capable of being stained, and is found prominently within macrophages specifically designated tingible body macrophages. On the other hand, B cells expressing surface immunoglobulin with high affinity for antigen are selected to proliferate and differentiate to memory B cells or plasma cells.38 As well as promoting activation of B cells, CD4+ T cells, and CD8+ T cells, the T-cell limb of the primary immune response may generate circulating CD4+ and CD8+ memory T cells.54,55
Following release of specific antibody, antigen-antibody complexes may form and become sequestered on the surface of follicular dendritic cells within the germinal centers. These antigen-antibody complexes produce a coating of small, beadlike, immune complex–coated bodies called iccosomes. Iccosomes may be presented to CD4+ T cells by B cells and dendritic cells. Iccosomes also appear to assist in anamnestic recall of high levels of antibody following reentry of antigen in the host.56 T-cell and B-cell memory functions and self-tolerance depend upon persistence of antigen.54,57,58 and 59
PERIPHERAL LYMPHOID TISSUES
MUCOSA-ASSOCIATED LYMPHOID TISSUES
The MALT are diffusely organized aggregates of lymphocytes that protect the respiratory and gastrointestinal epithelium. The lymphoid aggregates associated with the respiratory epithelium are sometimes referred to as the bronchial-associated lymphoid tissue. The lymphoid aggregates associated with the intestinal epithelium are sometimes referred to as the gut-associated lymphoid tissue.60 These tissues include the tonsils, adenoids, appendix, and specialized structures called Peyer’s patches found in the ileum, and they collect antigen from the epithelial surfaces of the gastrointestinal tract.
Solitary lymph nodules with follicular and germinal center structures occur in the mucosa and submucosa of the respiratory tract, the gastrointestinal tract (particularly within the ileum), the urinary tract, and the vagina. During states of chronic inflammation, lymphoid nodules may form as a localized center of lymphocytes with marked follicular activity. Waldeyer’s ring of pharyngeal lymphoid tissues and Peyer’s patches in the ileum contain prominent aggregated nodular lymphoid tissue. No capsule or efferent or afferent lymphatic vessels are present in these accessory lymphoid tissues.
These MALT are rich in plasma cells and eosinophils. The plasma cells are a source of secretory immunoglobulin that is transferred into the lumina of the bronchi and gastrointestinal tract. The majority of plasma cells in the mucosa of the bronchi and gut contain IgA. IgA is released from the plasma cell and then combines with a secretory piece synthesized within the mucosal epithelium to become secretory IgA (see Chap. 83). Secretory IgA then is secreted across the microvilli of mucosal epithelium into the lumen, where it may prevent colonization of mucosal membranes by pathogens. Lymphoid nodules along mucosa-lined tracts serve as precursors of IgA-producing cells. These nodules form a barrier against many microorganisms and antigens. Microfolds overlying specialized epithelial cells in the gut transport antigenic material by pinocytosis, with subsequent immunization and IgA secretion.
PEYER’S PATCHES
Peyer’s patches are the most important and highly organized of the gut-associated lymphoid tissues. They are found in the lamina propria of the small intestine (beneath the mucosa near the ileocolonic junction) and consist of up to 50 or more lymphoid nodules covered by a single layer of columnar epithelium. They are well developed in youth and regress with age. Antigens from the intestinal epithelium are collected by specialized epithelial cells called M cells, allowing for generation of specific immune responses against intestinal pathogens.61 Peyer’s patches are the sites at which B cells differentiate, in response to these antigens, into the plasma cells found within the intestine.62
TONSILS
The tonsils are the major component of Waldeyer’s ring of pharyngeal lymphoid tissues. They are covered by variable epithelial surfaces that have deep, branching depressions called crypts. Fused lymphatic nodules lie adjacent to the crypts, and germinal centers are prominent. A pseudocapsule of condensed connective tissue surrounds the tonsils, and septae within the structures form lobulations. Together with the other lymphoid tissues of Waldeyer’s ring, the tonsils provide the initial barrier to pathogens entering the oral pharynx.
CHAPTER REFERENCES

1.
Hasselbalch H, Jeppesen DL, Ersbøll AK, Engelmann MD, Nielsen MB: Thymus size evaluated by sonography: A longitudinal study on infants during the first year of life. Acta Radiol 38:222, 1997.

2.
Pawelec G, Adibzadeh M, Solana R, Beckman I: The T cell in the ageing individual. Mech Ageing Dev 93:35, 1997.

3.
Haynes BF, Hale LP: The human thymus: A chimeric organ comprised of central and peripheral lymphoid components Immunol Res 18:175, 1998.

4.
Hasselbalch H, Jeppesen DL, Ersbøll AK, Lisse IM, Nielsen MB: Sonographic measurement of thymic size in healthy neonates: Relation to clinical variables. Acta Radiol 38:95, 1997.

5.
Hasselbalch H, Jeppesen DL, Engelmann MD, Michaelsen KF, Nielsen MB: Decreased thymus size in formula-fed infants compared with breastfed infants. Acta Paediatr 85:1029, 1996.

6.
Röpke C: Thymic epithelial cell culture. Microsc Res Tech 38:276, 1997.

7.
Cifone MG, Migliorati G, Parroni R, et al: Dexamethasone-induced thymocyte apoptosis: Apoptotic signal involves the sequential activation of phosphoinositide-specific phospholipase C, acidic sphingomyelinase, and capsases. Blood 93:2282, 1999.

8.
Rijhsinghani AG, Thompson K, Bhatia SK, Waldschmidt TJ: Estrogen blocks early T cell development in the thymus. Am J Reprod Immunol 36:269, 1996.

9.
Ayala A, Herdon CD, Lehman DL, Ayala CA, Chaudry IH: Differential induction of apoptosis in lymphoid tissues during sepsis: Variation in onset, frequency, and the nature of the mediators. Blood 87:4261, 1996.

10.
Laufer TM, Glimcher LH, Lo D: Using thymus anatomy to dissect T cell repertoire selection. Semin Immunol 11:65, 1999.

11.
Blackman M, Kappler J, Marrack P: The role of the T cell receptor in positive and negative selection of developing T cells. Science 248:1335, 1990.

12.
Müller-Hermelink HK, Wilisch A, Schultz A, Marx A: Characterization of the human thymic microenvironment: Lymphoepithelial interaction in normal thymus and thymoma. Arch Histol Cytol 60:9, 1997.

13.
Takahama Y, Shores EW, Singer A: Negative selection of precursor thymocytes before their differentiation into CD4+CD8+ cells. Science 258:653, 1992.

14.
Oukka M, Colucci-Guyon E, Tran PL, et al: CD4 T cell tolerance to nuclear proteins induced by medullary thymic epithelium. Immunity 4:545, 1996.

15.
Romero-Torres R: The true splenic blood supply and its surgical applications. Hepato-Gastroenterology 45:885, 1998.

16.
Paul R, Bielmeier J, Breul J, Nathrath WBJ, Hartung R: Accessory spleen of the spermatic cord. Urol Ausg A 36:262, 1997.

17.
Takayama T, Shimada K, Inoue K, Wakao F, Yamamoto J, Kosuge T: Intrapancreatic accessory spleen. Lancet 344:957, 1994.

18.
Harris GN, Kase DJ, Bradnock H, McKinley MJ: Accessory spleen causing a mass in the tail of the pancreas: MR imaging findings. Am J Roentgenol 163:1120, 1994.

19.
Ota T, Tei M, Yoshioka A, et al: Intrapancreatic accessory spleen diagnosed by technetium-99m heat-damaged red blood cell SPECT. J Nucl Med 38:494, 1997.

20.
Churei H, Inoue H, Nakajo M: Intrapancreatic accessory spleen: Case report. Abdom Imaging 23:191, 1998.

21.
Lauffer JM, Baer HU, Maurer CA, Wagner M, Zimmermann A, Buchler MW: Intrapancreatic accessory spleen: A rare cause of a pancreatic mass. Int J Pancreatol 25:65, 1999.

22.
Sprogøe-Jakobsen S, Sprogøe-Jakobsen U: The weight of the normal spleen. Forensic Sci Int 88:215, 1997.

23.
Prassopoulos P, Daskalogiannaki M, Raissaki M, Hatjidakis A, Gourtsoyiannis N: Determination of normal splenic volume on computed tomography in relation to age, gender, and body habitus. Eur Radiol 7:246, 1997.

24.
Watanabe Y, Todani T, Noda T, Yamamoto S: Standard splenic volume in children and young adults measured from CT images. Surg Today 27:726, 1997.

25.
Rodrigues Júnior AJ, Rodrigues CJ, Germano MA, Rasera Júnior I, Cerri GG: Sonographic assessment of normal spleen volume. Clin Anat 8:252, 1995.

26.
Weiss L: The cell: The blood and hematopoietic tissues, in Histology: Cell and Tissue Biology, 6th ed, p 3. Urban & Schwarzenburg, Baltimore, 1988.

27.
Tarlinton D: Germinal centers: Form and function. Curr Opin Immunol 10:245, 1998.

28.
Dunn-Walters DK, Isaacson PG, Spencer J: Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MgZ) B cells suggests that the MgZ of human spleen is a reservoir of memory B cells. J Exp Med 182:559, 1995.

29.
Spencer J, Perry ME, Dunn-Walters DK: Human marginal-zone B cells. Immunol Today 19:421, 1998.

30.
Tierens A, Delabie J, Michiels L, Vandenberghe P, De Wolf-Peeters C: Marginal-zone B cells in the human lymph node and spleen show somatic hypermutations and display clonal expansion. Blood 93:226, 1999.

31.
Weiss L: Barrier cells in the spleen. Immunol Today 12:24, 1991.

32.
Weiss L, Geduldig U, Weidanz W: Mechanisms of splenic control of murine malaria: Reticular cell activation and the development of a blood-spleen barrier. Am J Anat 176:251, 1986.

33.
Smith NC, Fell A, Good MF: The immune response to asexual blood stages of malaria parasites. Chem Immunol 70:144, 1998.

34.
Rizzo LV, Secord EA, Tsiagbe VK, et al: Components essential for the generation of germinal centers. Dev Immunol 6:325, 1998.

35.
Hollowood K, Goodlad JR: Germinal centre cell kinetics. J Pathol 185:229, 1998.

36.
Varade WS, Insel RA: Isolation of germinal center-like events from human spleen RNA: Somatic hypermutation of a clonally related VH6DJH rearrangement expressed with IgM, IgG, and IgA. J Clin Invest 91:1838, 1993.

37.
Han S, Zheng B, Takahashi Y, Kelsoe G: Distinctive characteristics of germinal center B cells. Semin Immunol 9:255, 1997.

38.
Przylepa J, Himes C, Kelsoe G: Lymphocyte development and selection in germinal centers. Curr Top Microbiol Immunol 229:85, 1998.

39.
Burton GF, Masuda A, Heath SL, Smith BA, Tew JG, Szakal AK: Follicular dendritic cells (FDC) in retroviral infection: Host/pathogen perspectives. Immunol Rev 156:185, 1997.

40.
Gulbranson-Judge A, Casamayor-Palleja M, MacLennan IC: Mutually dependent T and B cell responses in germinal centers. Ann NY Acad Sci 815:199, 1997.

41.
Butcher EC, Williams M, Youngman K, Rott L, Briskin M: Lymphocyte trafficking and regional immunity. Adv Immunol 72:209, 1999.

42.
Warnock RA, Askari S, Butcher EC, von Andrian UH: Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J Exp Med 187:205, 1998.

43.
Greenfield EA, Nguyen KA, Kuchroo VK: CD28/B7 costimulation: A review. Crit Rev Immunol 18:389, 1998.

44.
Van Parijs L, Abbas AK: Homeostasis and self-tolerance in the immune system: Turning lymphocytes off. Science 280:243, 1998.

45.
Malvey EN, Telander DG, Vanasek TL, Mueller DL: The role of clonal anergy in the avoidance of autoimmunity: Inactivation of autocrine growth without loss of effector function. Immunol Rev 165:301, 1998.

46.
Mosmann TR, Li L, Hengartner H, Kagi D, Fu W, Sad S: Differentiation and functions of T cell subsets. Ciba Found Symp 204:148, 1997.

47.
Mosmann TR, Li L, Sad S: Functions of CD8 T-cell subsets secreting different cytokine patterns. Semin Immunol 9:87, 1997.

48.
Takatsu K: Cytokines involved in B-cell differentiation and their sites of action. Proc Soc Exp Biol Med 215:121, 1997.

49.
Seder RA, Gazzinelli RT: Cytokines are critical in linking the innate and adaptive immune responses to bacterial, fungal, and parasitic infection. Adv Intern Med 44:353, 1999.

50.
Grewal IS, Flavell RA: CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol 16:111, 1998.

51.
van Kooten C, Banchereau J: Functions of CD40 on B cells, dendritic cells and other cells. Curr Opin Immunol 9:330, 1997.

52.
Vora KA, Ravetch JV, Manser T: Insights into the mechanisms of antibody-affinity maturation and the generation of the memory B-cell compartment using genetically altered mice. Dev Immunol 6:305, 1998.

53.
Liu YJ, de Bouteiller O, Fugier-Vivier I: Mechanisms of selection and differentiation in germinal centers. Curr Opin Immunol 9:256, 1997.

54.
Doherty PC, Topham DJ, Tripp RA: Establishment and persistence of virus-specific CD4+ and CD8+ T cell memory. Immunol Rev 150:23, 1996.

55.
Callan MF, Annels N, Steven N, et al: T cell selection during the evolution of CD8+ T cell memory in vivo. Eur J Immunol 28:4382, 1998.

56.
Liu YJ, Grouard G, de Bouteiller O, Banchereau J: Follicular dendritic cells and germinal centers. Int Rev Cytol 166:139, 1996.

57.
Choe J, Kim HS, Armitage RJ, Choi YS: The functional role of B cell antigen receptor stimulation and IL-4 in the generation of human memory B cells from germinal center B cells. J Immunol 159:3757, 1997.

58.
Freitas AA, Rocha B: Peripheral T cell survival. Curr Opin Immunol 11:152, 1999.

59.
Slifka MK, Ahmed R: Long-lived plasma cells: A mechanism for maintaining persistent antibody production. Curr Opin Immunol 10:252, 1998.

60.
Isaacson PG, Spencer J: Low-grade B-cell lymphoma of gut-associated lymphoid tissue (GALT): A model of the structure and migration pathways of the B-cell component of normal human GALT. Digestion 46:274, 1990.

61.
Porta C, James PS, Phillips AD, Savidge TC, Smith MW, Cremaschi D: Confocal analysis of fluorescent bead uptake by mouse Peyer patch follicle-associated M-cells. Exp Physiol 77:929, 1992.

62.
Dunn-Walters DK, Isaacson PG, Spencer J: Sequence analysis of human IgV(H) genes indicates that ileal lamina propria plasma cells are derived from Peyer’s patches. Eur J Immunol 27:463, 1997.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

CHAPTER 4 STRUCTURE OF THE MARROW AND THE HEMATOPOIETIC MICROENVIRONMENT

CHAPTER 4 STRUCTURE OF THE MARROW AND THE HEMATOPOIETIC MICROENVIRONMENT
Williams Hematology

CHAPTER 4 STRUCTURE OF THE MARROW AND THE HEMATOPOIETIC MICROENVIRONMENT

CAMILLE N. ABBOUD
MARSHALL A. LICHTMAN

Hematopoietic Location

Embryogenesis and Early Stem Cell Development

Histogenesis
Marrow Structure

Vasculature

Innervation

Sinus Architecture and Cellular Organization

Bone Cells

Macrophages and Lymphocytes

Extracellular Matrix

Hematopoietic Cells
Cell Adhesion and Homing

Integrins

Immunoglobulin Superfamily

Lectins (Selectins)

Sialomucins

Hyaladherin

Other Adhesion Molecules

Cellular Homing

Cell Proliferation and Maturation
Cellular Release
Stem Cell Circulation
Chapter References

The marrow, located in the medullary cavity of bone, is the sole site of effective hematopoiesis in human beings. It produces about 6 billion cells per kilogram of body weight per day. Hematopoietically active (red) marrow regresses after birth until late adolescence, after which time it is focused in the lower skull, vertebrae, shoulder and pelvic girdles, ribs, and sternum. Fat cells replace hematopoietic cells in the bones of the hands, feet, legs, and arms (yellow marrow). Fat comes to occupy about 50 percent of the space of red marrow in the adult, and further fatty metamorphosis continues slowly with aging. In very old individuals, a gelatinous transformation of fat to a mucoid material may occur (white marrow). Yellow marrow can revert to hematopoietically active marrow if prolonged demand is present, such as hemolytic anemia. Thus, hematopoiesis can be expanded by increasing the volume of red marrow and decreasing the development (transit) time from progenitor to mature cell.
The marrow stroma consists principally of a network of sinuses that originate at the endosteum from cortical capillaries and terminate in collecting vessels that enter the systemic venous circulation. The trilaminar sinus wall is composed of endothelial cells; an underdeveloped, thin basement membrane; and adventitial reticular cells that are fibroblasts capable of transforming into adipocytes. The endothelium and reticular cells are sources of hematopoietic cytokines. Hematopoiesis takes place in the intersinus spaces and is controlled by a complex array of stimulatory and inhibitory cytokines, cell-to-cell contacts, and the effects of extracellular matrix components on proximate cells. In this unique environment, lymphohematopoietic stem cells differentiate into all the blood cell lineages. Mature cells are produced and released to maintain steady-state blood cell levels. The system can also go into overdrive to meet increased demands for additional cells as a result of blood loss, hemolysis, inflammation, immune cytopenias, and other causes. Stem cells can leave and reenter marrow as part of their normal circulation. Their extramedullary circulation can be increased by exogenous cytokines and chemokines.
The evolutionary pressures that led to hematopoiesis being confined to the medullary cavity of bone is unclear, but advances in knowledge of the chemical links between the two tissues may provide the answers.

Acronyms and abbreviations that appear in this chapter include: AGM, aorta-gonad-mesonephros; b-FGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; ECMs, extracellular matrix proteins; ELAM-1, endothelial leukocyte adhesion molecule 1; GAGs, glycosaminoglycans; G-CSF, granulocyte colony stimulating factor; GCSFR, G-CSF receptor; GM-CSF, granulocyte-macrophage colony stimulating factor; HCA, hematopoietic cell antigen; HCAM, homing cell adhesion molecule; IAP, integrin-associated protein; ICAM-1, intercellular adhesion molecule 1; LIF, leukemia inhibitory factor; M-CSF, macrophage colony stimulating factor; MIP-1, macrophage inflammatory protein 1; MMP-9, matrix metalloproteinase 9; NK, natural killer; ODF, osteoclast differentiation and activation factor; OPG, osteoprotegrin; PCLP1, podocalyxin-1; PDGF, platelet-derived growth factor; PRR2, poliovirus receptor-related 2 protein; PSGL-1, P-selectin glycoprotein ligand; SDF-1, stroma-derived factor 1; SHP-1, Src homology 2 domain-bearing protein tyrosine phosphatase 1; TGF-b, transforming growth factor beta; TSP, thrombospondin; IIICS, type III connecting segment; VAP-1 vascular adhesion protein 1; VCAM-1, vascular cell adhesion molecule 1; VEGF, vascular endothelial growth factor; VLA-4, very late antigen 4.

The marrow, one of the largest organs in the human body, is the principal site for blood cell formation. In the normal adult its daily production amounts to about 2.5 billion red cells, 2.5 billion platelets, and 1.0 billion granulocytes per kilogram of body weight. The rate of production is adjusted to actual needs and can be varied from nearly zero to many times normal.1 Until the late nineteenth century, blood cell formation was thought to be the prerogative of the lymph nodes or the liver and spleen. In 1868 Neuman2 and Bizzozero3 independently observed nucleated blood cells in material squeezed from the ribs of human cadavers and proposed that the marrow is the major source of blood cells.4 The first in vivo marrow biopsy was probably done in 1876 by Mosler,5 who used a regular wood drill to obtain marrow particles from a patient with leukemia. Fifty years passed before Arinkin’s studies in 1929 established marrow aspiration as a safe, easy, and useful technique.6
Kinetic studies of marrow cells, using radioisotopes and in vitro cultures, have shown that cell lines consist of mature end cells with a finite functional life span, capable of limited proliferation before their full maturation but without the capacity for self-renewal. Sustained cellular production, on the other hand, depends on the presence of pools of primordial cells capable both of differentiation and of self-replication.7 The most primitive pool consists of pluripotential stem cells with the capacity for continuous self-renewal. The more mature pools consist of differentiated unipotential progenitor cells with their maturation restricted to single cell lines and with no capacity for self-renewal. The proliferative activity of these pools involves humoral feedback from peripheral target tissues8 as well as cell-to-cell interactions within the microenvironment of the marrow.9,10 The marrow stroma has evolved to provide a unique structural and chemical environment to support the survival, differentiation, and proliferation of pluripotential (lymphohematopoietic) stem cells. These stem cells can be identified and isolated using a unique array of surface antigen-receptor expression, especially CD34 and Thy-1, but lacking CD38 and CD33.11,12,13 and 14 Isolated cell populations enriched in stem cells can be quantified using in vitro progenitor assays15,16,17 and 18 and surrogate in vivo long-term repopulating assays in severely immunodeficient mice and xenogeneic animal models19,20,21 and 22 (see Chap. 14).
HEMATOPOIETIC LOCATION
EMBRYOGENESIS AND EARLY STEM CELL DEVELOPMENT
The yolk sac and later the fetal liver are sites of early erythropoiesis and contain cells with multilineage differentiation capabilities beginning at day 8 of gestation (yolk sac).23 Non-yolk-sac regions such as the paraaortic splanchnopleura give rise to B-cell progenitors when transplanted into mice with severe combined immunodeficiency.24 The aorta-gonad-mesonephros region (AGM) contains pluripotential stem cells during embryogenesis.25 Stem cells in the AGM region appear before the fetal liver, indicating the importance of this mesodermal region of the embryo in stem cell migration. Early lymphoid precursors have been identified in the day 8 yolk sac26 and the body of embryos beginning at the 10- to 12-somite stage.27 The earliest repopulating lymphohematopoietic stem cells in the day 9 yolk sac have been detected in vivo, using primary conditioned newborn mice,28 and in vitro.29
The early inductive microenvironment for pluripotential stem cells elaborates KIT ligand, encoded by the Sl locus; a later transition from early independent to the late KIT-ligand-dependent fetal hematopoiesis in the embryo occurs.30 Similarly, KIT-negative stem cells have been shown to give rise to KIT-positive cells with pluripotential stem cell activity.31 Murine embryonic stem cells require multiple growth factors such as leukemia inhibitory factor (LIF), KIT ligand, and basic fibroblast growth factor (b-FGF) acting in concert.32,33 Direct interactions and soluble growth factors from AGM stromal34 or endothelial cells35 and marrow-derived stromal cells improve the survival of primitive hematopoiesis.34,35 and 36 This action is exerted via intimate cell-cell interactions of CD34-positive stem cells, which are HLA-DR-negative and uncommitted, with adventitial reticular cells.36
Locally expressed cytokines may lead to differences in the functions of the stromal cells of early blood islands27,32 and those of marrow or spleen.37 Morphologic studies of marrow recovering from aplasia show that early hematopoiesis is localized to the endosteum and vascular endothelium.38 The intimate relationship of angiogenesis and early hematopoiesis is validated by the demonstration that AGM-derived single cells at day 10.5 postcoitum express the receptor tyrosine kinase, TEK, and give rise to hematopoietic cells in the presence of IL-3 and endothelial cells when exposed to angiopoietin-1, defining them as hemangioblasts.39 Podocalyxin-1 (PCLP1), a highly glycosylated protein with similarity to CD34, a high-endothelial venule ligand for L-selectin, has been found on AGM-derived hemangioblasts.40 These PCLP1-positive, CD45-negative cells give rise to hematopoietic cells and endothelial cells when cultured over stromal cells.40 Expression of the alpha4-integrin, in CD45-negative vascular E (VE)-cadherin-positive or -negative cells, defines the earliest precursor of hematopoietic cell lineage diverged from endothelial cells.41 Primitive stem cells obtained from human fetal liver or marrow reconstitute all lymphohe-matopoietic-derived cells and part of the stromal microenvironment in in vivo repopulation assays.42 These observations are consistent with the early derivation of hematopoietic, vascular, and stromal cells from a CD34-negative, vascular-endothelial cell growth factor 2 receptor (known as KDR) -positive, multipotential mesenchymal stem cell.43,44,45,46 and 47 These findings are also underscored by the identification of AC133-positive, CD34-negative, CD7-negative hematopoietic stem cells685 and by the presence of endothelial precursors in AC133-positive progenitor cells.686 The presence of long-term reconstituting hematopoietic stem cells in murine skeletal tissue,48 and in brain-derived neural cells,49 emphasizes the plasticity of these totipotential cells. (See Chap. 14.)
HISTOGENESIS
Cavities within bone occur in the human being at about the fifth fetal month and soon become the exclusive site for granulocytic and megakaryocytic proliferation. Erythropoietic activity at the time is confined to the liver, and it is not until the end of the last trimester that the microenvironment in the marrow becomes supportive of erythroblasts (Fig. 4-1). At birth, the bone cavities are the only sites of significant hematopoietic activity and are completely engorged with hematopoietic cells.50,51 The sequential appearance and disappearance of hematopoietic activity is governed by signaling via chemokine receptors (CXCR4) for stroma-derived factor 1 (SDF-1)52,53 and cellular adhesion molecule-ligand pair interactions, such as, alpha4-integrin with vascular cell adhesion molecule 1 (VCAM-1) or alpha4-integrin with fibronectin.54,55

FIGURE 4-1 Expansion and recession of hematopoietic activity in extramedullary and medullary sites.

By the fourth year of life, a significant number of fat cells have appeared in the diaphysis of the human long bones.56 These cells slowly replace hematopoietic elements and expand centripetally until, at about the age of 18 years, hematopoietic marrow is found only in the vertebrae, ribs, skull, pelvis, and proximal epiphyses of the femora and humeri. Direct measurements of the volume of bone cavities reveal that the bone cavity volume increases from 1.4 percent of body weight at birth to 4.8 percent in the adult,50,57 while the blood volume decreases from 8 percent of body weight in the newborn to about 7 percent in the adult.58 The expansion of marrow space continues throughout life, resulting in a further gradual increase in the amount of fatty tissue in all bone cavities, especially in the long bones.59,60 The preference of hematopoietic tissue for centrally located bones has been ascribed to higher central tissue temperature with greater vascularity.61 However, since complete reactivation of fatty marrow can occur in experimental animals in which hematopoietic expansion is induced, other factors must be involved.62,63
MARROW STRUCTURE
VASCULATURE
The blood supply comes from two major sources.64 The nutrient artery, the principal source, penetrates the cortex through the nutrient canal. In the marrow cavity, it bifurcates into ascending and descending medullary arteries from which radial branches travel to the inner face of the cortex. After repenetrating the endosteum, the radial vessels diminish in caliber to structures of capillary size that course within the canalicular system of the cortex. Here arterial blood from the nutrient artery mixes with blood that enters the cortical capillary system from the periosteal capillaries derived from muscular arteries. After reentering the marrow cavity, the cortical capillaries form a sinusoidal network. Hematopoietic cells are located in the intersinusoidal tissue spaces (Fig. 4-2). Some arteries have specialized, thin-walled segments that arise abruptly as continuations of arteries with walls of normal thickness.65 These vessels give off nearly perpendicular branches analogous to the arterial branching observed in the spleen and kidney, permitting volume compensation for changes in intramedullary pressure. In the marrow cavity, blood flows through a highly branching network of medullary sinuses. These sinuses collect into a large central sinus from which the blood enters the systemic venous circulation through emissary veins.

FIGURE 4-2 Schematic diagram of the circulation of the marrow. See text for further explanation.

Vascular networks consisting of cells expressing CD31, CD34, and CD105 (endoglin) but lacking intercellular adhesion molecule 1 (ICAM-1), ICAM-2, ICAM-3, or endothelial leukocyte adhesion molecule 1 (ELAM-1, E-selectin) can form also within the stroma of long-term marrow cultures, underscoring the intimate relationship of blood vessels to hematopoietic activity.66 A study of early hematopoiesis of human marrow from long bones (ages 6 to 28 weeks) has shown an absence of CD34-positive hematopoietic progenitors before onset of hematopoiesis, a predominance of CD68-positive cells mediating chondrolysis, and CD34-positive endothelial cells developing into specific vascular structures organized by endothelial cells and myoid cells.67 The vascular endothelial growth factor (VEGF) receptors found on CD34-positive cells46 and AGM primitive stem cells underscore that common ontogeny.68,69 Subsets of CD34-positive cells expressing the AC133 antigen and the human vascular endothelial receptor-2 define the functional endothelial precursor phenotype.687
INNERVATION
Myelinated and nonmyelinated nerve fibers are present in periarterial sheaths in marrow,70 where they are thought to regulate arterial vessel tone. Nerve terminals are distributed between layers of periarterial adventitial cells or localize next to arterial smooth muscle cells.71 Nonmyelinated fibers terminate in the hematopoietic spaces, implying that neurohumors elaborated from free-nerve terminals may affect hematopoiesis. Intimate cell-cell communication between sympathetic nerve cells and structural elements within the marrow sinuses occurs at less than 5 percent of nerve terminals that terminate within the hematopoietic parenchyma or on sinus walls. This anatomic unit, termed a neuroreticular complex, consists of efferent (autonomic) nerves and marrow stromal cells connected by gap junctions.71
Nerve growth factor receptor antibody reacts with adventitial reticular cells.72 Tachykinins have demonstrated stimulatory and inhibitory hematopoietic activities within the marrow microenvironment,73,74 and substance P stimulates CD34-positive cell proliferation by modulating stromal cell release of cytokines such as IL-3, IL-6, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor (GM-CSF), and KIT ligand.75,76 Interactions between neurokinins and cytokines such as platelet-derived growth factor and IL-1 result in fibroblast proliferation.77 Neurokinin 1 receptors for substance P are present on marrow vascular endothelium,78 and both noradrenergic and peptidergic innervation have been demonstrated in mouse marrow with dense fibers seen predominantly around blood vessels but also ramifying among marrow cells.79 Thus, adrenergic responses to stress may regulate marrow blood flow and cellular release directly80,81,82 and 83 or by altering endogenous nitric oxide levels within the marrow.84 Exposure of marrow mononuclear cells to hypoxia increases the expression of neurokin-2 receptor and alters the proliferation of myeloid and erythroid progenitors.85 Furthermore, the positive regulation of hematopoiesis by adrenergic agents after syngeneic marrow transplantation86 supports the concept of neural influences. This issue remains controversial. In one study, no neuronal regulation of marrow function was elicited after neonatal sympathectomy or hind limb denervation in mice.87
SINUS ARCHITECTURE AND CELLULAR ORGANIZATION
In mammals, hematopoiesis takes place in the extravascular spaces between marrow sinuses. The sinus wall is composed of a luminal layer of endothelial cells and an abluminal coat of adventitial reticular cells, which forms an incomplete outer lining (Fig. 4-3). A thin, interrupted basement lamina is present between these cell layers.

FIGURE 4-3 Transmission electron micrograph (TEM) of a mouse marrow sinus. The small arrow in the sinus lumen (L) indicates the perikaryon of an endothelial cell. Several endothelial cell junctions are present along the circumference of the sinus endothelial wall. Thus, the wall is composed of the cytoplasm of endothelial cells that overlap or interdigitate. Two adventitial reticular cells are identified by arrows at the top and upper left of the sinus. The cytoplasm of the adventitial reticular cells is discontinuous as it is followed around the sinus. Four cytoplasmic processes of adventitial reticular cells are indicated by arrows. Other, smaller processes of reticular cell cytoplasm can be found on close inspection of the sinus periphery and the hematopoietic spaces. The scattered rough endoplasmic reticulum and dense bodies are characteristic of the reticular cell cytoplasm. (Reprinted from Lichtman,70 with permission.)

ENDOTHELIAL CELLS
Endothelial cells are broad flat cells that completely cover the inner surface of the sinus.88,89 and 90 They form the major barrier and control the system for chemicals and particles entering and leaving the hematopoietic spaces, with overlapping or interdigitating unions permitting volume expansion.89 The endothelium of marrow sinusoids is actively endocytic and contains clathrin-coated pits, clathrin-coated vesicles, lysosomes, phagosomes, transfer tubules, and diaphragmed fenestrae.91,92 Particles are endocytosed by endothelial cells primarily through clathrin-coated pits.93
Such endocytic features are in keeping with studies demonstrating colony-stimulating factor receptors on endothelial cells94 and their shared antigenic determinants with macrophages.95,96 Marrow endothelial cells express von Willebrand factor antigen,97 type IV collagen, and laminin98; they also constitutively express two adhesion molecules: VCAM-1 and E-selectin.99 The distribution of sialic acid and other carbohydrates on the luminal surface of marrow sinus endothelium is discontinued at diaphragmed fenestrae and coated pits, suggesting that such sugars play a role in endothelial membrane function.93,100 Marrow microvascular endothelium can be isolated using the Ulex europeaus lectin, as well as CD34 monoclonal antibodies.101,102,103,104 and 105 Marked attenuation of endothelial cell cytoplasm, short of discontinuity, can occur such that a short length of cytoplasm thins to approach a double plasma membrane in thickness (fenestra with a diaphragm).106
There is reciprocal regulation of CD34 expression and adhesion molecules by vascular endothelial cells exposed to inflammatory stimuli such as IL-1, interferon-g, and tumor necrosis factor-a.107 Receptors for the complement component C1q are upregulated on marrow microvascular endothelium by inflammatory cytokines.108 Other receptors that may mediate marrow cellular trafficking include fractalkine, a novel endothelial membrane-bound chemokine with a mucin stalk, also upregulated by cytokines.109 Marrow sinusoidal endothelium specifically expresses sialylated CD22 ligands, which are homing receptors for recirculating B lymphocytes.110
ADVENTITIAL RETICULAR CELLS
The abluminal or adventitial surface of the vascular sinus is composed of reticular cells.88,111,112 The reticular cell bodies are contiguous with the sinus, forming part of its adventitial coat (Fig. 4-4). Their extensive branching cytoplasmic processes envelop the outer wall of the sinus to form an adventitial sheath. This sheath is interrupted and has been estimated to cover about two-thirds of the abluminal surface area of sinuses. The reticular cells synthesize reticular (argentophilic) fibers that, along with their cytoplasmic processes, extend into the hematopoietic compartments and form a meshwork on which hematopoietic cells rest. The cell bodies, their broad processes, and their fibers constitute the reticulum of the marrow.

FIGURE 4-4 Scanning electron micrograph (SEM) of rat marrow sinus. The floor of the lumen is labeled L. The arrow on the left indicates the cell body of an adventitial reticular cell, which is just beneath the endothelial cell layer. Reticular cell processes can be seen coursing between the sinus wall and the hematopoietic compartment. Several of these are indicated by small arrows. (Reprinted from Lichtman,70 with permission.)

Adventitial reticular cells have a high concentration of alkaline phosphatase in their membranes; express CD10, CD13, and class I HLA antigens88; react with the 6/19 and STRO-1 monoclonal antibodies114,115; and express all neurotrophin receptors including the low-affinity nerve growth factor receptor (p75LNGFR) and the Trk receptors (TrkA, TrkB, and TrkC)116 even though NGF is not a growth factor for STRO-1-derived stromal cells.117 These adventitial reticular cells can differentiate along the smooth-muscle pathway and contain alpha smooth-muscle actin, vimentin, laminin, fibronectin, and collagens I, III, and IV.118,119 Unlike embryonic fibroblasts,120 adventitial reticular cells are usually CD34-negative.89,119,121 Stromal cells display cell-cell contacts via connexin-43 gap junctions.122 These gap junctions are localized to areas of adherence of stromal cells and hematopoietic cells in marrow recovering from cytotoxic injury, underscoring the importance of direct cell-cell communication between progenitors and stromal cells during active hematopoiesis.123,124 Marrow-derived stromal cell lines display heterogeneity at the molecular—expression of cytokines such as KIT ligand, TPO, and Flt3, or differentiation regulatory genes such as human Jagged1 (hJagged1)—and functional—cobblestone formation, CD34+ cell proliferation—levels, with variable expression of ICAM-1, VCAM-1, and collagens I, III, and IV.125
More specialized contractile reticular “barrier cells” have been described in both spleen and marrow in mice after hematopoietic stress, such as malarial infection or administration of IL-1.126,127 Barrier cells increase in number and seem to enclose developing hematopoietic progenitors in these animals. These cells may regulate the release of precursors into the circulation.127 Human counterparts of barrier cells are alpha smooth-muscle-positive cells that appear in culture after 2 weeks and are represented by myoid cells lining sinuses at the abluminal side of endothelial cells in marrow biopsies.119 These cells also have been described in fetal marrow and are increased in areas of active marrow proliferation after inflammation.127
ADIPOCYTES
Adipocytes in marrow develop by lipogenesis in fibroblast-like cells, most likely the adventitial reticular cells (Fig. 4-5). Reticular cells in mouse marrow and human marrow can undergo transformation to fat cells in vitro and can transform into fibroblasts in culture by a process of lipolysis.88,129 Marrow fat cells are relatively resistant to lipolysis during starvation. Their proportion of saturated fatty acids is lower than in other fat deposits, but their composition depends to a certain extent on whether they are located in red, hematopoietically active, or yellow, hematopoietically inactive, marrow.129 Adipocytes express leptin, osteocalcin, and increased prolactin receptors during their differentiation, thereby promoting hematopoiesis and influencing osteogenesis.130,131,132 and 133 Adipocyte maturation in vitro is inhibited by stromal-derived cytokines such as IL-1 and IL-11.134,135 Marrow adipocyte leptin may modulate adjacent hematopoietic progenitor growth.129 Adipocyte differentiation by marrow stromal cells is inhibited by bone morphogenetic proteins136 and leptin,137 supporting the reciprocal regulation of osteogenesis and adipogenesis in the marrow microenvironment.

FIGURE 4-5 SEM of rat marrow. Several sinuses are evident. The exposed lumen of one branching sinus is labeled L. The short horizontal arrow points to the cytoplasm of a transected megakaryocyte. The longer, vertical arrow points to the remnant of a fat cell. The rat femoral marrow contains a modest number of fat cells. (Reprinted from Lichtman,70 with permission.)

STROMAL CELLS
Stromal cells are obtained from animal or human marrow and studied in cultures. They presumably are derived from fibroblasts. They have unique phenotypic and functional characteristics that allow them to nurture hematopoietic development in highly specialized microenvironmental niches.125 These cells express nerve growth factor receptor, VCAM-1, tenascin, endoglin, and collagens IV and VI but do not express intercellular adhesion molecules138; unlike marrow fibroblasts, marrow stromal cells fail to upregulate collagenase when exposed to IL-1.139 Stromal cells and cell lines differ in their capacities to support the growth of myeloid,140,141 and 142 pro-B,143,144 and T-cell precursors.145 This nurturing function is mediated by different combinations of early acting growth factors such as Flt3-ligand,146 KIT ligand,147 thrombopoietin,148,149,150 and 151 LIF,152 and IL-6,153 all released by stromal cells. Other interactions that regulate hematopoietic cell survival and differentiation are mediated by cell-cell contact via negative regulators of hematopoiesis such as transforming growth factor beta (TGF-b), which downregulates c-KIT expression147,154; the Notch/Jagged pathway, which inhibits myeloid differentiation155; and specific receptors (e.g., WNT protein family156 or angiogenins such as neuropilin-1157) and adhesion molecules (MUC18, CD164, and HCA) on stromal cells and hematopoietic CD34-positive cells.158,159 and 160
BONE CELLS
Osteoblasts, osteoclasts, and elongated flat cells with a spindle-shaped nucleus form the marrow endosteal lining.161 Resting endosteal cells express vimentin, tenascin, alpha smooth-muscle actin, osteocalcin, CD51, and CD56 and do not react with CD3, CD15, CD20, CD34, CD45, CD68, or CD117.162 Enriched CD56-positive, CD45-negative, CD34-negative endosteal cells grown in the presence of cytokines [insulin growth factor 1, basic fibroblast growth factor (b-FGF), KIT ligand, IL-3, and GM-CSF] do not give rise to hematopoietic cells, suggesting that they are not totipotent mesenchymal stem cells in these culture conditions.162 Cultured human bone cells have high levels of a1/b1, a3/b1, a5/b1, av/b5 integrins.163 Endosteal cells are a rich source of stem cells (using the in vivo CFU-S assay, see Chap. 14)164 and provide a homing niche for newly transplanted hematopoietic stem cells.165 Mesenchymal stem cells positive for the STRO-1 antibody can differentiate into adipocyte, chondrocytic, and osteogenic cells,166,167,168 and 169 and similar osteogenic potential is found in STRO-1-positive vascular pericytes.170 This process of mesenchymal stem cell to osteogenic differentiation is associated with the loss of the activated leukocyte adhesion molecule (CD166).171
OSTEOBLAST
Bone-forming osteoblast progenitor cells, like stromal precursors, reside in the CD34-negative, STRO-1-positive nonadherent marrow cell population.172,173 and 174 Bone morphogenetic protein 2, b-FGF, and TGF-b promote the growth and differentiation of these cells.172,175 Osteoblasts expand early hematopoietic progenitor survival in long-term cultures and secrete hematopoietic growth factors such as macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), GM-CSF, IL-1, and IL-6.176,177 Osteoblasts also produce hematopoietic cell-cycle inhibitory factors such as TGF-b, which may contribute to their intimate role in stem cell regulation within the marrow microenvironment.178 These cells can be transplanted in nonablated mouse179 and facilitate the engraftment of purified allogeneic hematopoietic stem cells, in keeping with their ability to support hematopoiesis.180 Subcapsular renal explants of bone are able to form a suitable hematopoietic microenvironment for early stem cells, underscoring the potential for osteoblasts to nurture hematopoiesis.181 Direct cell-cell communication has been shown in marrow123 as well as in osteoblastic cell networks,182 indicating a potential regulatory role for these anatomic gap junctions in hematopoiesis.122
OSTEOCLAST
Bone-resorbing osteoclasts are derived from hematopoietic progenitors (CD34-positive, STRO-1-negative) and branches from the monocyte-macrophage lineage early during differentiation.183,184 The essential role of M-CSF in osteoclastogenesis is demonstrated by the op/op mouse, which has osteopetrosis and congenital deficiency of M-CSF185 and which improves after M-CSF treatment.186 KIT ligand and M-CSF act synergistically on osteoclast maturation,187 and M-CSF is essential for the proliferation and maturation of osteoclast progenitors.188 The major form of secreted M-CSF is a proteoglycan.189 It binds to bone-derived collagens and can be extracted from the bone matrix,190 implying a local role for this factor in bone development and remodeling. Targeted disruption of oncogenes such as C-FOS191 and pp60 C-SRC192 prevents osteoclast differentiation leading to osteopetrosis. Osteoprotegrin (OPG), or osteoclastogenesis inhibitory factor, is a cytokine of the tumor necrosis factor receptor superfamily, which inhibits osteoclast differentiation.193 Osteoclast maturation requires osteoprotegrin ligand (TRANCE/RANKL), an osteoclast differentiation and activation factor (ODF) elaborated by stromal cells and osteoblasts.194 ODF together with M-CSF induces osteoclast formation without requiring stromal cells.195,196 and 197 Cross-linking antibodies to the adhesion receptor CD44 inhibit osteoclast formation in primary marrow cultures treated with 1 alpha 25-dihydroxyvitamin D3.198 Similarly, blocking the expression of cadherin-6 interferes with heterotypic interactions between osteoclasts and stromal cells, impairing their ability to support osteoclast formation.199 CD9, a tetraspan transmembrane adhesion protein on stromal cells, is known to influence myelopoiesis in long-term marrow cultures.200 Inhibition of stromal cell CD9-mediated signaling by a blocking antibody reduces ODF transcription, leading to reduced osteoclastogenesis.201 Such cell-cell cross-talk underscores the functional heterogeneity of hematopoietic inductive signals within the marrow microenvironment.
MACROPHAGES AND LYMPHOCYTES
Macrophages and lymphocytes form part of the marrow microenvironment through growth factor production (IL-3, MIP-1a) and cell-cell interactions with developing progenitors.70,88,202,203,204,205 and 206 Macrophages207 and lymphocytes140,208 are an integral part of the adherent monolayer found in long-term lymphohematopoietic cultures. Mature T and B lymphocytes and plasma cells are found near foci of granulopoiesis in the adherent layers of long-term cultures in humans.209 Marrow stroma can support thymocyte differentiation,210 and an early T-cell progenitor maturation pathway occurs in the marrow.211 Marrow stroma regulates B lymphopoiesis by different stromal cell niches and homing receptors (VCAM-1) and the production of cytokines such as Flt3 ligand, KIT ligand, IL-7, and TGF-b.212,213 and 214 Stromal cells facilitate the maturation of natural killer cells,215 an effect likely mediated by stromal-derived Flt3 ligand and IL-15.216
Stromal cells elaborate and respond to peptide growth factors such as platelet-derived growth factor (PDGF).217 PDGF upregulates M-CSF secretion by stromal cells, establishing a paracrine stimulatory loop between these two cell types.218 The addition of PDGF to macrophages expressing PDGF receptors upregulates interleukin-1 secretion and thereby activates primitive hematopoietic cells.219 Macrophages also modulate the structure and composition of the extracellular matrix and its fibronectin content.220 Marrow macrophage phenotype221 is regulated by adjoining stromal cell–accessory cell–derived colony-stimulating factors and cytokines,222 such as M-CSF upregulation of a4b1 and a5b1 integrin expression223 and Flt3 ligand-promoting macrophage outgrowth with B-cell-associated antigens.224 Macrophages express sialic-acid-binding receptors225 and play an integral role in erythropoiesis.226
EXTRACELLULAR MATRIX
Mesenchymal cells forming the cellular stroma in marrow are active in laying down a rich carpet of extracellular matrix proteins (ECMs)227 such as proteoglycans or glycosaminoglycans (GAGs),227,228 fibronectin,227,229 tenascin,227,230 collagen,227,230 laminin,98,230 hemonectin,231 and thrombospondin.227,232 Localizing signals are provided by stromal, ECM hematopoietic cell adhesive interactions,233,234 in concert with chemoattractant small molecules, the chemokines235 and cytokines, bound to heparin-like structures in the GAGs.236 These interactions form specialized niches that may facilitate lymphocytic (B and T) or lineage-specific development along the erythroid, myeloid, or megakaryocytic pathways.237,238 Other functions of these niches include stem cell survival239 and quiescence.237,240 Cytokines that are presented on the surface of stromal cells and matrix-binding chemokines and cytokines are shown in Table 4-1.236,241,242,243,244,245,246,247,248,249,250,251,252 and 253 Sl/Sld mice that have a deficient hematopoietic microenvironment as a result of a deficiency in KIT ligand147,254 processing or membrane presentation are anemic and have alterations in their extracellular matrix composition.255 The addition of hemonectin improves stem cell adhesion to a stromal line derived from Sl/Sld mice.256

TABLE 4-1 CELL MEMBRANE PRESENTATION AND MATRIX ASSOCIATION OF CYTOKINES AND CHEMOKINES

In long-term marrow cultures, collagen, fibronectin, and laminin are secreted early, and extracellular deposition of these proteins coincides with active hematopoiesis.255 GM-CSF is found to prominently stain adipocyte membranes.257 Cultures actively generating granulocyte-macrophage precursors produce M-CSF and GM-CSF and, to a lesser extent, KIT ligand and G-CSF within the adherent layer.258 GM-CSF, G-CSF, and b-FGF are detected on the surface of endothelial cells and fibroblasts, and GM-CSF localizes to the extracellular matrix as shown by double-labeling of heparan sulfate proteoglycans and GM-CSF.259 Negative regulators like TGF-b exert their effects early on long-term marrow cultures by limiting megakaryocyte progenitor and stem cell expansion.260
PROTEOGLYCANS
Proteoglycans are polyanionic macromolecules (heparan sulfate, dermatan, chondroitin sulfate, and hyaluronic acid) that are distributed on the surface of adventitial reticular cells as well as within the extracellular matrix.227,261 Heparan sulfate is the main cell-surface glycosaminoglycan in long-term marrow cultures, and chondroitin sulfate is the major secreted species.255,262 D-xylosides, which stimulate artificial sulfated glycosaminoglycan synthesis, cause an increase in chondroitin sulfate synthesis and hematopoietic cell production.262 Hyaluronic acid and chondroitin sulfate-containing proteoglycans are prominent in the adherent and nonadherent compartments of long-term marrow cultures.261 Heparin-containing and heparan-sulfate-containing proteoglycans interact with laminin and type IV collagen263 and may play a role in cell-cell interactions, cytokine presentation, and cell differentiation.264,265,266 and 267 They also mediate progenitor binding to stroma, along with other extracellular matrix molecules such as fibronectin.268,269,270,271 and 272
Another important lymphocyte-progenitor cell-associated proteoglycan, CD44, uses hyaluronate as a ligand and promotes stromal adhesive interactions.208,273 A binding site for lymphocyte CD44 on the carboxy-terminal heparin-binding domain of fibronectin is present,274 and neutralizing antibodies to CD44 inhibit hematopoiesis in long-term marrow cultures.275 Cytokines (GM-CSF, IL-3, and KIT ligand) rapidly induce CD44 expression and increase CD44-mediated adhesion of CD34-positive hematopoietic progenitors to hyaluronan.276 Chondroitin sulfates A and B mediate monocyte and B-cell activation via a CD44-dependent pathway,277 while hyaluronate, the CD44 ligand, enhances hematopoiesis by releasing IL-1 (CD44-dependent) and IL-6 (CD44-independent pathway), supporting the important role of this proteoglycan receptor in hematopoiesis.278 Heparan sulfate mediates IL-7-dependent lymphopoiesis249 and modulates hematopoiesis and stromal cell-matrix remodeling282 by anchoring both hepatocyte growth factor250,279 and b-FGF.280,281 and 282 Marrow stromal cell surface heparan-sulfate-containing proteoglycans consist mainly of syndecan-3 and -4 and glypican-1, while the major extracellular matrix-associated form is perlecan.283 Syndecan-3 is expressed in marrow stromal cells as a variant form with a core protein of 50 to 55 kDa, suggesting it may play a role in hematopoiesis.283 Perlecan promotes b-FGF receptor binding and mitogenesis and is able to bind GM-CSF.277,284 Heparan sulfate expression is also induced in early erythroid differentiation of multipotential hematopoietic stem cells.285 Glypican-4, another member of this family, has been found on marrow stromal cells and progenitor cells.286 Syndecan-1 expression in B lymphoid cells is reduced by IL-6, which may imply similar regulatory pathways in other cell types.287 Biglycan, a matrix glycoprotein sc1 with homology to osteo-nectin, and the molecule SIM selectively increase IL-7-dependent pro-liferation of B cells.288 Interactions of B cells with other components of the immune system are mediated by syndecan-4, which facilitates the formation of dendritic processes289 and regulates focal adhesion, stress fiber formation, and cell migration.290 Taken together, these observations underscore the major contribution of proteoglycans in the formation of specialized microenvironmental niches to promote lineage-specific hematopoiesis.
FIBRONECTIN
Fibronectin localizes at sites of attachment of hematopoietic cells and marrow stromal cells in vitro,229,291 at sites of interaction between these cells and developing granulocytes or monocytes.292 Early erythroid progenitors attach to the cell-binding domain of fibronectin,293,294 and this association can be inhibited by blocking antibodies to the fibronectin integrin receptors a5b1 and a4bA1.295 Adhesion of hematopoietic progenitor cells to stroma is mediated in part by fibronectin,268,296 and this binding can be enhanced by protein kinase C activators such as phorbol esters, suggesting the involvement of integrin receptors in this process.297,298 and 299 The alternatively spliced form of fibronectin (type III connecting segment, IIICS) is expressed uniquely within the marrow microenvironment122,299 and associates with the a4b1 integrin receptor on hematopoietic stem cells.300 Additional IIICS fibronectin variants have been detected in marrow stroma, providing for a fine control using mRNA splicing of progenitor–stem cell interactions.301 Fibronectin adhesion to peptide domains, such as the CS1 domain (which activates alpha4 integrins) or stromal cells, has dual effects of stimulation as well as inhibition of hematopoietic progenitor growth.302,303,304 and 305
The integrins very late antigen 4 (VLA-4) and VLA-5, as well as CD44, cooperate to promote these fibronectin adhesive interactions.302,306,307 and 308 Cytokines such as IL-3, KIT ligand, and thrombopoietin augment the magnitude of the fibronectin-mediated hematopoietic progenitor cell adhesion and migration.309,310,311 and 312 Fibronectin facilitates the maturation of CD34-positive progenitor-derived dendritic cells313 and is involved in the adhesion of mature cells like megakaryocytes,314,315 mast cells,316 chemokine-activated T lymphocytes,317 eosinophils,318 and neutrophils.319 Fibronectin is required for the expression of gelatinase in macrophages320 and regulates the cytokine release by M-CSF-activated macrophages321 and chondrocytes.322 These interactions of fibronectin and its integrin counterreceptors on hematopoietic cells are associated with activation of the sodium-hydrogen exchanger and result in improved cell survival or stimulation.323
TENASCIN
Tenascin is an extracellular matrix glycoprotein family consisting of three members: tenascin-C, tenascin-R (restrictin), and tenascin-X.230,324 Tenascin-C is expressed on the surface of stromal cells in the marrow and, like fibronectin and collagen III, is found in the microenvironment surrounding maturing hematopoietic cells.227,325 In a long-term marrow culture system (Whitlock-Witte), the thiol 2-mercaptoethanol induced the expression of tenascin-C and improved lymphoid-lineage differentiation.326 Glucocorticoids, on the other hand, promote myeloid differentiation in long-term marrow cultures and downregulate tenascin expression.327 Tenascin-C has distinct functional domains that promote hematopoietic cell adhesion to stroma or extracellular matrix proteins, or mediate a strong mitogenic signal to marrow mononuclear cells.328 In tenascin-C-deficient mutant mice, the colony-forming capacity of marrow is markedly decreased.329 Long-term marrow cultures from these tenascin-deficient animals result in a decreased progenitor cell output.329 Addition of tenascin-C to these cultures restores hematopoietic cell production.329 Mutant tenascin-C-deficient animals also display decreased fibronectin in their marrow, suggesting a possible mechanistic interaction between tenascin-C and fibronectin in the marrow microenvironment.330 These studies underscore the important role of extracellular matrix proteins such as fibronectin and tenascin-C in hematopoiesis.
COLLAGEN
Collagen type I and type III are associated with microvascular walls, whereas type IV collagen is confined to basal lamina beneath endothelial cells.96,255,331 Marrow-derived capillary networks grow in collagen gel cultures,332 and inhibition of collagen synthesis reduces hematopoiesis in vitro,333 underscoring the importance of the underlying matrix in reconstituting an intact hematopoietic microenvironment.262,334 Erythroid and granulocytic progenitors adhere to collagen type I in vitro,335 and a low-molecular-weight collagen has been described in lithium-stimulated marrow cultures,336 emphasizing the effects of cytokines on matrix composition and stromal support of hematopoiesis.220,337 Marrow-derived fibroblasts and stromal cells synthesize collagens I, III, IV, V, and VI.338 Collagen VI is a strong cytoadhesive component of the marrow microenvironment. It binds von Willebrand factor.339 Collagen type XIV, another fibril-associated collagen, promotes hematopoietic cell adhesion of myeloid and lymphoid cell lines.340 Collagen-induced, intracellular calcium-mediated signaling events occur in mega-karyocytes.341 In situ immunolocalization of ECMs in murine marrow showed that collagen types I, IV, and fibronectin localize to the endosteum.342 The distinct spatial distribution of these matrix proteins underscores their role in the preferential homing of engrafted hematopoietic stem cells to marrow.165
LAMININ
A multidomain glycoprotein with mitogenic and adhesive sites, laminin is a major component of the extracellular matrix and basement membranes.227,343 Laminin interacts with collagen type IV and basement membrane components such as proteoglycans and entactin344 and thus can regulate leukocyte chemotaxis.345,346 Similarly, CD34-positive granulocytic progenitors,347 mature monocytes,348 and neutrophils349 adhere to laminin. Its role within the cytomatrix may be to strengthen adhesive interactions with integrin receptors, a5b1 (VLA-5) and a6b1 (VLA-6), on hematopoietic cells.350,351 VLA-6 mediates mast cell adhesion to laminin,352 while the Lutheran blood group glycoproteins serve as laminin receptors on erythroid cells.353 A 67-kDa laminin receptor has been identified on acute myeloid leukemia cells displaying monocytic differentiation. Laminins are heterodimers composed of alpha, beta, and gamma polypeptides. Laminin-1 (a1b1g1) is not expressed in marrow, which expresses laminin-2 (a2,b1,g1), laminin-8 (a4b1g1), and laminin-10 (a5b1g1).355 Laminins containing the a5 chain bind to multipotential hematopoietic cells (FDCP-mix cells), in contrast to laminin-1 heterodimers.355 Stromal cells in cultures as well as cytokine-expanded CD34-positive cells also express laminin b2, which is found in the pericellular space in marrow and intracellularly in megakaryocytes.354,356 Laminin promotes the M-CSF-dependent proliferation of marrow-derived macrophages and macrophage cell lines. This effect is partially mediated via an a6 integrin subunit.357
HEMONECTIN
Hemonectin, a 60-kDa glycoprotein, mediates the attachment of granulocytes to marrow.231 This protein is expressed in hematopoietic tissues as they develop in murine embryos.358 Hemonectin is related to the plasma glycoprotein fetuin.359 Granulocytic adhesion to marrow-derived hemonectin is mediated by galactose and mannose.360 The exact nature of this molecule and its receptor has yet to be identified, hence the role of hemonectin in the marrow hematopoietic microenvironment remains unclear.
THROMBOSPONDIN
Thrombospondin (TSP) is a 450-kDa multifunctional extracellular matrix protein, initially identified in platelet a granules. TSP has domains that interact with collagen and fibronectin and may participate in stem cell lodgement.361 Receptors on hematopoietic and nonhematopoietic cells can interact with thrombospondin, including CD36362,363 and 364 and a protein, CLA-1, of the CD36/LIMP II gene family.365 Perlecan mediates the binding of thrombospondin to endothelial cells.366 The TSP receptor CD36 is expressed during erythroid (CFU-E stage) and megakaryocytic maturation.367 TSP has a dual range of activities from suppression of megakaryopoiesis,368 to an early stimulatory effect on early hematopoietic stem cells,271 erythropoiesis,369 and natural killer cells.370 The inhibition of megakaryocytopoiesis is partially reversed by a low-molecular-weight heparin, suggesting a role for the N-terminal heparin-binding domain in this interaction.368 Other modulatory effects like the natural killer cell expansion, are a consequence of TSP’s ability to activate latent TGF-b.370,371 All-trans retinoic acid-induced granulocytic differentiation of HL-60 cells is associated with an increase in TSP secretion. This process is delayed by a blocking anti-TSP antibody.372 TSP decreases the proliferation and promotes the differentiation of HL-60 cells; these effects are not mediated by latent TGF-b activation.372 A 140-kDa fragment of TSP binds b-FGF and has antiangiogenic properties.373 Endothelial cell TSP expression is inhibited by proangiogenic inflammatory cytokines such as IL-1 and TNF-a.374 TSP stimulates matrix metalloproteinase-9 activity in endothelial cells375 and is chemotactic to monocytes and neutrophils.376 These multiple cellular and microenvironmental interactions underscore TSP’s importance in hematopoietic stem cells homing and differentiation.
VITRONECTIN
Vitronectin, also known as serum spreading factor, is a 75-kDa protein present in plasma, platelets, and connective tissue.230 Vitronectin, a major cytoadhesive glycoprotein, binds to specific integrin receptors (avb3) on fibroblasts, endothelial cells, and mature hematopoietic cells,378 namely, megakaryocytes,379 mast cells,380 bone cells381 such as osteoblasts and osteoclasts,382,383 monocyte-macrophages,384,385 neutrophils, and platelets.386 Transendothelial migration of monocytes and neutrophils is mediated through the aVb3 vitronectin receptor.385,386 Metargidin (ADAM-15) is a type I transmembrane glycoprotein (ADAM, a disintegrin and metalloprotease domain) that binds the avb3 receptor on a monocytic cell line.387 It uses a different integrin receptor (a5b1) to mediate adhesion of a lymphoid cell line, underscoring the complexity of cell adhesive interactions in different hematopoietic cells. The aVb3 vitronectin receptor cooperates with TSP and CD36 in the recognition and phagocytosis of apoptotic cells.388,389 and 390 Vitronectin and a platelet-derived GAG, serglycin, augment mega-karyocyte proplatelet formation.391,392 and 393 Soluble vitronectin inhibits b-FGF-mediated endothelial cell adhesion by interfering with its interaction with the avb3 receptor.394 Cytotoxic T lymphocytes,395 g/d lymphocytes396 and natural killer cells,397 utilize the aVb3 vitronectin receptor as a costimulatory molecule mediating activation signals and cell proliferation. The TSP receptor integrin-associated protein CD47, together with the aVb3 vitronectin receptor, mediate monocyte activation and cytokine release after interacting with soluble CD23.398 Hence, vitronectin appears to contribute mainly to terminal megakaryocyte maturation and platelet formation, while exerting a major role in apoptotic cell clearance, cellular activation, and trafficking to areas of inflammation, bone remodeling, and angiogenesis.
HEMATOPOIETIC CELLS
The hematopoietic cells lie in cords or wedges between the vascular sinuses. Erythroblasts are arranged against the outside surface of the vascular sinuses in distinctive clusters, erythroblastic islands,399 which consist of one or more concentric circles of erythroblasts closely surrounding a macrophage. The inner erythroblastic cells are less mature than the peripheral ones. The central macrophage sends out extensive slender membranous processes that envelop each erythroblast and may phagocytize defective erythroblasts and extruded nuclei.400 The optimal microenvironmental niche for the terminal erythroid maturation into erythroblasts and erythrocytes consists of closely associated fibroblasts, macrophages, and endothelial cells.401 Erythropoiesis is stimulated by stromal cell-derived activin A,402,403 a member of the TGF-b family, while mesodermal erythroid islands are induced by stromal cell-derived growth factors acting in concert, BMP-4 plus activin A or b-FGF.404 The additional ability of b-FGF and HGF to enhance erythropoiesis405,406 underscores the complex cell-cell interactions required for steady-state erythropoiesis in vivo.
Megakaryocytes also lie directly outside the vascular wall407 in normal and myeloproliferative diseases,408 while granulocytes mature deeper in the hematopoietic cords, away from the vascular sinuses. Such discrete spatial structural distribution may be determined by specific adhesive interactions and the provision of specific growth factors for a given cell lineage.88,231,409 The intimate relation of megakaryocyte to sinus endothelium is explained by their expression of CXCR4, the receptor for the marrow endothelial cell-derived chemokine (SDF-1).410 SDF-1 increases transendothelial migration of megakaryocytes and, unlike thrombopoietin, enhances platelet formation.411,412 Thrombopoiesis is also regulated by locally produced synergistic cytokines such as IL-11,413 KIT ligand,414 IL-6,153,415 LIF,152,416 thrombopoietin,148,417 and extracellular matrix proteins.315,391 Stem cells and granulocytic progenitor cells are concentrated in the subcortical regions of the hematopoietic cords.418
Lymphocytes and macrophages concentrate around arterial vessels, near the center of the hematopoietic cords. Computer-assisted three-dimensional reconstruction analysis of human marrow confirms the megakaryocyte apposition against the sinus wall and the position of granulocytic cells along the wall of the central arteriole.419 Erythropoietic cells located mainly around the sinus wall form a continuous network or cord instead of separate “islands.” On this basis, the unitary structure of marrow has been defined as a hematopoietic cord with a central arteriole and surrounded by sinuses.419 A similar structure termed a hematon serves as a multicellular functional unit of marrow and contains adipocytes, stromal elements, macrophages, and hematopoietic stem cells in a compact spheroid.420
Macrophages are a source of stem cell stimulators, such as IL-1, and inhibitors, such as macrophage inflammatory protein (MIP) 1 alpha and tumor necrosis factor a, and play an important role in local control of hematopoiesis.421,422 and 423 Stromal cells and accessory cells are needed for optimum hematopoietic cell development.424 Signals regulating the pluripotential hematopoietic stem cells are not entirely defined but require intimate cell-cell contact for signaling through cytokine-chemokine receptors, integrin receptors, alone or together with heparan sulfate or chondroitin-sulfate-containing glycoproteins.
This regulatory paradigm is underscored by several studies: (1) a neutralizing antibody to KIT, while able to abrogate myelopoiesis in stromal–stem cell cocultures, did not affect stem cell survival425; (2) stromal-cell-derived BMPs (BMP-2, -4, -7) regulate the proliferation and differentiation of CD34-positive, CD38-negative, lineage-negative cells, with high amounts of BMP-2 and -7 inhibiting proliferation and maintaining repopulating capacity, while BMP-4 at higher concentrations extends the survival of these repopulating cells ex vivo426; (3) several adhesion receptors of the sialomucin family mediate inhibitory signals to limit stem cell expansion or differentiation427; (4) direct contact of enriched CD34-positive, lineage-negative cells and stroma induces a soluble factor that increases primitive hematopoietic cell production.428
CELL ADHESION AND HOMING
Hematopoietic stem-progenitor cells (mostly expressing the CD34 antigen12) have multiple adhesion receptors, allowing them to attach to cellular and matrix components within the marrow sinusoidal spaces,295,296,297,298,299 and 300 thereby facilitating their homing and lodgement in the marrow, and providing the close cell-cell contacts required for cell survival and regulated steady-state proliferation.427,429 Adhesive receptors and their ligands, present on hematopoietic stem-progenitor cells, and components of the hematopoietic microenvironment, are shown in Table 4-2. Six subgroups of receptors, the integrins,299,430 immunoglobulins,427,431 lectins (selectins),432,433 sialomucins,434,435 hyaladherin (CD44, H-CAM),436,437 and other receptors such as CD38 (ADP-ribosyl cyclase),438 CD144 439,440 (cadherin), and CD157 (BST-1),441 are shown, listing mostly interactions involving CD34-positive cells and progenitors.429,442 Thus receptor-ligand interactions that regulate the trafficking of mature leukocytes are not included exhaustively.443

TABLE 4-2 HEMATOPOIETIC AND MICROENVIRONMENT ADHESION RECEPTORS AND THEIR LIGANDS

INTEGRINS
Members of this family are divalent cation-requiring heterodimeric proteins (17 a and 8 b subunits), and they mediate important cellular functions including embryonic development, cell differentiation, and adhesive interactions between hematopoietic cells and inflammatory cells and surrounding vascular and stromal microenvironment.299,444 They are subdivided based on the b-chain composition, and as shown in Table 4-2, a chains can associate with more than one b-chain subunit. The principal integrin receptors of the b1 subgroup involved in hematopoietic stem cells endothelial and stromal interactions are a4b1 (VLA-4), a5b1 (VLA-5), and aLb2 (LFA-1) of the b2 subgroup. a4b1-based stromal adhesion events in vitro,445 or in vivo,446 alone or in conjunction with the integrin-associated protein (IAP, CD47)447 regulate erythropoiesis. This receptor also mediates selective granulopoiesis over established marrow stromal cells in cooperation with PECAM-1 (CD31), an immunoglobulin superfamily member,448 and is essential for pre-B cell growth and differentiation over stromal cells expressing IL-7, KIT ligand, and Flt3 ligand.449,450,451 and 452 An acquired defect in stromal function, characterized by a deficiency in VCAM-1 and IL-7 expression,453,454,455,456 and 457 accounts for the delayed B lymphoid reconstitution seen after marrow transplantation.
Integrins also are signaling molecules,458,459 and after engaging their ligands, or subsequent to activation by monoclonal antibodies, multiple events (tyrosine phosphorylation of focal adhesion kinase, paxillin, and ERK-2) are triggered (inside-out signaling), culminating with RAS activation.460,461,462,463 and 464 Integrin receptor cross-talk465 with other adhesive receptor members, such as the immunoglobulin superfamily [natural killer cell-T cell (aLb2 /DYNAM-1), CD34-positive-endothelial cell PECAM-1,466,467,468 and 469 or selectins470], results also from outside-in signaling events that regulate receptor-binding affinity451,471 and mediates inhibitory signals for erythroid, myeloid, and lymphoid progenitor growth.472,473,474,475 and 476 Also, integrin-binding to their counterreceptors, such as a4b1/VCAM-1477 or a4b1/FN,312 in early CD34-positive progenitors, is associated with a decreased rate of apoptosis. Unchecked tyrosine kinase activation, as is the case in chronic myeloid leukemia cells,478 alters integrin affinity and allows the cells to egress from the marrow.479 Inhibition of the Abl kinase activity directly,480 or indirectly, using alpha interferon,481 restores the adhesive properties of these progenitors.
IMMUNOGLOBULIN SUPERFAMILY
The immunoglobulin superfamily233 designates a group of molecules containing one or more amino acid repeats also found in immunoglobulins and consists of PECAM-1 (CD31), ICAM-3/R (CD50) and ICAM-1 (CD54), LFA-3 (CD58), ICAM-2 (CD102), VCAM-1 (CD106), KIT (CD117),482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502 and 503 and PRR2, a molecule related to CD155, which serves as a poliovirus receptor.503 (See Table 4-2.) VCAM-1 is upregulated by inflammatory cytokines (IL-4, IL-13).500,501 Immunoglobulin-like adhesion molecules also include NCAM, a neural adhesion molecule that binds lymphocytes but not hematopoietic progenitors; Thy-1, a stem cell antigen MHC classes I and II; and CD2, CD4, and CD8.233 (See Table 4-2.)
LECTINS (SELECTINS)
Homing of stem cells requires lectin receptors with galactosyl and mannosyl specificities.504,505 The selectins are a family of adhesion molecules, each containing type C lectin structures. The leukocyte selectin (L-selectin, CD62L) is expressed on hematopoietic stem-progenitors506 and mediates adhesive interactions with other receptors (addressins), such as the CD34 sialomucin present on specialized endothelium, using sialylated fucosyl-glucoconjugates. (See Table 4-2.) The CD34 receptor on stem cells, however, does not bind L-selectin,506 as a putative L-selectin ligand yet to be defined exists on these cells. The selectin family also contains CD62E, which is an E-selectin constitutively expressed on marrow sinusoidal endothelium, and regulates the transmigration of leukocytes as well as CD34-positive stem cell homing. The third member of this family is P-selectin, which is found on platelets and is able to bind hematopoietic stem cells, using a mucin receptor, the P-selectin glycoprotein ligand (PSGL-1), which binds to all three selectins. (See Table 4-2.) These proteins are responsible for leukocyte rolling over endothelial surfaces and tethering, thereby allowing integrin-mediated firm adhesion to the endothelium to form, and mediating cellular homing events using specialized high endothelial venule lymphocyte homing sites.507,508,509,510,511,512,513,514,515 and 516
SIALOMUCINS
The mucin family includes the CD34 stem cell antigen,517,518 not an L-selectin ligand on these cells,519 and CD43, an antiadhesion large glycoprotein (leukosialin)520 able to regulate hematopoietic progenitor survival.521 Both CD34 and CD43 signal via tyrosine kinases when capping their surface receptors517,522,523 and, in the case of CD43, clustering of cytoskeleton with CD44 and ICAM-2.522 CD162 (PSGL-1) is important in cell trafficking and stem cell homing,524,525,526,527,528,529 and 530 CD164 (MGC-24v), another sialomucin receptor,531 transmits inhibitory signals to stem-progenitor cells like CD162 and CD34.233 Lastly, CD166, the hematopoietic cell antigen (HCA, ALCAM), forms homodimers (CD166) and heterodimers with CD6.532
HYALADHERIN
The fifth subgroup shown in Table 4-2 is the cartilage-related proteoglycan, CD44, also known as the lymphocyte homing cell adhesion molecule (HCAM). This adhesion receptor is expressed on hematopoietic stem-progenitor cells and facilitates their homing and adhesion to marrow in concert with VLA-4 and ICAM-1, -3. CD44 has several isoforms expressed in normal and tumor tissues. The CD44 variant v10 has been shown to regulate hematopoietic progenitor mobilization, underscoring its importance in mediating cellular matrix-stromal cell adhesion.533,534,535 and 536
OTHER ADHESION MOLECULES
CD38 is a newly recognized adhesion receptor that binds the CD31 receptor and matrix hyaluronan. It is expressed on early T and B cells and subsets of CD34-positive hematopoietic progenitors.537,538 Cadherins are large molecules involved in cell-cell junctions and vascular integrity. CD144, E-cadherin, is expressed on CD34-positive progenitors as well as marrow stroma and endothelial cells, thereby providing another pathway for stem cell lodgement.539 The stromal adhesion receptor BST-1, CD157, is an ADP-ribosyl cyclase, with similarity to CD38. CD157 is expressed on marrow stroma, T and B cells, and myeloid cells and promotes pre-B cell adhesion and growth.540,541,542 and 543
CELLULAR HOMING
The control of lymphocyte and leukocyte cellular trafficking544,545 is a multistep process that involves: (1) selectin-mediated tethering and rolling over vascular endothelial cells expressing in a tissue-specific distribution selectin-binding sialomucins like GlyCAM-1 on lymphatic tissue high endothelial venules,546 MAdCAM-1 on Peyer’s patch endothelium,547 the peripheral lymph node addressin PNAd,548 and the vascular adhesion protein 1 (VAP-1)549 molecule (both mediating CD8 T-lymphocyte migration)547; (2) a triggering step, at sites of inflammation, by short-acting signals such as platelet activating factor,550 cytokine551,552 or chemokine-activating553,554; integrins; (3) tight adhesion and spreading of cells over endothelial surfaces mediated by the immunoglobulin receptors (ICAM-1, -2, VCAM-1)555,556 and 557; (4) CD31-mediated diapedesis,558 in concert with selectin-mediated tethering at vascular endothelial cell junctions.559 Other molecules can promote rolling of cells, such as tenascin,560 and cooperation between different adhesion receptors is frequently seen during the transmigration process.561
Chemokines bind heparan sulfate proteoglycans and thereby play a central role in directing cellular trafficking at sites of inflammation244,245,562 and, in the case of SDF-1,247 regulate cellular trafficking under steady-state conditions. Fractalkine, an endothelial transmembrane mucin-chemokine hybrid molecule, is strategically placed on the surface of activated endothelium and mediates the rapid capture, firm adhesion, and activation under physiologic flow of circulating monocytes, resting or IL-2-activated CD8 lymphocytes, and natural killer (NK) cells.563 The cytokines, TNF-a, and IL-1 upregulate fractalkine, in keeping with the need to recruit effector cells rapidly at sites of inflammation.564 Tissue-restricted chemokines modulate hematopoietic cell adhesive interactions by providing local activation signals, thereby enhancing the specificity of cellular trafficking.235
Unlike lymph nodes, no specific marrow sinusoidal addressins have been defined. A study comparing the adhesive capacity of human marrow or umbilical-cord-derived endothelial cell lines565 did not show any major differences in CD34-positive progenitor adhesion. This interaction is blocked to varying degree by combinations of monoclonal antibodies against a4b1, CD18, and/or E-selectin.565 These findings support the concept of a complex stem cell homing and lodgement process that relies on several short-range signals–adhesive interactions between homing CD34-positive cells and marrow sinusoidal endothelial cells.566,567
Thus, stem cell homing and lodgement to the marrow appears to rely on the distinct characteristics of marrow endothelium and stroma and intrinsic properties of hematopoietic stem and progenitor cells. First, the marrow sinusoidal endothelial cells express constitutively E-selectin, and upon activation both E-selectin and P-selectin are upregulated568; they also express VCAM-1,569,570 while the homing CD34-positive progenitors express PSGL-1 (CD162), a highly glycosylated sialomucin that binds all selectins,524,525 as well as the integrin receptor a4b1, which engages VCAM-1.451 Secondly, L-selectin on CD34-positive progenitors571 may influence the engraftment process by providing a carbohydrate interaction with sinus cavity E-selectin510 and with underlying stroma; L-selectin may also improve progenitor survival as shown by its ability to improve the clonogenic potential of CD34-positive cells.572,573 While the sinusoidal endothelial cells rarely express PSGL-1 (CD162), they display other L-selectin ligands such as chondroitin sulfate574 and heparan sulfate proteoglycans,575 in addition to VEGF-driven E-selectin.576 Thirdly, stromal cells and endothelial cells elaborate SDF-1, a potent chemokine known to enhance integrin activation,576 mediate endothelial CD34-positive cell arrest under flow,577 and enhance CD34-positive cell transmigration.570,576 The fourth element in this complex homing process is based upon the constitutive stromal cell expression of VCAM-1,573 leading to a4b1 integrin-mediated firm adhesion to marrow stroma,573 of a4b1-positive early reconstituting hematopoietic stem cells41 and CD34-positive progenitor cells.54,55
Additional homing signals could result from a5b1 integrin binding to fibronectin,297,311 CD44 binding to cytomatrix hyaluronan,276 ubiquitin binding sites on stroma cells interacting with progenitors,578 and L-selectin interacting with PCLP1.40,516 This heterotypic adhesion occurs because both CD34-positive stem cells and endothelial cells express this receptor/ligand pair. Other immunoglobulin superfamily receptors, PECAM-1 (CD31), ICAM-1, -2 (CD54, CD102), and CD117, also participate in the stem cell lodgement process.427 CD117 (KIT) can interact with membrane-bound KIT ligand to promote adhesion as well as cross-activate other integrin receptors.427
Another homotypic adhesion receptor in this family is the human poliovirus receptor-related 2 protein (PRR2), which is expressed on endothelial cells at the intercellular junctions and on the majority of CD34-positive cells, as well as precursors differentiating along the myelomonocytic and megakaryocytic lineages (CD33- and CD41-positive).503 PRR2 isoforms can homodimerize or heterodimerize, on the cell surface of endothelial cells, in a fashion similar to PECAM-1 (CD31)-mediated aggregation. The latter PECAM-1-signaling events involve phosphorylation of tyrosine on the receptor’s intracytoplasmic tail and by recruitment and activation of Src homology 2 domain-bearing protein tyrosine phosphatase 1 (SHP-1) and SHP-2.579
This cellular trafficking model is supported by experiments in mutant mice deficient in E and P selectins,573 showing decreased marrow progenitor homing in vivo. Similar results are obtained after the administration of blocking antibodies to VLA-4-VCAM-1566,567 or to SDF-1-CXCR4.579 These events result in decreased stromal–stem cell adhesion451 and in diminished CD34-positive cell–endothelial cell transmigration,53,570 leading to impaired homing of transplanted stem cells.580
CELL PROLIFERATION AND MATURATION
The earliest stem cells are pluripotential and capable of differentiation to either lymphopoietic or hematopoietic multipotential stem cells (Chap. 14). These pluripotential stem cells and progenitor cells are in a dormant state12 and are able to withstand the normal hypoxic milieu within the marrow sinusoidal spaces.581 Hematopoietic stem-progenitor cells are prevented from unchecked proliferation by matrix-associated negative regulators such as BMPs426 and TGF-b,582,583 and 584 alone or with locally induced inhibitory chemokines like MIP-1a585 and MCP-1.586,587 Direct inhibitory signals are also triggered by stromal-hematopoietic progenitor binding using sialomucins such as CD34,427 CD162,512 and CD164.159
Later unipotential progenitor cells respond to lineage-specific cytokines and mature into precursor cells that may undergo four or five cell divisions before terminating in functional blood cells (Chap. 14). Hematopoietic growth factors and cytokines are produced locally by stromal cells and other cellular elements of marrow. Such factors as KIT ligand are expressed in a membrane-bound form,147 bind to proteoglycans and heparan sulfate moieties within the cytomatrix, and mediate hematopoietic cell attachment, where they are presented in an active form to receptor-bearing hematopoietic progenitors.284,588 (See “Extracellular Matrix” and Table 4-1.) Cellular attachment to the marrow cytomatrix is an active process leading to signaling and activation of focal adhesion kinases within regions of integrin receptor clustering.589 These properties explain the ability of stromal cells to promote the self-renewal of stem cells590 and inhibit apoptosis of hematopoietic cells.591,592,593 and 594
After maturation of committed progenitor cells, the erythroid and granulocytic blast cells undergo four to five mitotic divisions, while the megakaryocytic blast cells divide perhaps once and then undergo five or six endomitotic (nuclear) divisions. The number of precursor cells in the marrow of humans has been calculated primarily through the study of marrow films and sections relating differential counts of marrow samples to their content of injected radioactive iron. A number of assumptions and approximations need to be made,595 but the summary data given in Table 4-3 agree well with many other observations on the cellular content and kinetics of normal marrows.

TABLE 4-3 NORMAL PRECURSOR CELL KINETICS

CELLULAR RELEASE
Cell migration occurs between adventitial cells but through endothelial cell channels that develop at the time of cell transit. Migrating cells make the hole that develops in the endothelial cell cytoplasm. A number of releasing factors have been implicated in the initiation of marrow egress. The best characterized are those for granulocytes, which include G-CSF,596,597 GM-CSF,598 the C3e component of complement,599 zymosan-activated plasma-containing complement fragments,600 glucocorticoid hormones,601 androgenic steroids,602 and endotoxin.603 Cellular migration is under the complex control of a family of small cytokines termed chemokines with overlapping tissue and target cell specificity, allowing them to regulate effector cell trafficking throughout the body. The chemokine superfamily has several branches based on the cysteine motifs: the “C-X-C” family (platelet factor 4, IL-8, melanocyte growth-stimulating activity/groa, neutrophil activating protein 2, and granulocyte chemotactic protein 2), all mediating neutrophil migration and activation, and the “C-C” family (MIP-1a and b, RANTES, and MCP-1, -2, -3, -4, -5) mediating mostly monocyte and in some cases lymphocyte chemotaxis.235,564 Neutrophils residing in the marrow venous sinusoids are rapidly released into the circulation by IL-8.604 Eosinophil and eosinophil progenitors are recruited from marrow selectively in allergic states, after exposure to IL-5,605 by the chemokines eotaxin606 or RANTES.607 In both systems, migration is inhibited by blocking the b2 integrin CD18, underscoring the importance of integrin activation as well as surface proteolytic activation in mediating transendothelial migration.607,608 Similarly, SDF-1 and KIT ligand cooperate to enhance hematopoietic progenitor chemotaxis.609 Table 4-4 has a detailed listing of chemokine receptors as well as cellular targets and ligands interacting with each receptor subgroup.610,611,612,613 and 614 Chemokines-receptors active on CD34-positive cells are shown in bold font.

TABLE 4-4 CHEMOKINE RECEPTORS, INTERACTING CHEMOKINE LIGANDS, AND CELLULAR SPECIFICITY

Releasing factors for reticulocytes and platelets have been more difficult to identify and may also be of less biological significance, since early release of these cells has little impact on the large pool of circulating cells. Erythropoietin therapy in uremic patients accelerates the egress of reticulocytes.615 Adventitial reticular cell cytoplasm is a barrier to the reticulocytes on the abluminal surface of the endothelium.616 Phlebotomy, phenylhydrazine-induced hemolytic anemia, and erythropoietin result in marked reduction of the adventitial cell cover of the sinus, a process that is thought to facilitate cell egress through the endothelium.617
To leave the marrow, the reticulocyte depends on a pressure gradient across the membrane to drive it through the pore616,617 (see Fig. 4-6). The pressures within the marrow sinuses are pulsatile, and pressures sufficient to cause egress may be transient.618 Anemia and the administration of erythropoietin markedly increase blood flow to marrow and bone,83,619 while G-CSF increases blood flow to marrow only.620 This effect is not blocked by denervation83 and may explain the egress of cells after G-CSF administration.620

FIGURE 4-6 Composite TEM of reticulocytes in egress. (A) Small protrusion of marrow reticulocyte into sinus lumen (L). (B) A reticulocyte in egress with about half the cell in the sinus lumen. (C) A reticulocyte virtually completely in the sinus. Egress occurs through a migration pore which is parajunctional in position (arrows point to endothelial cell junctions). (Reprinted from Lichtman and Waugh,400 with permission.)

Electron micrographs of leukocytes partially translocated across endothelium indicate that marked deformation of these cells occurs as they penetrate the cytoplasm of the endothelial cell to enter the sinus lumen.621 As with reticulocytes, egress occurs adjacent to junctions of endothelial cells.400 The nucleus of the granulocyte, usually segmented, does not require as marked a deformation to traverse the migration pore as do the nuclei of monocytes and lymphocytes.621 The immature granulocytes in marrow are anchored to adventitial reticular cells through lectinlike adhesion molecules. Gradual loss of these molecules (e.g., shedding of L-selectin) during maturation or after activation, could permit movement toward the sinus wall.622 Transient changes in surface glycoproteins (upregulation of a-2,6 sialylation of CD11b and CD18) of maturing marrow myeloid cells lead to decreased stromal and fibronectin adhesion and may favor contact with endothelium and cell egress.623 Activated neutrophils can adhere under flow using the VLA-4 integrin pathway.624 Neutrophil egress occurs mostly at the endothelial cell borders and is entirely P selectin mediated.625 C5a and G-CSF administration recruit neutrophils by altering integrins (low CD11a with G-CSF) and decreased L-selectin expression (with both agents).626,627 Similar findings obtained in mice lacking two or all three selectins underscore the essential role selectins play in neutrophil recruitment.628
The release of platelets is initiated by megakaryocytes that invaginate the abluminal surface of the marrow sinus endothelial cell until a pore is made. Cytoplasm flows through this pore into the marrow sinus and is eventually separated from the body of the megakaryocyte, resulting in a multiplatelet fragment or proplatelet.407,629 The proplatelets often are stringbean-shaped structures and are found in the marrow sinus lumen. Eventually they fragment into single platelets.391,392 and 393 Megakaryocyte nuclei are left in marrow after platelet release and are degraded and phagocytized there.630 The entry of either nuclear remnants or entire megakaryocytes with residual cytoplasm has been observed in both normal individuals631 and patients with marrow disorders.632 The latter regulatory events are mediated by the chemokine SDF-1411 and by c-Mpl ligand.412
Occasional immature granulocytes and megakaryocyte nuclei or whole megakaryocytes are present in cell concentrates of normal blood.631 Nucleated red cells rarely escape from the marrow under normal conditions. The absence of circulating erythroblasts may also relate to the capacity of the spleen to sequester and enucleate circulating erythroblasts. The late myelocytes and metamyelocytes have the capacity to move, respond to chemoattractants, and deform, albeit less well than the mature neutrophils, and thus may occasionally exit marrow by normal mechanisms. The invasion of marrow by neoplastic cells or the replacement of marrow by fibrous tissue is associated with an increased prevalence of immature cells in the circulation. Damage to the architecture of marrow with a breakdown of the integrity of sinus walls may allow cells to enter the circulation less discriminately. Tumor cells elaborate chemoattractive cytokines (chemokines), and this explains their ability to facilitate cell egress from marrow.633
The intramedullary expression of SDF-1 and KIT ligand may allow stem cells to localize to that space.634 KIT ligand upregulates CXCR4 expression on CD34-positive cells, enhancing their chemotactic response, while mobilized blood CD34-positive progenitors have a defective response to SDF-1.635 CXCR4 is expressed on early lymphohematopoietic progenitors,636 providing a model in which mobilized CD34-positive cells have alterations in their adhesion repertoire and chemotactic capacities, allowing them to leave their sinusoidal niches to the peripheral circulation.637 Enhanced hematopoietic progenitor mobilization is also seen when the chemokine MIP-2 is combined with G-CSF.638
The homing and egress processes require the interaction between separate adhesion pathways on hematopoietic stem and progenitor cells and marrow endothelium and stroma, as seen in a mouse model using blocking antibodies to a4b1 and CD44.639 Marrow stem cell homing depends on the a4b1/VCAM-1 adhesion pathway, while CD44 affects homing to marrow and spleen. Inhibition of CD44 and/or a4b1adhesion rapidly mobilized stem cells.639 The CS1 domain FN fragment did not mobilize progenitors, and antibody to a5b1 did not alter homing.640 G-CSF augments the mobilizing action of a4b1/VCAM-1 integrin-blocking antibodies in primates,641 while c-KIT signaling cooperates with this integrin-based mobilization process,642 confirming the complexity of the stem cell egress process.643
STEM CELL CIRCULATION
Stem cells circulate in the blood and can reenter marrow and reestablish hematopoiesis in the marrow cords. Whole-body irradiation of an animal with shielding of a single bone results in the repopulation of the irradiated marrow, strongly implying transfer of stem cells from shielded marrow into irradiated marrow.644 Also, marrow or blood cells from a syngeneic or histocompatible allogeneic donor can reenter marrow and reconstitute hematopoiesis of an animal or human recipient.645 The expression of L-selectin,646 and CD44,647 in blood CD34-positive progenitors seems to correlate with faster engraftment and platelet recovery. Umbilical cord blood CD34-positive cells express L-selectin on their surface in higher amounts than steady-state adult blood progenitors, thereby displaying a preferential homing capacity to the marrow.648 High proliferative potential colony-forming cells in the CD34-positive, CD38-negative subgroup are detectable in the circulation, very early after allogeneic transplantation, coinciding with rapid recovery of blood counts and implying a role for in vivo stem cell recirculation leading to a sustained engraftment process.649
The entry of stem cells into the marrow is mediated by a lectin-sugar interaction650,651 and may be facilitated by alterations in the sinus endothelium induced by the conditioning therapy.652,653 However, c-KIT-positive primitive hematopoietic stem cells, when infused in a nonirradiated host model, home more efficiently to areas of marrow, spleen, lung, and thymus than after sublethal irradiation.654 Unpurified marrow cells labeled with the membrane dye PKH-2 appear to be governed by a nonspecific seeding process rather then by a selective homing signal,655 suggesting that stem cells display adhesive and chemotactic properties that allow them to preferentially seek marrow endothelial sinusoidal spaces. Indeed, marrow endothelial cells under the influence of VEGF constitutively express E-selectin and VCAM-1 and elaborate chemotactic signals such as SDF-1 to attract CD34-positive cells.656,657 Similar findings have been seen when the in vivo homing of long-term repopulating stem cells is analyzed in a serial marrow transplantation model.658
Blood stem cell mobilization for marrow transplantation has been facilitated by improvements in CD34 cell collection and processing659 and the growing availability of recombinant cytokines660 such as G-CSF, GM-CSF, Flt3 ligand, KIT ligand, IL-3, interleukin-7, and thrombopoietin, all of which enhance the release of stem cells into the circulation.661,662,663,664,665,666 and 667 The KIT ligand receptors are downregulated in certain hematopoietic cell lines exposed to growth factors.668 This explains the propensity of KIT ligand to mobilize stem cells, since it can alter receptor affinity and/or density and thus decrease the anchorage of stem cells to the membrane-bound KIT ligand on marrow stromal cells.147,637
As discussed earlier, both CD44-mediated adhesion and a4b1/VCAM-1 interactions affect hematopoietic stem cell egress and homing.539,643 Antibodies directed to the CD44v10 isoform release hematopoietic progenitors into the circulation.536 Moreover, intracellular pools of hyaluronate receptor (RHAMM) and CD44 have been identified in early stem cells (CD34-positive, CD45-low/medium). Steady-state marrow CD34-positive progenitors have larger intracellular CD44 and intracellular RHAMM pools then do cells obtained from G-CSF mobilized blood collections which show a depleted intracellular RHAMM compartment.669 Progenitor adhesion is blocked by anti-CD44 and anti-b1 integrin antibodies, whereas motility is inhibited by antibodies to b1 integrin and RHAMM, suggesting a reciprocal role between these two molecules during stem cell trafficking.
A working model of stem cell egress can be divided into five events shown in Table 4-5. This complex process does not rely on any one feature of stem cells and the marrow microenvironment; rather, the process assumes a continuous series of interactions affecting blood flow,620 adventitial reticular cell-microvascular endothelial cell contraction,670 altered integrin, selectin, cytokine and cytoskeletal receptor expression,669 or functional activation. Chemokines such as IL-8 can efficiently mobilize hematopoietic stem cells.671 IL-8, a potent activator of neutrophil integrin function, causes shedding of L-selectin and degranulation, exposing nearby matrix components to proteolytic enzymes such as elastase and gelatinase B, known also as matrix metalloproteinase 9 (MMP-9).235,433,610 Antibodies against gelatinase B inhibit stem cell mobilization in this model.672 Also, G-CSF administration in vivo is accompanied by a surge in IL-8 that may potentiate stem cell release.673 This action is an indirect one, since long-term repopulating stem cells mobilized by IL-8 do not express aLb1,674 while anti-aLb1 antibody administration blocks IL-8-induced stem cell egress.675

TABLE 4-5 FACTORS REGULATING MARROW STEM CELL EGRESS

Another example of cooperation between cytokines and chemoattractants is provided by the study of G-CSF receptor (GCSFR)-deficient neutrophils, showing that a functional GCSFR is needed for b integrin activation.676 In that GCFR knockout model, Flt3 ligand mobilizes progenitors, whereas IL-8 fails to do so.677 Indeed, a functional GCFR is needed to activate b2 integrins and mediate the IL-8 activation process, with subsequent gelatinase B release.678 The inhibitory effects of anti-aLb1 antibodies and the requirement for a functional G-CSF receptor imply that this mobilization process involves intramedullary activation of neutrophils, leading to enhanced stem cell egress.675 This localized proteolysis (elastase, gelatinase B) is necessary for active cell migration679 and is enhanced by cooperating signals from IL-8-, G-CSF-activated neutrophils adhering to matrix heparan sulfates.680,681,682 and 683 In addition, CD34-positive progenitor cells elaborate gelatinase A and B, a process also augmented by cytokines.684
Hence, stem cell egress is affected by gelatinase expression coupled with altered integrin-, hyuloronan-based anchorage-migration (a4b1-VCAM-1, CD44), by cytokine enhanced blood flow, and by E-selectin-chemokine driven transendothelial migration. This model (see Table 4-5) also takes into account the ability of antibody to gelatinase B, and to b2 integrin, to block the IL-8 mobilization cascade. Integrin signaling and cross-talk with CD44, and the localized production of cytokines (such as KIT ligand, Flt3 ligand, G-CSF, thrombopoietin), create a complex matrix of interactions resulting in upmodulation (or downregulation) of CD34 active chemokine-chemokine receptors (SDF-1/CXCR4, IL-8/CXCR2, RANTES/CCR1, MIP-1a/CCR1, and SLC/CCR7), thereby setting the stage for multiple stem cell mobilization strategies.
CHAPTER REFERENCES

1.
Testa NG, Molineux G: Haemopoiesis: a Practical Approach. IRL Press/Oxford University Press, New York, 1993.

2.
Neuman E: Ueber die Bedeutung des Knochenmarks für die Blutbildung. Cbl Med Wiss 6:689, 1868.

3.
Bizzozero G: Sulla fungione ematopoietica del midollo delle ossa. Gazz Med Ital-Lomb, vol 46, 1868.

4.
Neuman E: Du Role de la möelle des os dans la formation du sang. CR Acad Sci (Paris), 68:1112, 1869.

5.
Mosler F: Klinische Symptome und Therapie der medullalären Leukemi. Berl Klin Wochenschr 13:233, 1876.

6.
Arinkin MJ: Die intravitale Untersuchungsmetodik des Knochenmarks. Folia Haematol (Leipz) 38:233, 1929.

7.
Lajtha LG: The common ancestral cell, in Blood Pure and Eloquent, edited by MM Wintrobe, p 81. McGraw-Hill, New York, 1980.

8.
Erslev AJ: Feedback circuits in the control of stem cell differentiation. Am J Pathol 65:629, 1971.

9.
Trentin JJ: Determination of bone marrow stem cell differentiation by stroma hemopoietic inductive microenvironment (HIM). Am J Pathol 65:621, 1971.

10.
Zipori D: The renewal and differentiation of hemopoietic stem cells. FASEB J 6:2691, 1992.

11.
Simmons DL, Satterthwaite AB, Tenen DG, Seed B: Molecular cloning of a cDNA encoding CD34, a sialomucin of human hematopoietic stem cells. J Immunol 148:267, 1992.

12.
Ogawa M: Differentiation and proliferation of hematopoietic stem cells. Blood 81:2844, 1993.

13.
Craig W, Kay R, Cutler RL, Lansdorp PM: Expression of Thy-1 on human hematopoietic progenitor cells. J Exp Med 177:1331, 1993.

14.
Goodell MA, Rosenzweig M, Kim H, et al: Dye efflux studies suggest the existence of CD34-negative/low hematopoietic stem cells in multiple species. Nat Med 3:1337, 1997.

15.
Bertoncello I, Bradford GB: Surrogate assays for hematopoietic stem cell activity, in Colony-Stimulating Factors: Molecular and Cellular Biology, edited by JM Garland, PJ Quesenberry, DJ Hilton, pp 35–47. Marcel Dekker, New York, 1997.

16.
Sato T, Laver JH, Ogawa M: Reversible expression of CD34 by murine hematopoietic stem cells. Blood 94:2548, 1999.

17.
Fujisaki T, Berger MG, Rose-John S, Eaves CJ: Rapid differentiation of a rare subset of adult human Lin- CD34- CD38- cells stimulated by multiple growth factors in vitro. Blood 94:1926, 1999.

18.
Punzel M, Wissink SD, Miller JS, et al: The myeloid-lymphoid initiating cell (ML-IC) assay assesses the fate of multipotent human progenitors in vitro. Blood 93:3750, 1999.

19.
Bahtia M, Bonnet D, Murdoch B, et al: A newly discovered class of human hematopoietic cells with SCID- repopulating activity. Nat Med 4:1038, 1998.

20.
Kim DK, Fujiki Y, Fukushima T, et al: Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells. Stem Cells 17:286, 1999.

21.
Novelli EM, Ramirez M, Civin CI: Human hematopoietic stem/progenitor cells generate CD5+ B lymphoid cells in NOD/SCID mice. Stem Cells 17:242, 1999.

22.
Zanjani ED, Almeida-Porada G, Flake AW: The human/sheep xenograft model: a large animal model of human hematopoiesis. Int J Hematol 63:179, 1996.

23.
Moore MAS: Embryologic and phylogenetic development of the haemopoietic system. Adv Biosci 16:87, 1975.

24.
Godin IE, Garcia-Porrero JA, Coutinho A, et al: Para-aortic splanchnopleura from early mouse embryos contains B1a cell progenitors. Nature 364:67, 1993.

25.
Medvinski A, Dzierzak EA: Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86:897, 1996.

26.
Yoder MC, Hiatt K, Dutt P, et al: Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity 7:335, 1997.

27.
Cumano A, Furlonger C, Paige CJ: Differentiation and characterization of B-cell precursors detected in the yolk sac and embryo body of embryos beginning at the 10- to 12-somite stage. Proc Natl Acad Sci USA 90:6429, 1993.

28.
Yoder MC, Hiatt K, Mukherjee P: In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus. Proc Natl Acad Sci USA 94:6776, 1997.

29.
Palis J, Starr M, Koniski A, Yoder MC: Temporal and spatial emergence of high proliferative potential colony forming cells (HPP-CFC) during mammalian embryogenesis. Blood 94(suppl 1):32a, 1999.

30.
Ogawa M, Nishikawa S, Yoshinaga K, et al: Expression and function of c-Kit in fetal hemopoietic progenitor cells: transition from the early c-Kit-independent to the late c-Kit-dependent wave of hematopoiesis in the murine embryo. Development 117:1089, 1993.

31.
Ortiz M, Wine JW, Lohrey N, et al: Functional characterization of a novel hematopoietic stem cell and its place in the c-Kit maturation pathway in bone marrow cell development. Immunity 10:173, 1999.

32.
Matsui Y, Zsebo K, Hogan BL: Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70:841, 1992.

33.
Conquet F, Brulet P: Developmental expression of myeloid leukemia inhibitory factor gene in preimplantation blastocysts and in extraembryonic tissue of mouse embryos. Mol Cell Biol 10:3801, 1990.

34.
Xu MJ, Tsuji K, Ueda T, et al: Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. Blood 92:2032, 1998.

35.
Ohneda O, Fennie C, Zheng Z, et al: Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonad-mesonephros region-derived endothelium. Blood 92:908, 1998.

36.
Verfaille CM: Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation. Blood 82:2045, 1993.

37.
Fukushima N, Nishina H, Koishihara Y, Ohkawa H: Enhanced hematopoiesis in vivo and in vitro by splenic stromal cells derived from the mouse with recombinant granulocyte colony-stimulating factor. Blood 80:1914, 1992.

38.
Islam A, Glomski C, Henderson ES: Endothelial cells and hematopoiesis: a light microscopic study of fetal, normal, and pathologic human bone marrow in plastic-embedded sections. Anat Rec 233:440, 1992.

39.
Hamaguchi I, Huang X-L, Takakura N, et al: In vitro hematopoietic and endothelial cell development from cells expressing TEK receptor in murine aorta-gonad-mesonephros region. Blood 93:1549, 1999.

40.
Hara T, Nakano Y-K, Tanaka M, et al: Identification of podocalyxin-like protein 1 as a novel cell surface marker for hemangioblasts in the murine aorta-gonad-mesonephros region. Immunity 11:567, 1999.

41.
Ogawa M, Kizumoto M, Nishikawa S, et al: Expression of a4-integrin defines the earliest precursor of hematopoietic cell lineage diverged from endothelial cells. Blood 93:1168, 1999.

42.
Almeida-Porada GD, Hoffman R, Manalo P, et al: Detection of human cells in human/sheep chimeric lambs with in vitro human stroma-forming potential. Exp Hematol 24:482, 1996.

43.
Osawa M, Hanada K-I, Hamada H, et al: Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273:242, 1996.

44.
Zanjani ED, Almeida-Porada G, Livingston AG, et al: Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells. Exp Hematol 26:353, 1998.

45.
Dieterlen-Lievre F: Hematopoiesis: progenitors and their genetic program. Curr Biol 8:R727, 1998.

46.
Ziegler BL, Valtieri M, Almeida-Porada G, et al: KDR receptor: a key marker defining hematopoietic stem cells. Science 285:1553, 1999.

47.
Nakamura Y, Ando K, Chargui J, et al: Ex vivo generation of CD34+ cells from CD34- hematopoietic cells. Blood 94:4053, 1999.

48.
Jackson KA, Mi T, Goodell MA: Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci USA 96:14482, 1999.

49.
Bjornson CR, Rietze RL, Reynolds BA, et al: Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 283:534, 1999.

50.
Hudson G: Bone marrow volume in the human foetus and newborn. Br J Haematol 11:446, 1965.

51.
Rosse C, Kraemer MJ, Dillon TL, et al: Bone marrow cell populations of normal infants: the predominance of lymphocytes. J Lab Clin Med 89:1225, 1977.

52.
Nagasawa T, Hirota S, Tachibana K, et al: Defects of B-cell lymphopoiesis and bone marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382:635, 1996.

53.
Imai K, Kobayashi M, Wang J, et al: Selective transendothelial migration of hematopoietic progenitor cells: a role in homing of progenitor cells. Blood 93:149, 1999.

54.
Arroyo AG, Yang JT, Rayburn H, et al: a4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo. Immunity 11:555, 1999.

55.
Roy V, Verfaille CM: Expression and function of cell adhesion molecules on fetal liver, cord blood and bone marrow hematopoietic progenitors: implications for anatomical localization and developmental stage specific regulation of hematopoiesis. Exp Hematol 27:302, 1999.

56.
Custer RP, Ahlfeldt FE: Studies on the structure and function of the bone marrow. J Lab Clin Med 17:960, 1932.

57.
Mechanik N: Untersuchange über das Gewicht des Knochenmarks des Menschen. Z Ges Anat 79:58, 1926 (summarized by RE Ellis, Phys Med Biol 5:255, 1961).

58.
Gregersen MI, Rawson RA: Blood volume. Physiol Rev 39:307, 1969.

59.
Christy M: Active marrow distribution as a function of age in humans. Phys Med Biol 26:389, 1981.

60.
Babyn PS, Ranson M, McCarvelle ME: Normal bone marrow signal characteristics and fatty conversion. Med Clin North Am 6:473, 1998.

61.
Huggins C, Blocksom BH Jr: Changes in outlying bone marrow accompanying a local increase in temperature within physiologic limits. J Exp Med 64:253, 1936.

62.
Maniatis A, Tavassoli M, Crosby WH: Factors affecting the conversion of yellow to red marrow. Blood 37:581, 1971.

63.
Crosby WH: Experience with injured and implanted bone marrow: relation of function to structure, in Hemopoietic Cellular Proliferation, edited by F Stohlman Jr, p 87. Grune & Stratton, New York, 1970.

64.
Brookes M: The Blood Supply of Bone. Butterworth, London, 1971.

65.
Tavassoli M: Arterial structure of the bone marrow in rabbits with special reference to thin walled arteries. Acta Anat (Basel) 90:608, 1974.

66.
Wilkins BS, Jones DB: Vascular networks within the stroma of human long-term bone marrow cultures. J Pathol 177:295, 1995.

67.
Charbord P, Tavian M, Humeau L, Peault B: Early ontogeny of the human marrow from long bones: an immunohistochemical study of hematopoiesis and its microenvironment. Blood 87:4109, 1996.

68.
Eichman A, Corbel C, Nataf V, et al: Ligand-dependent development of the endothelial and hematopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2. Proc Natl Acad Sci USA 94:141, 1997.

69.
Marshall CJ, Moore RL, Thorogood P, et al: Detailed characterization of the human aorta-gonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer. Dev Dyn 215:139, 1999.

70.
Lichtman MA: The ultrastructure of the hemopoietic environment of the marrow: a review. Exp Hematol 9:391, 1981.

71.
Yamazaki K, Allen TD: Ultrastructural morphometric study of efferent nerve terminals on murine bone marrow stromal cells, and the recognition of a novel anatomical unit: the “neuro-reticular complex.” Am J Anat 187:261, 1990.

72.
Cattoretti G, Schiro R, Orazi A, et al: Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro. Blood 81:1726, 1993.

73.
Rameshwar P, Gascon P: Induction of negative hematopoietic regulators by neurokinin-A in bone marrow stroma. Blood 88:98, 1996.

74.
Rameshwar P, Gascon P: Substance P (SP) mediates production of stem cell factor and interleukin-1 in bone marrow stroma: potential autoregulatory role for these cytokines in SP receptor expression and induction. Blood 86:482, 1995.

75.
Rameshwar P, Gascon P: Hematopoietic modulation by the tachykinins. Acta Haematol 98:59, 1997.

76.
Hiramoto M, Aizawa S, Iwase O, et al: Stimulatory effects of substance P on CD34 positive cell proliferation and differentiation in vitro are mediated by the modulation of stromal cell function. Int J Mol Med 1:347, 1998.

77.
Rameshwar P, Poddar A, Zhu G, Gascon P: Receptor induction regulates the synergistic effects of substance P with IL-1 and platelet-derived growth factor on the proliferation of bone marrow fibroblasts. J Immunol 158:3417, 1997.

78.
Greeno EW, Mantyh P, Vercellotti GM, Moldow CF: Functional neurokin 1 receptors for substance P are expressed by human vascular endothelium. J Exp Med 177:1269, 1993.

79.
Tabarowski Z, Gibson-Berry K, Felten SY: Noradrenergic and peptidergic innervation of mouse femur bone marrow. Acta Histochem 98:453, 1996.

80.
Afran AM, Broome CS, Nicholls SE, et al: Bone marrow innervation regulates cellular retention in the murine haematopoietic system. Br J Haematol 98:569, 1997.

81.
Iversen PO, Stokland A, Rolstad B, Benestad HB: Adrenaline-induced leucocytosis: recruitment of blood cells from rat spleen, bone marrow and lymphatics. Eur J Appl Physiol 68:219, 1994.

82.
Tang Y, Shankar R, Gamelli R, Jones S: Dynamic norepinephrine alterations in bone marrow: evidence of functional innervation. J Neuroimmunol 96:182, 1999.

83.
Iversen PO: Blood flow to the haemopoietic bone marrow. Acta Physiol Scand 159:269, 1997.

84.
Iversen PO, Nicolaysen G, Benestad HB: Endogenous nitric oxide causes vasodilatation in rat bone marrow, bone, and spleen during accelerated hematopoiesis. Exp Hematol 22:1297, 1994.

85.
Quinlan DP Jr, Rameshwar P, Qian J, et al: Effect of hypoxia on the hematopoietic and immune modulator preprotachykinin-I. Arch Surg 133:1328, 1998.

86.
Maestroni GJM, Conti A, Pedrinis E: Effect of adrenergic agents on hematopoiesis after syngeneic bone marrow transplantation in mice. Blood 80:1178, 1992.

87.
Benestad HB, Strom-Gundersen I, Iversen PO, et al: No neuronal regulation of murine bone marrow function. Blood 91:280, 1998.

88.
Abboud CN, Liesveld JL, Lichtman MA: The architecture of marrow and its role in hematopoietic cell lodgement, in The Hematopoietic Microenvironment, edited by MW Long, MS Wicha, pp 2-20. Johns Hopkins University Press, Baltimore and London, 1993.

89.
Tavassoli M, Shaklai M: Absence of tight junctions in endothelium of marrow sinuses: possible significance for marrow cell egress. Br J Haematol 41:303, 1979.

90.
Abboud CN: Human bone marrow microvascular endothelial cells: elusive cells with unique structural and functional properties. Exp Hematol 23:1, 1995.

91.
Bankston PW, DeBruyn PPH: The permeability to carbon of the sinusoidal lining cells of the embryonic rat liver and rat bone marrow. Am J Anat 141:281, 1974.

92.
Lichtman MA, Packman CH, Constine LS: Molecular and cellular traffic across the marrow sinus wall, in Blood Cell Formation: The Role of Hemopoietic Microenvironment, edited by M. Tavassoli, pp 87–140. Humana Press, Clifton, NJ, 1989.

93.
Kataoka M, Tavassoli M: Identification of lectin-like substances recognizing galactosyl residues of glycoconjugates on the plasma membrane of marrow sinus endothelium. Blood 65:1163, 1985.

94.
Bussolino F, Colotta F, Bocchietto E, et al: Recent developments in the cell biology of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor: Activities on endothelial cells. Int J Clin Lab Res 23:8, 1993.

95.
Koch AE, Burrows JC, Domer PH, et al: Monoclonal antibodies defining shared human macrophage-endothelial antigens. Pathobiology 60:59, 1992.

96.
Penn PE, Jiang D-Z, Fei R-G, et al. Dissecting the hematopoietic microenvironment: IX. Further characterization of murine bone marrow stromal cells. Blood 81:1205, 1993.

97.
Hasthorpe S, Bogdanovski M, Rogerson J, Radley JM: Characterization of endothelial cells in murine long-term marrow culture: Implication for hemopoietic regulation. Exp Hematol 20:386, 1992.

98.
Perkins S, Fleischman RA: Stromal cell progeny of murine bone marrow fibroblast colony-forming units are clonal endothelial-like cells that express collagen IV and laminin. Blood 75:620, 1990.

99.
Schweitzer KM, Drager AM, van der Valk P, et al: Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues. Am J Pathol 148:165, 1996.

100.
DeBruyn PPH, Michelson S: Changes in the random distribution of sialic acid at the surface of the myeloid sinus endothelium resulting from the presence of diaphragmed fenestrae. J Cell Biol 82:708, 1979.

101.
Masek LC, Sweetenham JW, Whitehouse JMA, Schumacher U: Immuno-, lectin-, and enzyme-histochemical characterization of human bone marrow endothelium. Exp Hematol 22:1203, 1994.

102.
Kuemmel TA, Thiele J, Hafenrichter EG, et al: Distribution of lectin binding sites in human bone marrow. Identification by use of ultrastructural postembedding technique. J Submicrosc Cytol Pathol 28:537, 1996.

103.
Garlanda C, Berthier R, Garin J, et al: Characterization of MEC 14.7, a new monoclonal antibody recognizing mouse CD34: a useful reagent for identifying and characterizing blood vessels and hematopoietic precursors. Eur J Cell Biol 73:368, 1997.

104.
Rafii S, Shapiro F, Rimarachin J, et al: Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor adhesion. Blood 84:10, 1994.

105.
Bazzoni G, Dejana E, Lampugnani MG: Endothelial adhesion molecules in the development of the vascular tree: the garden of forking paths. Curr Opin Cell Biol 11:573, 1999.

106.
DeBruyn PPH, Michelson S, Becker RP: Endocytosis, transfer tubules and lysosomal activity in myeloid sinusoidal endothelium. J Ultrastruct Res 53:133, 1975.

107.
Delia D, Lampugnani MG, Resnati M, et al: CD34 expression is regulated reciprocally with adhesion molecules in vascular cells in vitro. Blood 81:1001, 1993.

108.
Guo WX, Ghebrehiwet B, Weksler B, et al: Up-regulation of endothelial cell binding proteins/receptors for complement component C1q by inflammatory cytokines. J Lab Clin Med 133:541, 1999.

109.
Imai T, Hieshima K, Haskell C, et al: Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91:521, 1997.

110.
Nitschke L, Floyd H, Ferguson DJ, Crocker PR: Identification of CD22 ligands on bone marrow sinusoidal endothelium implicated in CD22-dependent homing of recirculating B cells. J Exp Med 189:1513, 1999.

111.
Weiss L, Chen L-T: The organization of hemopoietic cords and vascular sinuses in bone marrow. Blood Cells 1:617, 1975.

112.
Leblond PF, Chamberlain JK, Weed RI: Scanning electron microscopy of erythropoietin-stimulated bone marrow. Blood Cells 1:639, 1975.

113.
Lichtman MA: The relationship of stromal cells to hemopoietic cells in marrow, in Long-Term Bone Marrow Culture, edited by DG Wright, JS Greenberger, pp 3–26. Liss, New York, 1984.

114.
Abboud CN, Duerst RE, Frantz CN, et al: Lysis of human fibroblast colony-forming cells and endothelial cells by monoclonal antibody (6-19) and complement. Blood 68:1196, 1986.

115.
Simmons PJ, Torok-Storb B: Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 78:55, 1991.

116.
Labouyrie E, Dubus P, Groppi A, et al: Expression of neurotrophins and their receptors in human bone marrow. Am J Pathol 154:405, 1999.

117.
Gronthos S, Simmons PJ: The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood 85:929, 1995.

118.
Charbord P, Lerat H, Newton I, et al: The cytoskeleton of stromal cells from human bone marrow cultures resembles that of cultured smooth muscle cells. Exp Hematol 18:276, 1990.

119.
Galmiche MC, Koteliansky VE, Briere J, et al: Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway. Blood 82:66, 1993.

120.
Brown J, Greaves MF, Molgaard HV: The gene encoding the stem cell antigen, CD34, is conserved in mouse and expressed in haemopoietic progenitor cell lines, brain, and embryonic fibroblasts. Int Immunol 3:175, 1991.

121.
Simmons PJ, Torok-Storb B: CD34 expression by stromal precursors in normal adult bone marrow. Blood 78:2848, 1991.

122.
Dorshkind K, Green L, Godwin A, Fletcher WH: Connexin-43-type gap junctions mediate communication between bone marrow stromal cells. Blood 82:38, 1993.

123.
Rosendaal M, Green CR, Rahman A, Morgan D: Up-regulation of the connexin43+ gap junction network in haemopoietic tissue before the growth of stem cells. J Cell Sci 107:29, 1994.

124.
Krenacs T, Rosendaal M: Connexin43 gap junctions in normal, regenerating, and cultured mouse bone marrow and in human leukemias: their possible involvement in blood formation. Am J Pathol 152:993, 1998.

125.
Torok-Storb B, Iwata M, Graf L, et al: Dissecting the marrow microenvironment. Ann NY Acad Sci 872:164, 1999.

126.
Weiss L: Barrier cells in the spleen. Immunol Today 12:24, 1991.

127.
Weiss L, Geduldig U: Barrier cells: stromal regulation of hematopoiesis and blood cell release in normal and stressed murine bone marrow. Blood 78:975, 1991.

128.
Schmitt-Gräff A, Skalli O, Gabbiani G: Alpha-smooth muscle actin is expressed in a subset of bone marrow stromal cells in normal and pathological conditions. Virchows Archiv [B] 57:291, 1989.

129.
Tavassoli M: Fatty evolution of marrow and the role of adipose tissue in hematopoiesis, in Handbook of the Hemopoietic Microenvironment, edited by M Tavassoli, pp 157–187. Humana Press, Clifton, NJ, 1989.

130.
Laharrague P, Larrouy D, Fontanilles AM, et al: High expression of leptin by human bone marrow adipocytes in primary cultures. FASEB J 12:747, 1998.

131.
Benayahu D, Shamay A, Wientroub S: Osteocalcin (BGP), gene expression, and protein production by marrow stromal adipocytes. Biochem Biophys Res Commun 13:442, 1997.

132.
McAveny KM, Gimble JM, Yu-Lee L: Prolactin receptor expression during adipocyte differentiation of bone marrow stroma. Endocrinology 137:5723, 1996.

133.
Gimble JM, Robinson CE, Wu X, Kelly KA: The function of adipocytes in the bone marrow stroma: an update. Bone 19:421, 1996.

134.
Delikat S, Harris RJ, Galvani DW: IL-1 beta inhibits adipocyte formation in human long-term bone marrow culture. Exp Hematol 21:31, 1993.

135.
Keller DC, Du XX, Srour EF, et al: Interleukin-11 inhibits adipogenesis and stimulates myelopoiesis in human long-term marrow cultures. Blood 82:1428, 1993.

136.
Gimble JM, Morgan C, Kelly K, et al: Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells. J Cell Biochem 58:393, 1995.

137.
Thomas T, Gori F, Khosla S, et al: Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes. Endocrinology 140:1630, 1999.

138.
Wilkins BS, Jones DB: Immunophenotypic characterization of stromal cells in aspirated human bone marrow samples. Exp Hematol 26:1061, 1998.

139.
Takahashi GW, Moran D, Andrews DF III, Singer JW: Differential expression of collagenase by human fibroblasts and bone marrow stromal cells. Leukemia 8:305, 1994.

140.
Liesveld JL, Abboud CN, Duerst RE, et al: Characterization of human marrow stromal cells: role in progenitor cell binding and granulopoiesis. Blood 73:1794, 1989.

141.
Aiuti A, Friedrich C, Sieff CA, Gutierrez-Ramos J-C: Identification of distinct elements of the stromal microenvironment that control human hematopoietic stem/progenitor cell growth and differentiation. Exp Hematol 26:143, 1998.

142.
Li J, Sensebe L, Herve P, Charbord P: Nontransformed colony-derived stromal cell lines from normal human marrows: III. The maintenance of hematopoiesis from CD34+ cell populations. Exp Hematol 25:582, 1997.

143.
Osmond DG, Kim N, Manoukina R, et al: Dynamics and localization of early B-lymphocyte precursor cells (pro-B cells) in the bone marrow of scid mice. Blood 79:1695, 1992.

144.
Moreau I, Duvert V, Caux C, et al: Myofibroblastic stromal cells isolated from human bone marrow induce the proliferation of both early myeloid and B lymphoid cells. Blood 82:2396, 1993.

145.
Tamir M, Eren R, Globerson A, et al: Selective accumulation of lymphocyte precursor cells mediated by stromal cells of hemopoietic origin. Exp Hematol 18:332, 1990.

146.
Lisovsky M, Braun SE, Ge Y, et al: Flt3-ligand production by human bone marrow stromal cells. Leukemia 10:1012, 1996.

147.
Besmer P: Kit-ligand-stem cell factor, in Colony-Stimulating Factors: Molecular and Cellular Biology, edited by JM Garland, PJ Quesenberry, DJ Hilton, pp 369–404. Marcel Dekker, New York, 1997.

148.
Kaushansky K: Thrombopoietin and the hematopoietic stem cell. Blood 92:1, 1998.

149.
Solar GP, Kerr WG, Zeigler FC, et al: Role of c-mpl in early hematopoiesis. Blood 92:4, 1998.

150.
Guerriero A, Worford L, Holland HK, et al: Thrombopoietin is synthesized by bone marrow stromal cells. Blood 90:3444, 1997.

151.
Matsunaga T, Kato K, Miyazaki H, Ogawa M: Thrombopoietin promotes the survival of murine hematopoietic long-term reconstituting cells: comparison with the effects of FLT3/FLK-2 ligand and interleukin-6. Blood 92:452, 1998.

152.
Waring PM: Leukemia inhibitory factor, in Colony-Stimulating Factors: Molecular and Cellular Biology, edited by JM Garland, PJ Quesenberry, DJ Hilton, pp 467–513. Marcel Dekker, New York, 1997.

153.
Sui X, Tsuji K, Ebihara Y, et al: Soluble interleukin-6 (IL-6) receptor with IL-6 stimulates megakaryopoiesis from human CD34+ cells through glycoprotein (gp)130 signaling. Blood 93: 2525, 1999.

154.
Heberlein C, Friel J, Laker C, et al: Downregulation of c-kit (stem cell factor receptor) in transformed hematopoietic precursor cells by stroma cells. Blood 93:554, 1999.

155.
Walker L, Lynch M, Silverman S, et al: The Notch/Jagged pathway inhibits proliferation of human hematopoietic progenitors in vitro. Stem Cells 17:162, 1999.

156.
Van Den Berg DJ, Sharma AK, Bruno E, Hoffman R: Role of members of the Wnt gene family in human hematopoiesis. Blood 92:89, 1998.

157.
Tordjman R, Ortega N, Coulombel L, et al: Neuropilin-1 is expressed on bone marrow stromal cells: a novel interaction with hematopoietic cells? Blood 94:2301, 1999.

158.
Filshie RJ, Zannettino AC, Makrynikola V, et al: MUC18, a member of the immunoglobulin superfamily, is expressed on bone marrow fibroblasts and a subset of hematological malignancies. Leukemia 12:414, 1998.

159.
Zannettino ACW, Buhring H-J, Niutta S, et al: The sialomucin CD164 (MCG-24v) is an adhesive glycoprotein expressed by human hematopoietic progenitors and bone marrow stromal cells that serves as a potent negative regulator of hematopoiesis. Blood 92:2613, 1998.

160.
Cortes F, Deschaseauz F, Uchida N, et al: HCA, an immunoglobulin-like adhesion molecule present on the earliest human hematopoietic precursor cells, is also expressed by stromal cells in blood-forming tissues. Blood 93:826, 1999.

161.
Deldar A, Lewis H, Weiss L: Bone lining cells and hematopoiesis: an electron microscopic study of canine bone marrow. Anat Rec 213:187, 1985.

162.
Sillaber C, Walchshofer S, Mosberger I, et al: Immunophenotypic characterization of human bone marrow endosteal cells. Tissue Antigens 53:559, 1999.

163.
Saito T, Albelda SM, Brighton CT: Identification of integrin receptors on cultured human bone cells. J Orthop Res 12:384, 1994.

164.
Gong J: Endosteal marrow: a rich source of hematopoietic stem cells. Science 199:1443, 1978.

165.
Nilsson SK, Dooner MS, Tiarks CY, et al: Potential and distribution of transplanted hematopoietic stem cells in a nonablated mouse model. Blood 89:4013, 1997.

166.
Park SR, Oreffo RO, Triffitt JT: Interconversion potential of cloned human marrow adipocytes in vitro. Bone 24:549, 1999.

167.
Dennis JE, Merriam A, Awadallah, A et al: A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res 14:700, 1999.

168.
Pittenger MF, Mackay AM, Beck SC, et al: Multilineage potential of adult human mesenchymal stem cells. Science 284:143, 1999.

169.
Oyajobi BO, Lomri A, Hott M, Marie PJ: Isolation and characterization of human clonogenic osteoblast progenitors immunoselected from fetal bone marrow stroma using STRO-1 monoclonal antibody. J Bone Miner Res 14:351, 1999.

170.
Doherty MJ, Ashton BA, Walsh S, et al: Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res 13:828, 1999.

171.
Bruder SP, Ricalton NS, Boynton RE et al: Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation. J Bone Miner Res 13:655, 1998.

172.
Long MW, Robinson JA, Ashcraft EA, Mann KG: Regulation of human bone marrow-derived osteoprogenitor cells by osteogenic growth factors. J Clin Invest 95:881, 1995.

173.
Gronthos S, Zannettino AC, Graves SE, et al: Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells. J Bone Miner Res 14:47, 1999.

174.
Stewart K, Walsh S, Screen J, et al: Further characterization of cells expressing STRO-1 in cultures of adult human bone marrow stromal cells. J Bone Miner Res 14:1345, 1999.

175.
Hanada K, Dennis JE, Caplan AI: Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. J Bone Miner Res 12:1606, 1997.

176.
Taichman RS, Emerson SG: The role of osteoblasts in the hematopoietic microenvironment. Stem Cells 16:7, 1998.

177.
Ahmed N, Khokher MA, Hassan HT: Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts. Stem Cells 17:92, 1999.

178.
Gehron Robey P, Young MF, Flanders KC, et al: Osteoblasts synthesize and respond to transforming growth factor-type b (TGF-beta) in vitro. J Cell Biol 105:457, 1987.

179.
Nilsson SK, Dooner MS, Weier HU, et al: Cells capable of bone production engraft from whole bone marrow transplants in nonablated mice. J Exp Med 189:729, 1999.

180.
El-Badri NS, Wang B-Y, Cherry, Good RA: Osteoblasts promote engraftment of allogeneic hematopoietic stem cells. Exp Hematol 26:110, 1998.

181.
Gurevitch O, Fabian I: Ability of the hemopoietic microenvironment in the induced bone to maintain the proliferative potential of early hemopoietic precursors. Stem Cells 11:56, 1993.

182.
Civitelli R, Beyer EC, Warlow PM, et al: Connexin43 mediates direct intercellular communication in human osteoblastic cell networks. J Clin Invest 91:1888, 1993.

183.
Matayoshi A, Brown C, DiPersio JF, et al: Human blood-mobilized hematopoietic precursors differentiate into osteoclasts in the absence of stromal cells. Proc Natl Acad Sci USA 93:10785, 1996.

184.
Roodman GD: Cell biology of the osteoclast. Exp Hematol 27:1229, 1999.

185.
Yoshida H, Hayashi S-I, Kunisada T, et al: The murine mutation osteopetrosis is in the coding region of the macrophage colony-stimulating factor gene. Nature 345:442, 1990.

186.
Wiktor-Jedrzejczak W, Urbanowska E, Aukerman SL, et al: Correction by CSF-1 of defects in the osteopetrotic op/op mouse suggests local, developmental, and humoral requirements for this growth factor. Exp Hematol 19:1049, 1991.

187.
Demulder A, Suggs SV, Zsebo KM, et al: Effects of stem cell factor on osteoclast-like cell formation in long-term human marrow cultures. J Bone Min Res 7:1337, 1992.

188.
Tanaka S, Takahashi N, Udagawa N, et al: Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors. J Clin Invest 91:257, 1993.

189.
Price LK, Choi HU, Rosenberg L, Stanley ER: The predominant form of secreted colony-stimulating factor-1 is a proteoglycan. J Biol Chem 267:2190, 1992.

190.
Ohtsuki T, Suzu S, Hatake K, et al: A proteoglycan form of macrophage colony-stimulating factor that binds to bone-derived collagens and can be extracted from bone matrix. Biochem Biophys Res Commun 190:215, 1993.

191.
Grigoriadis AE, Wang ZQ, Ceccini MG, et al: c-Fos a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266:443, 1994.

192.
Soriano P, Montgomery C, Geske R, Bradley A: Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64:693, 1991.

193.
Shalhoub V, Faust J, Boyle WJ, et al: Osteoprotegrin and osteoprotegrin ligand effects on osteoclast formation from human peripheral blood mononuclear cell precursors. J Cell Biochem 72:251, 1999.

194.
Takahashi N, Udagawa N, Suda T: A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun 256:449, 1999.

195.
Yasuda H, Shima N, Nakagawa N, et al: A novel molecular mechanism modulating osteoclast differentiation and function. Bone 25:109, 1999.

196.
Burgess TL, Qian Y, Kaufman S, et al: The ligand for osteoprotegrin (OPGL) directly activates mature osteoclasts. J Cell Biol 145:527, 1999.

197.
Hsu H, Lacey DL, Dunstan CR, et al: Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegrin ligand. Proc Natl Acad Sci USA 96:3540, 1999.

198.
Kania JR, Kehat-Stadler T, Kupfer SR: CD44 antibodies inhibit osteoclast formation. J Bone Miner Res 12:1155, 1997.

199.
Mbalaviele G, Nishimura R, Myoi A, et al: Cadherin-6 mediates the heterotypic interactions between the hemopoietic osteoclast cell lineage and stromal cells in a murine model of osteoclast differentiation. J Cell Biol 141:1467, 1998.

200.
Oritani K, Wu X, Medina K, et al: Antibody ligation of CD9 modifies production of myeloid cells in long-term cultures. Blood 87:2252, 1996.

201.
Tanio Y, Yamazaki H, Kunisada T, et al: CD9 molecule expressed on stromal cells is involved in osteoclastogenesis. Exp Hematol 27:853, 1999.

202.
Quesenberry PJ, Crittenden RB, Lowry P, et al: In vitro and in vivo studies of stromal niches. Blood Cells 20:97, 1994.

203.
Gibson FM, Scopes J, Daly S, et al: IL-3 is produced by normal stroma in long-term bone marrow cultures. Br J Haematol 90:518, 1995.

204.
Verfaillie CM, Catanzarro PM, Li WN: Macrophage inflammatory protein 1 alpha, interleukin-3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro. J Exp Med 179:643, 1994.

205.
Garland JM, Rudin CE: Introduction to the hematopoietic system, in Colony-Stimulating Factors: Molecular and Cellular Biology, edited by JM Garland, PJ Quesenberry, DJ Hilton, pp 1-33. Marcel Dekker, New York, 1997.

206.
Crocker PR, Morris L, Gordon S: Novel cell surface adhesion receptors involved in interactions between stromal macrophages and haematopoietic cells. J Cell Sci 9(suppl):185, 1988.

207.
Wang QR, Wolf NS: Dissecting the hematopoietic microenvironment: VIII. Clonal isolation and identification of cell types in murine CFU-F colonies by limiting dilution. Exp Hematol 18:355, 1990.

208.
Kincade PW: Cell interaction molecules and cytokines which participate in B lymphopoiesis. Baillieres Clin Haematol 5:575, 1992.

209.
Berneman ZN, Chen ZZ, van Bockstaele D, et al: The nature of the adherent hemopoietic cells in human long-term bone marrow cultures (HLTBMCs): presence of lymphocytes and plasma cells next to the myelomonocytic population. Leukemia 9:648, 1989.

210.
Tong J, Kishi H, Matsuda T, Muraguchi A: A bone marrow-derived stroma line, ST2, can support the differentiation of fetal thymocytes from CD4+ CD8+ double negative to the CD4+ CD8+ double positive differentiation stage in vitro. Immunology 97:672, 1999.

211.
Dejbakhsh-Jones S, Strober S: Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow. Proc Natl Acad Sci USA 96:14493, 1999.

212.
Kurosaka D, LeBien TW, Priby JAR: Comparative studies of different stromal cell microenvironments in support of human B-cell development. Exp Hematol 27:1271, 1999.

213.
Funk PE, Stephan RP, Witte PL: Vascular adhesion molecule-1-positive reticular cells express interleukin-7 and stem cell factor in the bone marrow. Blood 86:2661, 1995.

214.
Tang J, Nuccie BL, Ritterman I, et al: TGF-beta down-regulates stromal IL-7 secretion and inhibits proliferation of human B cell precursors. J Immunol 159:117, 1997.

215.
Tsuji JM, Pollack SB: Maturation of murine natural killer precursor cells in the absence of exogenous cytokines requires contact with bone marrow stroma. Nat Immun 14:44, 1995.

216.
Yu H, Fehniger TA, Fuschsuber P, Thiel KS, et al: Flt3 ligand promotes the generation of a distinct CD34(+) human natural killer cell progenitor that responds to interleukin-15. Blood 92:3647, 1998.

217.
Abboud SL: A bone marrow stromal cell line is a source and target for platelet-derived growth factor. Blood 81:2547, 1993.

218.
Abboud SL, Pinzani M: Peptide growth factors stimulate macrophage colony-stimulating factor in murine stromal cells. Blood 78:103, 1991.

219.
Yan XQ, Brady G, Iscove NN: Platelet-derived growth factor (PDGF) activates primitive hematopoietic precursors (pre-CFCmulti) by upregulating IL-1 in PDGF receptor-expressing macrophages. J Immunol 150:2440, 1993.

220.
Lerat H, Lissitzky JC, Singer JW, et al: Role of stromal cells and macrophages in fibronectin biosynthesis and matrix assembly in human long-term marrow cultures. Blood 82:1480, 1993.

221.
Baldus SE, Wickenhauser C, Stefanovic A, et al: Enrichment of human bone marrow mononuclear phagocytes and characterization of macrophage subpopulations by immunoenzymatic double staining. Histochem J 30:285, 1998.

222.
Wijffels JF, de Rover Z, Kraal G, Beelen RH: Macrophage phenotype regulation by colony-stimulating factors at bone marrow level. J Leukoc Biol 53:249, 1993.

223.
Shima M, Teitelbaum SL, Holers VM, et al: Macrophage-colony-stimulating factor regulates expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 by murine marrow macrophages. Proc Natl Acad Sci USA 92:5179, 1995.

224.
Dannaeus K, Johannisson A, Nilsson K, Jonsson JI: Flt3 ligand induces the outgrowth of Mac-1+ B22+ mouse bone marrow progenitor cells restricted to macrophage differentiation that coexpress early B cell-associated genes. Exp Hematol 27:1646, 1999.

225.
Munday J, Floyd H, Criker PR: Sialic acid binding receptors (siglecs) expressed by macrophages. J Leukoc Biol 66:705, 1999.

226.
Sadahira Y, Mori M: Role of the macrophage in erythropoiesis. Pathol Int 49:841, 1999.

227.
Klein G: The extracellular matrix of the hematopoietic microenvironment. Experimentia 51:914, 1995.

228.
Singer JW, Keating A, Wright TN: The human haemopoietic microenvironment, in Recent Advances in Haematology, edited by AV Hoffbrand, pp 1–24. Churchill Livingstone, London, 1985.

229.
Bentley SA, Tralka TS: Fibronectin-mediated attachment of hematopoietic cells to stromal elements in continuous bone marrow culture. Exp Hematol 11:129, 1983.

230.
Postlethwaite A, Kang AH: Fibroblasts and matrix proteins, in Inflammation Basic Principles and Clinical Correlates, 3rd ed, edited by JI Gallin, R Snyderman, pp 227–263. Lippincott Williams and Wilkins, Philadelphia, 1999.

231.
Campbell AD, Long MW, Wicha MS: Haemonectin: a bone marrow adhesion protein specific for cells of granulocytic lineage. Nature 329:445, 1987.

232.
Lawler J: The structural and functional properties of thrombospondin. Blood 67: 1197, 1986.

233.
Simmons PJ, Levesque JP, Zannettino AC: Adhesion molecules in haemopoiesis. Baillieres Clin Haematol 10:485, 1997.

234.
Verfaille CM: Adhesion receptors as regulators of the hematopoietic process. Blood 92:2609, 1998.

235.
Broxmeyer HE, Kim CH: Regulation of hematopoiesis in a sea of chemokine family members with a plethora of redundant activities. Exp Hematol 27:1113, 1999.

236.
Gordon MY: Extracellular matrix- and membrane-bound cytokines, in Colony-Stimulating Factors: Molecular and Cellular Biology, edited by JM Garland, PJ Quesenberry, DJ Hilton, pp 133–144. Marcel Dekker, New York, 1997.

237.
Long MW: Hematopoietic microenvironments, in Colony-Stimulating Factors: Molecular and Cellular Biology, edited by JM Garland, PJ Quesenberry, DJ Hilton, pp 117–132. Marcel Dekker, New York, 1997.

238.
Oritani K, Kanakura Y, Aoyama K, et al: Matrix glycoprotein SC1/ECM2 augments B lymphopoiesis. Blood 90:3404, 1997.

239.
Koller MR, Oxender M, Jensen TC, et al: Direct contact between CD34+ lin- cells and stroma induces a soluble activity that specifically increases primitive hematopoietic cell production. Exp Hematol 27:734, 1999.

240.
Varnum-Finney B, Purton LE, Yu M, et al: The Notch ligand, Jagged-1, influences the development of primitive hematopoietic precursor cells. Blood 91:4084, 1998.

241.
Hoogewerf AJ, Kuschert GS, Proudfoot AE, et al: Glycosaminoglycans mediate cell surface oligomerization of chemokines. Biochemistry 36:13570, 1997.

242.
Luster AD, Greenberg SM, Leder P: The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. J Exp Med 182: 219, 1995.

243.
Tanaka T, Adams DH, Hubscher S, et al: T-cell adhesion induced by proteoglycan immobilized cytokine MIP-1b. Nature 361:78, 1993.

244.
Chakravarty L, Rogers L, Quach T, et al: Lysine 58 and histidine 66 at the C-terminal alpha-helix of monocyte chemoattractant protein-1 are essential for glycosaminoglycan binding. J Biol Chem 273:29641, 1998.

245.
Spillman D, Witt D, Lindahl U: Defining the interleukin-8-binding domain of heparan sulfate. J Biol Chem 273:15487, 1998.

246.
Koopman W, Ediriwickrema C, Krangel MS: Structure and function of the glycosaminoglycan binding site of chemokine macrophage-inflammatory protein-1 beta. J Immunol 163:2120, 1999.

247.
Amara A, Lorthioir O, Valenzuela A, et al: Stromal cell derived factor-1 alpha associates with heparan sulfates through the first beta-strand of the chemokine. J Cell Biol Chem 274:23916, 1999.

248.
Wolff EA, Greenfield B, Taub DD, et al: Generation of artificial proteoglycans containing glycosaminoglycan-modified CD44. Demonstration of the interaction between rantes and chondroitin sulfate. J Biol Chem 274:2518, 1999.

249.
Lipscombe RJ, Nakhoul AM, Sanderson CJ, Coombe DR: Interleukin-5 binds to heparin/heparan sulfate. A model for an interaction with extracellular matrix. J Leukoc Biol 63:342, 1998.

250.
Borghesi LA, Yamashita Y, Kincade PW: Heparan sulfate proteoglycans mediate interleukin-7-dependent B lymphopoiesis. Blood 93:140, 1999.

251.
Lyon M, Deakin JA, Nakamura T, Gallagher JT: Interaction of hepatocyte growth factor with heparan sulfate. Elucidation of major heparan sulfate structural determinants. J Biol Chem 269:11216, 1994.

252.
Kiefer MC, Stephans JC, Crawford K, et al: Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor. Proc Natl Acad Sci USA 87:6985, 1990.

253.
Robledo MM, Ursa MA, Sanchez-Madrid F, Teixido J: Associations between TGF-beta1 receptors in human bone marrow stromal cells. Br J Haematol 102:804, 1998.

254.
Kapur R, Cooper R, Xiao X, et al: The presence of novel amino acids in the cytoplasmic domain of stem cell factor results in hematopoietic defects in the Steel17H mice. Blood 94:1915, 1999

255.
Gay RE, Prince CW, Zuckerman KS, Gay S: The collagenous hemopoietic microenvironment, in Handbook of the Hemopoietic Microenvironment, edited by M Tavassoli, pp 369–398. Humana Press, Clifton, NJ, 1989.

256.
Anklesaria P, Greenberger JS, Fitzgerald TJ, et al: Hemonectin mediates adhesion of engrafted murine progenitors to a clonal bone marrow stromal cell line from Sl/Sld mice. Blood 77:1691, 1991.

257.
De Wynter E, Allen T, Coutinho L, et al: Localization of granulocytic macrophage colony-stimulating factor in human long-term bone marrow cultures. Biological and immunocytochemical characterization. J Cell Sci 106:761, 1993.

258.
Deschaseaux ML, Herve P, Charbord P: The detection of colony-stimulating factors and steel factor in adherent layers of human long-term marrow cultures using reverse-transcriptase polymerase chain reaction. Leukemia 8:513, 1994.

259.
Liu J, de Wynter E, Testa NG, et al: Immunoelectron microscopic localization of growth factors and other markers of human long-term bone marrow cultures. Chin Med Sci J 11:129, 1996.

260.
Waegell WO, Higley HR, Kincade PW, Dasch JR: Growth acceleration and stem cell expansion in Dexter-type cultures by neutralization of TGF-beta. Exp Hematol 22:1051, 1994.

261.
Wight TN, Kinsella MG, Keating A, Singer JW: Proteoglycans in human long-term bone marrow cultures: Biochemical and ultrastructural analyses. Blood 67:1333, 1986.

262.
Allen TD, Dexter TM, Simmons PJ: Marrow biology and stem cells, in Colony Stimulating Factors, Molecular and Cellular Biology, edited by TM Dexter, JM Garland, NG Testa, Immunology Series, vol 49, pp 1–38. Marcel Dekker, New York, 1990.

263.
Yurchenco PD, Schittny JC: Molecular architecture of basement membranes. FASEB J 4:1577, 1990.

264.
Keating A, Gordon MY: Hierarchical organization of hematopoietic microenvironments: role of proteoglycans. Leukemia 2:766, 1988.

265.
Gordon MY, Riley GP, Clarke D: Heparan sulfate is necessary for adhesive interactions between human early hemopoietic progenitor cells and the extracellular matrix of the marrow microenvironment. Leukemia 2:804, 1988.

266.
Uhlman DL, Luikart SD: The role of proteoglycans in the adhesion and differentiation of hematopoietic cells, in The Hematopoietic Microenvironment, edited by MW Long, MS Wicha, pp 232–245. Johns Hopkins University Press, Baltimore and London, 1993.

267.
Bruno E, Luikart SD, Long MW, Hoffman R: Marrow-derived heparan sulfate proteoglycan mediates the adhesion of hematopoietic progenitor cells to cytokines. Exp Hematol 23:1212, 1995.

268.
Minguell JJ, Hardy C, Tavassoli M: Membrane-associated chondroitin sulfate proteoglycan and fibronectin mediate the binding of hemopoietic progenitor cells to stromal cells. Exp Cell Res 201:200, 1992.

269.
Han ZC, Bellucci S, Shen ZX, et al: Glycosaminoglycans enhance megakaryopoiesis by modifying the activities of hematopoietic growth regulators. J Cell Physiol 168:97, 1996.

270.
Gordon MY, Lewis Jl, Marley SB, et al: Stromal cells negatively regulate primitive haematopoietic progenitor cell activation via a phosphatidylinositol-anchored cell adhesion/signalling mechanism. Br J Haematol 96:647, 1997.

271.
Gupta P, Oegema TR Jr, Brazil JJ, et al: Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche. Blood 92:4641,1998.

272.
De Prato, Valentini P, Testi R, et al: Differential activity of glycosaminoglycans on colony-forming cells from cord blood. Preliminary results. Leuk Res 23:1015, 1999.

273.
Lewinsohn DM, Nagler A, Ginzton N, et al: Hematopoietic progenitor cell expression of the H-CAM (CD44) homing-associated adhesion molecule. Blood 75:589, 1990.

274.
Jalkanen S, Jalkanen M: Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin. J Cell Biol 116:817, 1992.

275.
Miyake K, Medina KL, Mayashi S-I, et al: Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures. J Exp Med 171:477, 1990.

276.
Legras S, Levesque JP, Charrad R, et al: CD44-mediated adhesiveness of human hematopoietic progenitors to hyaluronan is modulated by cytokines. Blood 89:1905, 1997.

277.
Rachmilewitz J, Tykocinski ML: Differential effects of chondroitin sulfates A and B on monocyte and B cell activation: evidence for B-cell activation via a CD44-dependent pathway. Blood 92:223, 1998.

278.
Khaldoyanidi S, Moll J, Karakhanova S, et al: Hyaluronate-enhanced hematopoiesis: two different receptors trigger the release of interleukin-1b and interleukin-6 from bone marrow macrophages. Blood 94:940, 1999.

279.
Weimar IS, Miranda N, Muller EJ, et al: Hepatocyte growth factor/scatter factor (HGF/SF) is produced by bone marrow stromal cells and promotes proliferation, adhesion and survival of human hematopoietic progenitor cells (CD34+). Exp Hematol 26:885, 1998.

280.
Pivak-Kroizman T, Lemmon MA, Dikic I, et al: Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell 79:1015, 1994.

281.
Ratajczak MZ, Ratajczak J, Slorska M, et al: Effect of basic (FGF-2) and acidic (FGF-1) fibroblast growth factors on early haematopoietic cell development. Br J Haematol 93:772, 1996.

282.
Sternberg D, Peled A, Shezen E, et al: Control of stroma-dependent hematopoiesis by basic fibroblast growth factor: stromal phenotypic plasticity and modified myelopoietic functions. Cytokines Mol Ther 2:29, 1996.

283.
Schofield KP, Gallagher JT, David G: Expression of proteoglycan core proteins in human bone marrow stroma. Biochem J 343:663, 1999.

284.
Klein G, Conzelmann S, Beck S, et al: Perlecan in human bone marrow: a growth-factor-presenting, but anti-adhesive, extracellular matrix component for hematopoietic cells. Matrix Biol 14:457, 1995.

285.
Drzeniek Z, Stoocker G, Siebertz B, et al: Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of multipotential hematopoietic stem cells. Blood 93:2884, 1999.

286.
Siebertz B, Stocker G, Drzeniek Z, et al: Expression of glypican-4 in haematopoietic-progenitor and bone-marrow-stromal cells. Biochem J 344:937, 1999.

287.
Sneed TB, Stanley DJ, Young LA, Sanderson RD: Interleukin-6 regulates expression of the syndecan-1 proteoglycan on B lymphoid cells. Cell Immunol 153:456, 1994.

288.
Oritani K, Kincade PW: Identification of stromal cell products that interact with pre-B cells. J Cell Biol 134:771, 1996.

289.
Yamashita Y, Oritani K, Miyoshi EK, et al: Syndecan-4 is expressed by B lineage lymphocytes and can transmit a signal for formation of dendritic processes. J Immunol 162:5940, 1999.

290.
Longley RL, Woods A, Fleetwood A, et al: Control of morphology, cytoskeleton and migration by syndecan-4. J Cell Sci 112:3421, 1999.

291.
Zukerman KS, Wicha MS: Extracellular matrix production by the adherent cells of long-term murine bone marrow cultures. Blood 61:540, 1983.

292.
Sorrel JM: Ultrastructural localization of fibronectin in bone marrow of the embryonic chick and its relationship to granulopoiesis. Cell Tissue Res 252:565, 1988.

293.
Tsai S, Patel V, Beaumont E, et al: Differential binding of erythroid and myeloid progenitors to fibroblasts and fibronectin. Blood 69:1587, 1987.

294.
Vuillet-Gaugler MH, Breton-Gorius J, Vainchenker W, et al: Loss of attachment to fibronectin with terminal human erythroid differentiation. Blood 75:865, 1990.

295.
Rosemblatt M, Vuillet-Gaugler MH, Leroy C, Coulombel L: Coexpression of two fibronectin receptors, VLA-4 and VLA-5, by immature human erythroblastic precursor cells. J Clin Invest 87:6, 1991.

296.
Liesveld JL, Winslow J, Kempski MC, et al: Adhesive interactions of normal and leukemic human CD34+ myeloid progenitors: Role of marrow stroma, fibroblasts and cytomatrix components. Exp Hematol 19:63, 1991.

297.
Kerst JM, Sanders JB, Slaper Cortenbach IC, et al: Alpha 4 beta 1 and alpha 5 beta 1 are differentially expressed during myelopoiesis and mediate the adherence of human CD34+ cells to fibronectin in an activation-dependent way. Blood 81:344, 1993.

298.
Ryan DH, Nuccie BL, Abboud CN, Winslow JM: Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells. J Clin Invest 88:995, 1991.

299.
Hynes RO: Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11, 1992.

300.
Williams DA, Rios M, Stephens C, Patel VP: Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 352:438, 1991.

301.
Schofield KP, Humphries MJ: Identification of fibronectin IIICS variants in human bone marrow stroma. Blood 93:410, 1999.

302.
Verfaillie CM, Benis A, Iida J, et al: Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin: cooperation between the integrin alpha 4 beta 1 and the CD44 adhesion receptor. Blood 84:1802, 1994.

303.
Hassan HT, Sadovinkova E Yu, Drize NJ, et al: Fibronectin increases both non-adherent cells and CFU-GM while collagen increases adherent cells in human normal long-term bone marrow cultures. Haematologica (Budapest) 28:77, 1997.

304.
Yokota T, Oritani K, Mitsui H, et al: Growth-supporting activities of fibronectin on hematopoietic stem/progenitor cells in vitro and in vivo: structural requirements for fibronectin activities of CS1 and cell-binding domains. Blood 91:3263, 1998.

305.
Hurley RW, McCarthy JB, Verfaillie CM: Direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation. J Clin Invest 96:511, 1995.

306.
Goltry KL, Patel VP: Specific domains of fibronectin mediate adhesion and migration of early murine erythroid progenitors. Blood 90:138, 1997.

307.
Van der Loo JC, Xiao X, McMillin D, et al: VLA-5 is expressed by mouse and human long-term repopulating hematopoietic cells and mediates adhesion to extracellular matrix protein fibronectin. J Clin Invest 102:1051, 1998.

308.
Robledo MM, Sanz-Rodrigues F, Hidalgo A, Teixido J: Differential use of very late antigen-4 and -5 integrins by hematopoietic precursors and myeloma cells to adhere to transforming growth factor-beta-1-treated bone marrow stroma. J Biol Chem 273:12056, 1998.

309.
Schofield KP, Rushton G, Humphries MJ, et al: Influence of interleukin-3 and other growth factors on alpha4beta1 integrin-mediated adhesion and migration of human hematopoietic progenitor cells. Blood 90:1858, 1997.

310.
Levesque JP, Haylock DN, Simmons PJ: Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors. Blood 88:1168, 1996.

311.
Cui L, Ramsfjell V, Borge OJ, et al: Thrombopoietin promotes adhesion of primitive human hemopoietic cells to fibronectin and vascular cell adhesion molecule-1: role of activation of very late antigen (VLA)-4 and VLA-5. J Immunol 159:1961, 1997.

312.
Schofield KP, Humphries MJ, de Wynter E, et al: The effect of a4b1-integrin binding sequences of fibronectin on growth of cells from human hematopoietic progenitors. Blood 91:3230, 1998.

313.
Staquet MJ, Jacquet C, Dezutter-Dambuyant C, Schmitt D: Fibronectin upregulates in vitro generation of dendritic Langerhans cells from human cord blood CD34+ progenitors. J Invest Dermatol 109:738, 1997.

314.
Berthier R, Jacquier-Sarlin M, Schweitzer A, et al: Adhesion of mature polypoid megakaryocytes to fibronectin is mediated by beta 1 integrins and leads to cell damage. Exp Cell Res 242:315, 1998.

315.
Schick PK, Wojenski CM, He X, et al: Integrins involved in the adhesion of megakaryocytes to fibronectin and fibrinogen. Blood 92:2650, 1998.

316.
Krugger-Krasagakes S, Grutzkau A, Krasagakis K, et al: Adhesion of human mast cells to extracellular matrix provides a co-stimulatory signal for cytokine production. Immunology 98:253,1999.

317.
Lloyd AR, Oppenheim JJ, Kelvin DJ, Taub DD: Chemokines regulate T cell adherence to recombinant adhesion molecules and extracellular matrix proteins. J Immunol 156:932, 1996.

318.
Higashimoto I, Chihara J, Kawabata M, et al: Adhesion to fibronectin regulates expression of intercellular adhesion molecule-1 on eosinophilic cells. Int Arch Allergy Immunol 120(suppl 1):34, 1999.

319.
Xu X, Hakansson L: Simultaneous analysis of eosinophil and neutrophil adhesion to plasma and tissue fibronectin, fibrinogen, and albumin. J Immunol Methods 226:93, 1999.

320.
Xie B, Laouar A, Huberman E: Fibronectin-mediated cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation. The signaling role of protein kinase C-beta. J Biol Chem 273:11576, 1998.

321.
Kremlev SG, Chapoval AI, Evans R: Cytokine release by macrophages after interacting with CSF-1 and extracellular matrix proteins: characteristics of a mouse model of inflammatory responses in vitro. Cell Immunol 185:59, 1998.

322.
Yonezawa I, Kato K, Yagita H, et al: VLA-5-mediated interactions with fibronectin induces cytokine production by human chondrocytes. Biochem Biophys Res Commun 219:261, 1996.

323.
Rich IN, Brackmann I, Worthington-White D, Dewey MJ: Activation of sodium/hydrogen exchanger via the fibronectin-integrin pathway results in hematopoietic stimulation. J Cell Physiol 177:109, 1998.

324.
Klein G, Beck S, Muller CA: Tenascin is a cytoadhesive extracellular matrix component of the human hematopoietic microenvironment. J Cell Biol 123:1027, 1993.

325.
Chiquet-Ehrismann R, Matsuoka Y, Hofer U, et al: Tenascin variants: differential binding to fibronectin and distinct distribution in cell cultures and tissues. Cell Regul 2:927, 1991.

326.
Sakai T, Ohta M, Kawakatsu H, et al: Tenascin-C induction in Whitlock-Witte culture: a relevant role of the thiol moiety in lymphoid-lineage differentiation. Exp Cell Res 217:395, 1995.

327.
Ekblom M, Fassler R, Tomasini-Johansson B, et al: Downregulation of tenascin expression by glucocorticoids in bone marrow stromal cells and in fibroblasts. J Cell Biol 123:1037, 1993.

328.
Seiffert M, Beck SC, Schermutzki F, et al: Mitogenic and adhesive effects of tenascin-C on human hematopoietic cells are mediated by various functional domains. Matrix Biol 17:47, 1998.

329.
Ohta M, Sakai T, Saga Y, et al: Suppression of hematopoietic activity in tenascin-C-deficient mice. Blood 91:4074, 1998.

330.
Mackie EJ, Tucker RP: The tenascin-C knockout revisited. J Cell Sci 112:3847, 1999.

331.
Bentley SA: Collagen synthesis by bone marrow stromal cells: a quantitative study. Br J Haematol 50:491, 1982.

332.
Mori M, Sadahira Y, Kawasaki S, et al: Formation of capillary networks from bone marrow cultured in collagen gel. Cell Struct Funct 14:393, 1989.

333.
Zukerman KS, Rhodes RK, Goodrum DD, et al: Inhibition of collagen deposition in the extracellular matrix prevents the establishment of a stroma supportive of hematopoiesis in long-term murine bone marrow cultures. J Clin Invest 75:970, 1985.

334.
Zukerman KS, Prince CW, Gay S: The hemopoietic extracellular matrix, in Handbook of the Hemopoietic Microenvironment, edited by M Tavassoli, pp 399–432. Humana Press, Clifton, NJ, 1989.

335.
Koenigsmann M, Griffin JD, DiCarlo J, Cannistra SA: Myeloid and erythroid progenitor cells from normal bone marrow adhere to collagen type I. Blood 79:657, 1992.

336.
Waterhouse EJ, Quesenberry PJ, Balian G: Collagen synthesis by murine bone marrow cell culture. J Cell Physiol 127:397, 1987.

337.
Charbord P, Tamayo E, Saeland S, et al: Granulocyte-macrophage colony-stimulating factor (GM-CSF) in human long-term bone marrow cultures: Endogenous production in the adherent layer and effect on exogenous GM-CSF on granulomonopoiesis. Blood 78:1230, 1991.

338.
Chichester CO, Fernández M, Minguel JJ: Extracellular matrix gene expression by human bone marrow stroma and by marrow fibroblasts. Cell Adhesion Commun 1:93, 1993.

339.
Klein G, Muller CA, Tillet E, et al: Collagen type VI in the human bone marrow microenvironment: a strong cytoadhesive component. Blood 86:1740, 1995.

340.
Klein G, Kibler C, Schermutzki F, et al: Cell binding properties of collagen type XIV for human hematopoietic cells. Matrix Biol 16:307, 1998.

341.
Briddon SJ, Melford SK, Turner M, et al: Collagen mediates changes in intracellular calcium in primary mouse megakaryocytes through syk-dependent and -independent pathways. Blood 93: 3847, 1999.

342.
Nilsson SK, Debatis ME, Dooner MS, et al: Imunofluorescence characterization of key extracellular matrix proteins in murine bone marrow in situ. J Histochem Cytochem 46:371, 1998.

343.
Kleinman HK, Weeks BS: Laminin: structure, function and receptors. Curr Opin Cell Biol 1:964, 1989.

344.
Senior RM, Gresham HD, Griffin GL, et al: Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin. J Clin Invest 90:2251, 1992.

345.
Bryant G, Rao CN, Brentani M, et al: A role for the laminin receptor in leukocyte chemotaxis. J Leukocyte Biol 41:220, 1987.

346.
Lundgren-Akerlund E, Olofsson AM, Berger E, Arfors KE: CD11b/CD18-dependent polymorphonuclear leucocyte interaction with matrix proteins in adhesion and migration. Scand J Immunol 37:569, 1993.

347.
Liesveld JL, Ryan DH, Kempski MC, et al: Quantitation of the binding of human CD34 positive myeloid progenitors to marrow stroma fibroblasts, and components of the extracellular matrix, in Hematopoiesis, edited by SC Clark, DW Golde, UCLA Symposia on Molecular and Cellular Biology New Series, pp 157–169. Wiley-Liss, New York, 1990.

348.
Tobias JW, Bern MM, Netland PA, Zetter BR: Monocyte adhesion to subendothelial components. Blood 69:1265, 1987.

349.
Bohnsack JF, Akiyama SK, Damsky CH, et al: Human neutrophil adherence to laminin in vitro: evidence for a distinct neutrophil integrin receptor for laminin. J Exp Med 171:1221, 1990.

350.
Bohnsack JF: CD11/CD18-independent neutrophil adherence to laminin is mediated by the integrin VLA-6. Blood 79:1545, 1992.

351.
Thompson HL, Matsushima K: Human polymorphonuclear leucocytes stimulated by tumour necrosis factor-alpha show increased adherence to extracellular matrix proteins which is mediated via the CD11b/18 complex. Clin Exp Immunol 90:280, 1992.

352.
Fehlner-Gardiner C, Uniyal S, von Ballestrem C, et al: Integrin VLA-6 (alpha 6 beta 1) mediates adhesion of mouse bone marrow-derived mast cells to laminin. Allergy 51:650, 1996.

353.
El-Nemer W, Gane P, Colin Y, et al: The Lutheran blood group glycoproteins, the erythroid receptors for laminin, are adhesion molecules. J Biol Chem 273:16686, 1998.

354.
Monturi N, Selleri C, Risitano AM, et al: Expression of the 67-kDa laminin receptor in acute myeloid leukemia cells mediates adhesion to laminin and is frequently associated with monocytic differentiation. Clin Cancer Res 5:1465, 1999.

355.
Gu Y, Sorokin L, Durbeej M, et al: Characterization of bone marrow laminins and identification of a5-containing laminins as adhesive proteins for multipotent hematopoietic FDCP-mix cells. Blood 93:2533, 1999.

356.
Vogel W, Kanz L, Brugger W, et al: Expression of laminin b2 chain in normal human bone marrow. Blood 94:1143, 1999.

357.
Ohki K, Kohashi O: Laminin promotes proliferation of bone marrow-derived macrophages and macrophage cell lines. Cell Struct Funct 19:63, 1994.

358.
Peters C, OShea KS, Campbell AD, et al: Fetal expression of hemonectin: An extracellular matrix hematopoietic cytoadhesion molecule. Blood 75:357, 1990.

359.
White H, Totty N, Panayotou G: Haemonectin, a granulocytic-cell-binding protein, is related to the plasma glycoprotein fetuin. Eur J Biochem 213:523, 1993.

360.
Sullenbarger BA, Petitt MS, Chong P, et al: Murine granulocytic cell adhesion to bone marrow hemonectin is mediated by mannose and galactose. Blood 86:135, 1995.

361.
Long MW, Dixit VM: Thrombospondin functions as a cytoadhesion molecule for human hematopoietic progenitor cells. Blood 75:2311, 1990.

362.
Stomski FC, Gani JS, Bates RC, Burns GF: Adhesion to thrombospondin by human embryonic fibroblasts is mediated by multiple receptors and includes a role for glycoprotein 88 (CD36). Exp Cell Res 198:85, 1992.

363.
Li WX, Howard RJ, Leung LL: Identification of SVTCG in thrombospondin as the conformation-dependent, high affinity binding site for its receptor, CD36. J Biol Chem 268:16179, 1993.

364.
Suchard SJ, Burton MJ, Dixit VM, Boxer LA: Human neutrophil adherence to thrombospondin occurs through a CD11/CD18-independent mechanism. J Immunol 146:3945, 1991.

365.
Calvo D, Vega MA: Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family. J Biol Chem 268:18929, 1993.

366.
Vischer P, Feitsma K, Schon P, Volker W: Perlecan is responsible for thrombospondin 1 binding on the surface of cultured porcine endothelial cells. Eur J Cell Biol 73:332, 1997.

367.
Nakahata T, Okumura N: Cell surface antigen expression in human erythroid progenitors: erythroid and megakaryocytic markers. Leuk Lymphoma 13:401, 1994.

368.
Chen YZ, Incardona F, Legrand C, et al: Thrombospondin, a negative modulator of megakaryopoiesis. J Lab Clin Med 129:231, 1997.

369.
Dallalio G, van Laer A, Means RT: Effects of thrombospondin and CD36 on erythroid colony formation in vitro. Blood 94(suppl 1, part 2):163b, 1999.

370.
Pierson BA, Gupta K, Hu WS, Miller JS: Human natural killer cell expansion is regulated by thrombospondin-mediated activation of transforming growth factor-beta 1 and independent accessory cell-derived contact and soluble factors. Blood 87:180, 1996.

371.
Crawford SE, Stellmach V, Murphy-Ullrich JE, et al: Thrombospondin-1 is a major activator of TGF-beta 1 in vivo. Cell 93:1159, 1998.

372.
Touhami M, Fauvel-Lafeve F, Da Silva N, et al: Induction of thrombospondin-1 by all-trans retinoic acid modulates growth and differentiation of HL-60 myeloid leukemia cells. Leukemia 11:2137, 1997.

373.
Taraboletti G, Belotti D, Borsotti P, et al: The 140-kilodalton antiangiogenic fragment of thrombospondin-1 binds to basic fibroblast growth factor. Cell Growth Differ 8:471, 1997.

374.
Loganadane LD, Berge N, Legrand C, Fauvel-Lafeve F: Endothelial cell proliferation regulated by cytokines modulates thrombospondin-1 secretion into the subendothelium. Cytokine 9:740, 1997.

375.
Qian X, Wang TN, Rothman VL, et al: Thrombospondin-1 modulates angiogenesis in vitro by up-regulation of matrix metalloproteinase-9 in endothelial cells. Exp Cell Res 235:403, 1997.

376.
Mansfield PJ, Suchard SJ: Thrombospondin promotes both chemotaxis and haptotaxis of human peripheral blood monocytes. J Immunol 153:4219, 1994.

377.
Mansfield PJ, Suchard SJ: Thrombospondin promotes both chemotaxis and haptotaxis in neutrophil-like HL-60 cells. J Immunol 150:1959, 1993.

378.
Horton MA: The alphavbeta3 integrin “vitronectin receptor.” Int J Biochem Cell Biol 29:721, 1997.

379.
Molla A, Mossuz P, Berthier R: Extracellular matrix receptors and the differentiation of human megakaryocytes in vitro. Leuk Lymphoma 33:15, 1999.

380.
Shimizu Y, Irani AM, Brown EJ, et al: Human mast cells derived from fetal liver cells cultured with stem cell factor express a functional CD51/CD61 (alphavbeta3) integrin. Blood 86:930, 1995.

381.
Hughes DE, Salter DM, Dedhar S, Simpson R: Integrin expression in human bone. J Bone Miner Res 8:527, 1993.

382.
Mbalaviele G, Jaiswal N, Meng A, et al: Human mesenchymal stem cells promote human osteoclast differentiation from CD34+ bone marrow hematopoietic progenitors. Endocrinology 140:3736, 1999.

383.
Boissy P, Machuca I, Pfaff M, et al: Aggregation of mononucleated precursors triggers cell surface expression of alphavbeta3 integrin, essential to formation of osteoclast-like multinucleated cells. J Cell Sci 111:2563, 1998.

384.
Murphy JF, Bordet JC, Wyler B, et al: The vitronectin receptor (alphavbeta3) is implicated, in cooperation with P-selectin and platelet-activating factor, in the adhesion of monocytes to activated endothelial cells. Biochem J 304:537, 1994.

385.
Weerasinghe D, McHugh KP, Ross FP, et al: A role for the alphavbeta3 integrin in the transmigration of monocytes. J Cell Biol 142:595, 1998.

386.
Rainger GE, Buckley CD, Simmons DL, Nash GB: Neutrophils sense flow-generated stress and direct their migration through alphaVbeta3-integrin. Am J Physiol 276:H858, 1999.

387.
Nath D, Slocombe PM, Stephens PE, et al: Interactions of metargidin (ADAM-15) with alphavbeta3 and alpha5beta1 integrins on different haemopoietic cells. J Cell Sci 112:579, 1999.

388.
Savill J, Hogg N, Ren Y, Haslett C: Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J Clin Invest 90:1513, 1992.

389.
Fadok VA, Warner ML, Bratton DL, Henson PM: CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (alphavbeta3). J Immunol 161:6250, 1998.

390.
Rubartelli A, Poggi A, Zocchi MR: The selective engulfment of apoptotic bodies by dendritic cells is mediated by the alpha(v)beta3 integrin and requires intracellular calcium and extracellular calcium. Eur J Immunol 27:1893, 1997.

391.
Leven RM, Tablin F: Extracellular matrix stimulation of guinea pig megakaryocyte proplatelet formation in vitro is mediated through the vitronectin receptor. Exp Hematol 20:1316, 1992.

392.
Hunt P, Hokom MM, Hornkohl A, et al: The effect of platelet-derived glycosaminoglycan serglycin on in vitro proplatelet-like process formation. Exp Hematol 21:1295, 1993.

393.
Leven RM: Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading. J Cell Physiol 163:597, 1995.

394.
Rusnati M, Tanghetti E, Dell’Era P, et al: Alphavbeta3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells. Mol Cell Biol 8:2449, 1997.

395.
Ybarrondo B, O’Rouke AM, McCarthy JB, Mescher MF: Cytotoxic T-lymphocyte interaction with fibronectin and vitronectin: activated adhesion and cosignalling. Immunology 91:186, 1997.

396.
Roberts K, Yokoyama WM, Kehn PJ, Shevach EM: The vitronectin receptor serves as an accessory molecule for the activation of a subset of gamma/delta T cells. J Exp Med 173:231, 1991.

397.
Rabinowich H, Lin WC, Amoscato A, et al: Expression of vitronectin receptor on human NK cells and its role in protein phosphorylation, cytokine production, and cell proliferation. J Immunol 154:1124, 1995.

398.
Hermann P, Armant M, Brown E, et al: The vitronectin receptor and its associated CD47 molecule mediates proinflammatory cytokine synthesis in human monocytes by interactions with soluble CD23. J Cell Biol 144:767, 1999.

399.
Bessis M: L’ilot èrythroblastique, unitè fonctionelle de le moelle osseuse. Rev Hematol 13:8, 1958.

400.
Lichtman MA, Waugh RE: Red cell egress from the marrow: Ultrastructural and biophysical aspects, in Regulation of Erythropoiesis, edited by ED Zanjani, M Tavassoli, J Ascencao, pp 15–35. PMA, Great Neck, NY, 1989.

401.
Zuhrie SR, Wickramasinghe SN: Stromal cell-dependent terminal maturation of K562 erythroleukemia cells. Leuk Res 975, 1991.

402.
Yu AW, Shao LE, Frigon NL Jr, Yu J: Detection of functional and dimeric activin A in human marrow microenvironment. Implications for the modulation of erythropoiesis. Ann N Y Acad Sci 718:285, 1994.

403.
Mizugushi T, Kosaka M, Saito S: Activin A suppresses proliferation of interleukin-3-responsive granulocyte-macrophage colony-forming progenitors and stimulates proliferation and differentiation of interleukin-3-responsive erythroid burst-forming progenitors in peripheral blood. Blood 81:2891, 1993.

404.
Huber TL, Zhou Y, Mead PE, Zon LI: Cooperative effects of growth factors involved in the induction of hematopoietic mesoderm. Blood 92:4128, 1998.

405.
Koristschoner NP, Bartunek P, Knespel S, et al: The fibroblast growth factor receptor FGFR-4 acts as a ligand dependent modulator of erythroid cell proliferation. Oncogene 18:5904,1999.

406.
Iguchi T, Sogo S, Hisha H, et al: HGF activates signal transduction from EPO receptor on human cord blood CD34+/CD45+ cells. Stem Cells 17:82, 1999.

407.
Lichtman MA, Chamberlain JK, Simon W, et al: Parasinusoidal location of megakaryocytes in marrow: a determinant of platelet release. Am J Hematol 4:303, 1978.

408.
Thiele J, Galle R, Sander C, Fischer R: Interactions between megakaryocytes and sinus wall: an ultrastructural study of bone marrow tissue in primary (essential) thrombocythemia. J Submicrosc Cytol Pathol 23:595, 1991.

409.
Avraham H, Cowley S, Chi SY, et al: Characterization of adhesive interactions between human endothelial cells and megakaryocytes. J Clin Invest 91:2378, 1993.

410.
Riviere C, Subra F, Cohen-Solal K, et al: Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryopoiesis. Blood 93:1511, 1999.

411.
Hamada T, Mohle R, Hesselgesser J, et al: Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelet formation. J Exp Med 188:539, 1998.

412.
Ito T, Ishida Y, Kashiwagi R, Kuriya S: Recombinant human c-Mpl ligand is not a direct stimulator of proplatelet formation in mature human megakaryocytes. Br J Haematol 94:387, 1996.

413.
Bruno E, Briddell RA, Cooper RJ, Hoffman R: Effects of recombinant interleukin 11 on human megakaryocyte progenitor cells. Exp Hematol 19:378, 1991.

414.
Gordon MS, Hoffman R: Growth factors affecting human thrombopoiesis: potential agents for the treatment of thrombocythemia. Blood 80:302, 1992.

415.
Ishibashi T, Kimura H, Shikama Y, et al: Interleukin-6 is a potent thrombopoietic factor in vivo in mice. Blood 74:1241, 1989.

416.
Metcalf D, Hilton D, Nicola NA: Leukemia inhibitory factor can potentiate murine megakaryocyte production in vitro. Blood 77:2150, 1991.

417.
Kaushansky K: Thrombopoietin. N Engl J Med 339:746, 1998.

418.
Lambertsen RH, Weiss L: A model of intramedullary hemopoietic microenvironments based on stereologic study of the distribution of endoclonal colonies. Blood 63:287, 1984.

419.
Naito K, Tamahashi N, Chiba T, et al: The microvasculature of the human bone marrow correlated with the distribution of hematopoietic cells: a computer-assisted three-dimensional reconstruction study. Tohoku J Exp Med 166:439, 1992.

420.
Blazsek I, Misset JL, Benavides M, et al: Hematon, a multicellular functional unit in normal human bone marrow: structural organization, hemopoietic activity, and its relationship to myelodysplasia and myeloid leukemias. Exp Hematol 18:259, 1990.

421.
Wright EC, Pragnell IB: Stem cell proliferation inhibitors. Baillieres Clin Hematol 5:723, 1992.

422.
Jacobsen SEW, Ruscetti FW, Dubois CM, Keller JR: Tumor necrosis factor a directly and indirectly regulates hematopoietic progenitor cell proliferation: role of colony-stimulating factor receptor modulation. J Exp Med 175:1759, 1992.

423.
Rogers JA, Berman JW: A tumor necrosis factor-responsive long-term-culture-initiating cell is associated with the stromal layer of mouse long-term bone marrow cultures. Proc Natl Acad Sci USA 90:5777, 1993.

424.
Knospe WH, Husseini SG, Zipori D, Fried W: Hematopoiesis on cellulose ester membranes: XIII. A combination of cloned stromal cells is needed to establish a hematopoietic microenvironment supportive of trilineal hematopoiesis. Exp Hematol 21:257, 1993.

425.
Winerman JP, Nishikawa S, Muller-Sieburg CE: Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: Effect of stromal cells and c-kit. Blood 81:365, 1993.

426.
Bhatia M, Bonnet D, Wu D, et al: Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells. J Exp Med 189:1139, 1999.

427.
Simmons PJ, Zannettino A, Gronthos S, Leavesley D: Potential adhesion mechanisms for localization of haemopoietic progenitors to bone marrow stroma. Leuk Lymphoma 12:353, 1994.

428.
Koller MR, Oxender M, Jensen TC, et al: Direct contact between CD34+ lin- cells and stroma induces a soluble activity that specifically increases primitive hematopoietic cell production. Exp Hematol 27:734, 1999.

429.
Simmons PJ, Haylock DN, Levesque J-P: Influence of cytokines and adhesion molecules on hematopoietic stem cell development, in Ex Vivo Cell Therapy, edited by K Schindhelm, R Nordon, pp 51–83. Academic Press, San Diego, 1999.

430.
Coulombel L, Auffray I, Gaugler MH, Rosemblatt M: Expression and function of integrins on hematopoietic progenitor cells. Acta Haematol 97:13,1997.

431.
Wang J, Springer TA: Structural specializations of immunoglobulin superfamily members for adhesion to integrins and viruses. Immunol Rev 163:197, 1998.

432.
Kansas GS: Selectins and their ligands: current concepts and controversies. Blood 88:3259, 1996.

433.
Robinson LA, Steeber DA, Tedder TA: The selectins in inflammation, in Inflammation Basic Principles and Clinical Correlates, 3rd ed, edited by JI Gallin, R Snyderman, pp 571–583. Lippincott Williams and Wilkins, Philadelphia, 1999.

434.
Lasky LA: Sialomucin ligands for selectins: a new family of cell adhesion molecules. Princess Takamatsu Symp 24:81, 1994.

435.
Butcher EC, Picker LJ: Lymphocyte homing and homeostasis Science 272:60, 1996.

436.
Lesley J, Hyman R, Kincade PW: CD44 and its interaction with extracellular matrix. Adv Immunol 54:271, 1993.

437.
Borland G, Ross JA, Guy K: Forms and functions of CD44. Immunology 93:139, 1998.

438.
Deaglio S, Morra M, Mallone R, et al: Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member. J Immunol 160:395, 1998.

439.
Hynes RO: Specificity of cell adhesion in development: the cadherin superfamily. Curr Opin Genet Dev 2:621, 1992.

440.
Steinberg MS, McNutt PM: Cadherins and their connections: adhesion junctions have broader functions. Curr Opin Cell Biol 11:554, 1999.

441.
Okuyama Y, Ishihara K, Kimura N, et al: Human BST-1 expressed on myeloid cells functions as a receptor molecule. Biochem Biophys Res Commun 228:838, 1996.

442.
Liesveld JL, DiPersio JF, Abboud CN: Integrins and adhesive receptors in normal and leukemic CD34+ progenitor cells: potential regulatory checkpoints for cellular traffic. Leuk Lymphoma 14:19, 1994.

443.
Kishimoto TK, Baldwin ET, Anderson DC: The role of b2 integrins in inflammation, in Inflammation Basic Principles and Clinical Correlates, 3rd ed, edited by JI Gallin, R Snyderman, pp 537–569. Lippincott Williams and Wilkins, Philadelphia, 1999.

444.
Ruoslahti E: Integrins. J Clin Invest 87:1, 1991.

445.
Yanai N, Sekine C, Yagita H, Obinata M: Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis. Blood 83:2844, 1994.

446.
Hamamura K, Matsuda H, Takeuchi Y, et al: A critical role of VLA-4 in erythropoiesis in vivo. Blood 87:2513, 1996.

447.
Furasawa T, Yanai N, Hara T, et al: Integrin-associated protein (IAP, also termed CD47) is involved in stroma-supported erythropoiesis. J Biochem (Tokyo) 123:101, 1998.

448.
Iguchi A, Okuyama R, Koguma M, et al: Selective stimulation of granulopoiesis in vitro by established bone marrow stromal cells. Cell Struct Funct 22:357, 1997.

449.
Dittel BN, McCarthy JB, Wayner EA, LeBien TW: Regulation of human B-cell precursor adhesion to bone marrow stromal cells by cytokines that exert opposing effects on the expression of vascular cell adhesion molecule-1 (VCAM-1). Blood 81:2272, 1993.

450.
Ryan DH, Nuccie BL, Ritterman I, et al: Cytokine regulation of early human lymphopoiesis. J Immunol 152:5250, 1994.

451.
Oostendorp RA, Dormer P: VLA-4-mediated interactions between normal human hematopoietic progenitors and stromal cells. Leuk Lymphoma 24:423, 1997.

452.
Ryan DH, Nuccie BL, Ritterman I, et al: Expression of interleukin-7 receptor by lineage-negative human bone marrow progenitors with enhanced lymphoid proliferative potential and B-lineage differentiation capacity. Blood 89:929, 1997.

453.
Dittel BN, LeBien TW: Reduced expression of vascular cell adhesion molecule-1 on bone marrow stromal cells isolated from marrow transplant recipients correlates with a reduced capacity to support human B lymphopoiesis in vitro. Blood 86:2833, 1995.

454.
Novitzky N, Mohamed R: Alterations in both the hematopoietic microenvironment and the progenitor cell population follow the recovery from myeloablative therapy and bone marrow transplantation. Exp Hematol 23:1661, 1995.

455.
Funk PE, Stephan RP, Witte PL: Vascular cell adhesion molecule 1-positive reticular cells express interleukin-7 and stem cell factor in the bone marrow. Blood 86:2661, 1995.

456.
Funk PE, Kincade PW, Witte PL: Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates. Blood 83:361, 1994.

457.
Galotto M, Berisso G, Delfino L, et al: Stromal damage as a consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients. Exp Hematol 27:1460, 1999.

458.
Dedhar S: Integrins and signal transduction. Curr Opinion Hematol 6:37, 1999.

459.
Lowell CA, Berton G: Integrin signal transduction in myeloid leukocytes. J Leukoc Biol 65:313, 1999.

460.
Aplin AE, Howe A, Alahari SK, Juliano RL: Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules and selectins. Pharmacol Rev 50:197, 1998.

461.
Jarvis LJ, Maguire JE, LeBien TW: Contact between human bone marrow stromal cells and B lymphocytes enhances very late antigen-4/vascular cell adhesion molecule-1-independent tyrosine phosphorylation of focal adhesion kinase, paxillin, and ERK-2 in stromal cells. Blood 90:1626, 1997.

462.
Shibayama H, Anzai N, Braun SE, et al: H-Ras is involved in the inside-out signaling pathway of interleukin-3-induced integrin activation. Blood 93:1540, 1999.

463.
Levesque J-P, Simmons PJ: Cytoskeleton and integrin-mediated adhesion signaling in human CD34+ hemopoietic progenitor cells. Exp Hematol 27:579, 1999.

464.
Arai A, Nosaka Y, Kohsaka H, et al: CrkL activates integrin-mediated hematopoietic cell adhesion through the guanine nucleotide exchange factor C3G. Blood 93:3713, 1999.

465.
Porter JC, Hogg N: Integrin cross talk: activation of lymphocyte function-associated antigen-1 on human T cells alters alpha4beta1- and alpha5beta1-mediated function. J Cell Biol 138:1437, 1997.

466.
Shibuya A, Campbell D, Hannum C, et al: DYNAM-1, a novel adhesion molecule involved involved in the cytolytic function of T lymphocytes. Immunity 4:573, 1996.

467.
Shibuya K, Lanier LL, Phillips JH, et al: Physical and functional association of LFA-1 with DYNAM-1 adhesion molecule. Immunity 11:615, 1999.

468.
Rodriguez-Fernandez JL, Gomez M, Luque A, et al: The interaction of activated integrin lymphocyte function-associated antigen 1 with ligand intercellular adhesion molecule 1 induces activation and redistribution of focal adhesion kinase and proline-rich tyrosine kinase 2 in T lymphocytes. Mol Biol Cell 10:1891, 1999.

469.
Leavesley DI, Oliver JM, Swart BW, et al: Signals from platelet/endothelial cell adhesion molecule enhance the adhesive activity of the very late antigen-4 integrin of human CD34+ hematopoietic progenitor cells. J Immunol 153:4673, 1994.

470.
Vestweber D, Blanks JE: Mechanisms that regulate the function of the selectins and their ligands. Physiol Rev 79:181, 1999.

471.
Gotoh A, Ritchie A, Takahira H, Broxmeyer HE: Thrombopoietin and erythropoietin activate inside-out signaling of integrin and enhance adhesion to immobilized fibronectin in human growth-factor-dependent hematopoietic cells. Ann Hematol 75:207, 1997.

472.
Liesveld JL, Winslow JM, Frediani KE, et al: Expression of integrins and examination of their adhesive function in normal and leukemic hematopoietic cells. Blood 81:112, 1993.

473.
Ryan DH, Nuccie BL, Abboud CN: Inhibition of human bone marrow lymphoid progenitor colonies by antibodies to VLA integrins. J Immunol 149:3759, 1992.

474.
Sugahara H, Kanakura Y, Furitsu T, et al: Induction of programmed cell death in human hematopoietic cell lines by fibronectin via its interaction with very late antigen 5. J Exp Med 179:1757, 1994.

475.
Hurley RW, McCarthy JB, Wayner EA, Verfaillie CM: Monoclonal antibody crosslinking of the alpha 4 beta 1 integrin inhibits committed clonogenic hematopoietic progenitor proliferation. Exp Hematol 25:321, 1997.

476.
Oostendorp RA, Spitzer E, Reisbach G, Dormer P: Antibodies to the beta 1-integrin chain, CD44, or ICAM-3 stimulate adhesion of blast colony-forming cells and may inhibit their growth. Exp Hematol 25:345, 1997.

477.
Wang MW, Consoli U, Lane CM, et al: Rescue from apoptosis in early (CD34-selected) versus late (non-CD34-selected) human hematopoietic cells by very late antigen 4- and vascular cell adhesion molecule (VCAM) 1-dependent adhesion to bone marrow stromal cells. Cell Growth Differ 9:105, 1998.

478.
Bhatia R, Munthe HA, Verfaillie CM: Role of abnormal integrin-cytoskeletal interactions in impaired b1 integrin function in chronic myelogenous leukemia hematopoietic progenitors. Exp Hematol 27:1384, 1999.

479.
Verfaillie CM, Hurley R, Zhao RC, et al: Pathophysiology of CML: do defects in integrin function contribute to the premature circulation and massive expansion of the BCR/ABL positive clone? J Lab Clin Med 129:584,1997.

480.
Bahtia R, Munthe HA, Verfaillie CM: Tyrphostin AG957, a tyrosine kinase inhibitor with anti-BCR/ABL tyrosine kinase activity restores beta1 integrin-mediated adhesion and inhibitory signaling in chronic myelogenous leukemia hematopoietic progenitors. Leukemia 12:1708,1998.

481.
Bahtia R, Verfaillie CM: The effect of interferon-alpha on beta-1 integrin mediated adhesion and growth regulation in chronic myelogenous leukemia. Leuk Lymphoma 28:241, 1998.

482.
Sun QH, Paddock C, Visentin GP, et al: Cell surface glycosaminoglycans do not serve as ligands for PECAM-1. PECAM-1 is not a heparin-binding protein. J Biol Chem 273:11483, 1998.

483.
Muller WA, Randolph GJ: Migration of leukocytes across endothelium and beyond: molecules involved in the transmigration and fate of monocytes. J Leukoc Biol 66:698, 1999.

484.
Chiba R, Nakagawa N, Kurasawa K, et al: Ligation of CD31 (PECAM-1) on endothelial cells increases adhesive function of avb3 integrin and enhances b1 integrin-mediated adhesion of eosinophils to endothelial cells. Blood 94:1319, 1999.

485.
Duncan GS, Andrew DP, Takimoto H, et al: Genetic evidence for functional redundancy of platelet/endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficient mice reveal PECAM-1-dependent and PECAM-1-independent functions. J Immunol 162:3022, 1999.

486.
Nakada MT, Amin K, Christofidou-Solomidou M, et al: Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment. J Immunol 164:452, 2000.

487.
Arkin S, Naprstek B, Guarini L, et al: Expression of intercellular adhesion molecule-1 (CD54) on hematopoietic progenitors. Blood 77:948, 1991.

488.
Gunji Y, Nakamura M, Hagiwara T, et al: Expression and function of adhesion molecules on human hematopoietic stem cells: CD34+ LFA-1(neg) cells are more primitive than CD34+ LFA-1+ cells. Blood 80:429, 1992.

489.
Makgoba MW, Sanders ME, Ginther Luce GE, et al: ICAM-1, a ligand for LFA-1-dependent adhesion of B, T and myeloid cells. Nature 331:86, 1988.

490.
Rao SG, Chitnis VS, Deora A, et al: An ICAM-1-like cell adhesion molecule is responsible for CD34-positive haemopoietic stem cells adhesion to bone-marrow stroma. Cell Biol Int 20:255, 1996.

491.
Staunton DE, Dustin ML, Springer TA: Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1. Nature 339:61, 1989.

492.
Fawcett J, Holness CLL, Needham LA, et al: Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on resting leukocytes. Nature 360:481, 1992.

493.
Campanero MR, Sanchez-Mateos P, del Pozo MA, Sanchez-Madrid F: ICAM-3 regulates lymphocyte morphology and integrin-mediated T cell interactions with endothelial cell and extracellular matrix ligands. J Cell Biol 127:867, 1994.

494.
Wang JH, Smolyar A, Tan K, et al: Structure of a heterophilic adhesion complex between the human CD2 and CD58 (LFA-3) counterreceptors. Cell 97:791, 1999.

495.
Nielsen M, Gerwien J, Geisler C, et al: MHC class II ligation induces CD58 (LFA-3)-mediated adhesion in human T cells. Exp Clin Immunogenet 15:61, 1998.

496.
LeGuiner S, Le Drean E, Labarriere N, et al: LFA-3 co-stimulates cytokine secretion by cytotoxic T lymphocytes by providing a TCR-independent activation signal. Eur J Immunol 28:1322, 1998.

497.
Itzhaky D, Raz N, Hollander N: The glycosylphosphatidylinositol-anchored form and the transmembrane form of CD58 associate with protein kinases. J Immunol 60:4361,1998.

498.
Kirby AC, Cahen P, Porter SR, Olsen I: LFA-3 (CD58) mediates T-lymphocyte adhesion in chronic inflammatory infiltrates. Scand J Immunol 50:469, 1999.

499.
DE Waele M, Renmans W, Jochmans K, et al: Different expression of adhesion molecules on CD34+ cells in AML and B lineage ALL and their normal bone marrow counterparts. Eur J Haematol 63:192, 1999.

500.
McCarty JM, Yee EK, Deisher TA, et al: Interleukin-4 induces endothelial vascular cell adhesion molecule-1 (VCAM-1) by an NF-kappa b-independent mechanism. FEBS Lett 372:194, 1995.

501.
Bochner BS, Klunk DA, Sterbinsky SA, et al: IL-13 selectively induces vascular cell adhesion molecule-1 expression in human endothelial cells. J Immunol 154:799, 1995.

502.
Kinashi T, Springer TA: Regulation of cell-matrix adhesion by receptor tyrosine kinases. Leuk Lymphoma 18:203, 1995.

503.
Lopez M, Aoubala M, Jordier F, et al: The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule. Blood 92:4602, 1998.

504.
Aizawa S, Tavassoli M: In vitro homing of hemopoietic stem cells mediated by a recognition system with galactosyl and mannosyl specificities. Proc Natl Acad Sci USA 84:4485, 1987.

505.
Tavassoli M, Hardy CL: Molecular basis of homing of intravenously transplanted stem cells. Blood 76:1059, 1990.

506.
Sackstein R: Expression of an L-selectin ligand on hematopoietic progenitor cells. Acta Haematol 97:22, 1997.

507.
Karakantza M, Gibson FM, Cavenagh JD, et al: Sle(x) expression of normal CD34 positive bone marrow haemopoietic progenitor cells. Brit J Haematol 86:883, 1994.

508.
Tu L, Murphy PG, Li X, Tedder TF: L-selectin ligands expressed by human leukocytes are HECA-452 antibody-defined carbohydrate epitopes preferentially displayed by P-selectin glycoprotein ligand-1. J Immunol 161:1140, 1998.

509.
Mazo IB, Gutierrez-Ramos JC, Frenette PS, et al: Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1. J Exp Med 188:465, 1998.

510.
Zollner O, Lenter MC, Blanks JE, et al: L-selectin from human, but not mouse, neutrophils binds directly to E-selectin. J Cell Biol 136:707, 1997.

511.
Von Andrian UH, M-Rini C: In situ analysis of lymphocyte migration to lymph nodes. Cell Adhes Commun 6:85, 1998.

512.
Levesque J-P, Zannettino ACW, Pudney M, et al: PSGL-1-mediated adhesion of human hematopoietic progenitors to P-selectin results in suppression of hematopoiesis. Immunity 11:369, 1999.

513.
Van der Merwe PA: Leukocyte adhesion: High-speed cells with ABS. Curr Biol 9:R419, 1999.

514.
Vest weber D, Blanks JE: Mechanisms that regulate the function of the selectins and their ligands. Physiol Rev 79:181, 1999.

515.
Puri KD, Finger EB, Gaudernack G, Springer TA: Sialomucin CD34 is the major L-selectin ligand in human tonsil high endothelial venules. J Cell Biol 131:261, 1995.

516.
Sassetti C, Tangemann K, Singer MS, et al: Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: parallels to CD34. J Exp Med 187:1965, 1998.

517.
Tada J, Omine M, Suda T, Yamaguchi N: A common signaling pathway via Syk and Lyn tyrosine kinases generated from capping of the sialomucins CD34 and CD43 in immature hematopoietic cells. Blood 93:3723, 1999.

518.
Young PE, Baumhueter S, Lasky LA: The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development. Blood 85:96, 1995.

519.
Oxley SM, Sackstein R: Detection of an L-selectin ligand on a hematopoietic progenitor cell line. Blood 84:3299, 1994.

520.
Stockton BM, Cheng G, Manjunath N, et al: Negative regulation of T cell homing by CD43. Immunity 8:373, 1998.

521.
Bazil V, Brandt J, Chen S, et al: A monoclonal antibody recognizing CD43 (leukosialin) initiates apoptosis of human hematopoietic progenitor cells but not stem cells. Blood 87:1272, 1996.

522.
Yonemura S, Hirao M, Doi Y, et al: Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J Cell Biol 140:885, 1998.

523.
Anzai N, Gotoh A, Shibayama H, Broxmeyer HE: Modulation of integrin function in hematopoietic progenitor cells by CD43 engagement: possible involvement of protein tyrosine kinase and phospholipase C-g. Blood 93:3317, 1999.

524.
Spertini O, Cordey AS, Monai N, et al: P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells. J Cell Biol 135:523, 1996.

525.
Tracey JB, Rinder HM: Characterization of the P-selectin ligand on human hematopoietic progenitors. Exp Hematol 24:1494, 1996.

526.
Zannettino AC, Berndt MC, Butcher C, et al: Primitive human hematopoietic progenitors adhere to P-selectin (CD62P). Blood 85:3466, 1995.

527.
Blanks JE, Moll T, Eytner R, Vestweber D: Stimulation of P-selectin glycoprotein ligand-1 on mouse neutrophils activates beta 2-integrin mediated cell attachment to ICAM-1. Eur J Immunol 28:433, 1998.

528.
Yang J, Hirata T, Croce K, et al: Targeted gene disruption demonstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selectin-mediated but not E-selectin-mediated neutrophil rolling and migration. J Exp Med 190:1769, 1999.

529.
Fuhbridge RC, Kieffer JD, Armerding D, Kupper TS: Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells. Nature 389:978, 1997.

530.
Aigner S, Sthoeger ZM, Fogel M, et al: CD24, a mucin-type glycoprotein, is a ligand for P-selectin on human tumor cells. Blood 89:3385, 1997.

531.
Watt SM, Buhring HJ, Rappold I, et al: CD164, a novel sialomucin on CD34(+) and erythroid subsets, is located on human chromosome 6q21. Blood 92:849, 1998.

532.
Bowen MA, Aruffo A: Adhesion molecules, their receptors, and their regulation: analysis of CD6-activated leukocyte cell-adhesion molecule (ALCAM/CD166) interactions. Transplant Proc 31:795, 1999.

533.
Stamenkovin I, Aruffo A, Amiot M, Seed B: The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO J 10:343, 1991.

534.
Dougherty GJ, Lansdorp PM, Cooper DL, Humphries RK: Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte “homing” receptor expressed by hemopoietic cells. J Exp Med 174:1, 1991.

535.
Herrlich P, Zöller M, Pals ST, Ponta H: CD44 splice variants: Metastases meet lymphocytes. Immunol Today 14:395, 1993.

536.
Rosel M, Khaldoyanidi S, Zawadski V, Zoller M: Involvement of CD44 variant isoform v10 in progenitor cell adhesion and maturation. Exp Hematol 27:698, 1999.

537.
Funaro A, Malavasi F: Human CD38, a surface receptor, an enzyme, an adhesion molecule and not a simple marker. J Biol Regul Homeost Agents 13:54, 1999.

538.
Hoenstein AL, Stokinger H, Imhof BA, Malavasi F: CD38 binding to human myeloid cells is mediated by mouse and human CD31. Biochem J 330:1129, 1998.

539.
Turel KR, Rao SG: Expression of the cell adhesion molecule E-cadherin by the human bone marrow stromal cells and its probable role in CD34(+) stem cell adhesion. Cell Biol Int 22:641, 1998.

540.
Kaisho T, Ishikawa J, Oritani K, et al: BST-1, a surface molecule of bone marrow stromal cell lines that facilitates pre-B-cell growth. Proc Natl Acad Sci USA 91:5325, 1994.

541.
Hirata Y, Kimura N, Sato K, et al: ADP ribosyl cyclase activity of a novel bone marrow stromal cell surface molecule, BST-1. FEBS Lett 356:244, 1994.

542.
Vicari AP, Bean AG, Zlotnik A: A role for BP-3/BST-1 antigen in early T cell development. Int Immunol 8:183, 1996.

543.
Okuyama Y, Ishihara K, Kimura N, et al: Human BST-1 expressed on myeloid cells functions as a receptor molecule. Biochem Biophys Res Commun 228:838, 1996.

544.
Springer TA: Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301, 1994.

545.
Steeber DA, Tedder TF: Molecular basis of lymphocyte migration, in Inflammation Basic Principles and Clinical Correlates, 3rd ed, edited by JI Gallin, R Snyderman, pp 593–605. Lippincott Williams and Wilkins, Philadelphia, 1999.

546.
Imai Y, Lasky LA, Rosen SD: Sulphation requirement for GlyCAM-1, an endothelial ligand for L-selectin. Nature 361:555, 1993.

547.
Berg EL, McEvoy LM, Berlin C, et al: L-selectin-mediated lymphocyte rolling on MAdCAM-1. Nature 366:695, 1993.

548.
Lawrence MB, Berg EL, Butcher EC, Springer TA: Rolling of lymphocytes and neutrophils on peripheral node addressin and subsequent arrest on ICAM-1 in shear flow. Eur J Immunol 25:1025, 1995.

549.
Salmi M, Hellman J, Jalkanen S: The role of two distinct endothelial molecules, vascular adhesion protein-1 and peripheral lymph node addressin, in the binding of lymphocyte subsets to human lymph nodes. J Immunol 160:5629, 1998.

550.
Weber C, Springer TA: Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to alphaIIbbeta3 and stimulated by platelet-activating factor. J Clin Invest 100:2085, 1997.

551.
Kovach NL, Lin N, Yednock T, et al: Stem cell factor modulates avidity of alpha 4 beta 1 and alpha 5 beta 1 integrins expressed on hematopoietic cell lines. Blood 85:159, 1995.

552.
Levesque JP, Leavesley DI, Niutta S, et al: Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. J Exp Med 181: 1805, 1995.

553.
Weber C, Alon R, Moser B, Springer TA: Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis. J Cell Biol 134: 1063, 1996.

554.
Suehiro Y, Muta K, Umemura T, et al: Macrophage inflammatory protein 1alpha enhances in a different manner adhesion of hematopoietic progenitor cells from bone marrow, cord blood, and mobilized peripheral blood. Exp Hematol 27:1637,1999.

555.
Issekutz AC, Rowter D, Springer TA: Role of ICAM-1 and ICAM-2 and alternate CD11/CD18 ligands in neutrophil transendothelial migration. J Leukoc Biol 65:117, 1999.

556.
Meerschaert J, Furie MB: The adhesion molecules used by monocytes for migration across endothelium include CD11a/CD18, CD11b/CD18, and VLA-4 on monocytes and ICAM-1, VCAM-1, and other ligands on endothelium. J Immunol 154:4099, 1995.

557.
Weber C, Springer TA: Interaction of very late antigen-4 with VCAM-1 supports transendothelial chemotaxis of monocytes by facilitating lateral migration. J Immunol 161:6825, 1998.

558.
Yong KL, Watts M, Shaun TN, et al: Transmigration of CD34+ cells across specialized and non-specialized endothelium requires prior activation by growth factors and is mediated by PECAM-1 (CD31). Blood 91:1196, 1998.

559.
Muller WA: The role of PECAM-1 (CD31) in leukocyte emigration: studies in vitro and in vivo. J Leukoc Biol 57:523, 1995.

560.
Clark RA, Erickson HP, Springer TA: Tenascin supports lymphocyte rolling. J Cell Biol 137:755, 1997.

561.
Imhof BA, Weerasinghe D, Brown EJ, et al: Cross talk between alpha(v)beta3 and alpha4beta1 integrins regulates lymphocyte migration on vascular cell adhesion molecule 1. Eur J Immunol 27: 3242, 1997.

562.
Muller WA: Leukocyte-endothelial cell adhesion molecules in transendothelial migration, in Inflammation Basic Principles and Clinical Correlates, 3rd ed, edited by JI Gallin, R Snyderman, pp 585–592. Lippincott Williams and Wilkins, Philadelphia, 1999.

563.
Fong AM, Robinson LA, Steeber DA, et al: Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow. J Exp Med 188:1413, 1998.

564.
Bacon KB, Greaves DR, Dairaghi DJ, Schall TJ: The expanding universe of C, CX3C and CC chemokines, in The Cytokine Handbook, 3rd ed, edited by AW Thompson, pp 753–775. Academic Press, San Diego, 1998.

565.
Rood PML, Gerristen WR, Kramer D, et al: Adhesion of hematopoietic progenitor cell to human bone marrow or umbilical vein derived endothelial cell lines: a comparison. Exp Hematol 27:1306, 1999.

566.
Papayannopoulou T, Craddock C, Nakamoto B, et al: The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci USA 92:9647, 1995.

567.
Papayannopoulou T, Craddock C: Homing and trafficking of hematopoietic progenitor cells. Acta Hematol 97:97, 1997.

568.
Mazo IB, von Andrian UH: Adhesion and homing of blood-borne cells in bone marrow microvessels. J Leukoc Biol 66:25, 1999.

569.
Schweitzer KM, Vicart P, Delouis C, et al: Characterization of a newly established human bone marrow endothelial cell line: distinct adhesive properties for hematopoietic progenitors compared with human umbilical vein endothelial cells. Lab Invest 76:25, 1997.

570.
Mohle R, Bautz F, Rafii S, et al: The chemokine receptor CXCR-4 is expressed on CD34+ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor-1. Blood 91:4523, 1998.

571.
Mohle R, Murea S, Kirsch M, Haas R: Differential expression of L-selectin, VLA-4, from LFA-1 on CD34+ progenitor cells from bone marrow and peripheral blood during G-CSF-enhanced recovery. Exp Hematol 23:1535, 1995.

572.
Koenig JM, Baron S, Luo D, et al: L-selectin expression enhances clonogenesis of CD34+ cord blood progenitors. Pediatr Res 45:867, 1999.

573.
Frenette PS, Subbarao S, Mazo IB, et al: Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow. Proc Natl Acad Sci USA 95:14423, 1998.

574.
Derry CJ, Faveeuw C, Mordsley KR, Ager A: Novel chondroitin sulfate-modified ligands for L-selectin on lymph node high endothelial venules. Eur J Immunol 29:419, 1999.

575.
Norgard-Sumnicht K, Varki A: Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups. J Biol Chem 270:12012, 1995.

576.
Naiyer AJ, Jo DY, Ahn J, et al: Stromal derived factor-1-induced chemokinesis of cord blood CD34(+) cells (long-term culture-initiating cells) through endothelial cells is mediated by E-selectin. Blood 94:4011, 1999.

577.
Peled A, Grabovsky V, Habler L, et al: The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow. J Clin Invest 104:1199, 1999.

578.
Parakh KA, Kannan K: Demonstration of a ubiquitin binding site on murine haemopoietic progenitor cells: implications of ubiquitin in homing and adhesion. Br J Haematol 84:212, 1993.

579.
Hua CT, Gamble JR, Vadas MA, Jackson DE: Recruitment and activation of SHP-1 protein-tyrosine kinase phosphatase by human platelet endothelial cell adhesion molecule-1 (PECAM-1). Identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates. J Biol Chem 273:28332, 1998.

580.
Peled A, Petit I, Kollet O, et al: Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 283:845, 1999.

581.
Cipolleschi MG, Dello Sbarba P, Olivotto M: The role of hypoxia in the maintenance of hematopoietic stem cells. Blood 82:2031, 1993.

582.
Jacobsen SE, Ruscetti FW, Ortiz M, et al: The growth response of Lin-Thy1+ hematopoietic progenitors to cytokines is determined by the balance between synergy of multiple stimulators and negative cooperation of multiple inhibitors. Exp Hematol 22:985, 1994.

583.
Tang J, Nuccie BL, Ritterman I, et al: TGF-beta down-regulates stromal IL-7 secretion and inhibits proliferation of human B cell precursors. J Immunol 159:117, 1997.

584.
Sakamaki S, Hirayama Y, Matsunaga T, et al: Transforming growth factor-b1 (TGF-b1) induces thrombopoietin from bone marrow stromal cells which stimulates the expression of TGF-b receptor on megakaryocytes and, in turn, renders them susceptible to suppression by TGF-b itself with high specificity. Blood 94:1961, 1999.

585.
Cashman JD, Clark-Lewis I, Eaves AC, Eaves CJ: Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model. Blood 94:3722, 1999.

586.
Gautam SC, Noth CJ, Janakiraman N, et al: Induction of chemokine mRNA in bone marrow stromal cells: modulation by TGF-beta 1 and IL-4. Exp Hematol 23:482, 1995.

587.
Cashman JD, Eaves CJ, Sarris AH, Eaves AC: MCP-1, not MIP-1alpha, is the endogenous chemokine that cooperates with TGF-beta to inhibit the cycling of primitive normal but not leukemic (CML) progenitors in long-term human marrow cultures. Blood 92:2338, 1998.

588.
Kinashi T, Springer TA: Steel factor and c-kit regulate cell matrix adhesion. Blood 83:1033, 1994.

589.
Juliano RL, Haskill S: Signal transduction from the extracellular matrix. J Cell Biol 120:577, 1993.

590.
Issaad C, Croisille L, Katz A, et al: A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38– progenitor cells in long-term cultures and semisolid assays. Blood 81:2916, 1993.

591.
Sachs L, Lotem J: Control of programmed cell death in normal and leukemic cells: new implications for therapy. Blood 82:15, 1993.

592.
Gibson LF, Piktel D, Narayanan R, et al: Stromal cells regulate bcl-2 and bax expression in pro-B cells. Exp Hematol 24:628, 1996.

593.
Liesveld JL, Harbol AW, Abboud CN: Stem cell factor and stromal cell co-culture prevent apoptosis in a subculture of the megakaryoblastic cell line, UT-7. Leuk Res 20:591, 1996.

594.
Borge OJ, Ramsfjell V, Cui L, Jacobsen SE: Ability of early acting cytokines to directly promote survival and suppress apoptosis of human primitive CD34+CD38- bone marrow cells with multilineage potential at the single-cell level: key role of thrombopoietin. Blood 90:2282, 1997.

595.
Finch CA, Harker LA, Cook JD: Kinetics of the formed elements of human blood. Blood 50:699, 1977.

596.
Yong KL: Granulocyte colony-stimulating factor (G-CSF) increases neutrophil migration across vascular endothelium independent of an effect on adhesion: comparison with granulocyte-macrophage colony-stimulating factor (GM-CSF). Br J Haematol 94:40, 1996.

597.
Ulich TR, del Castillo J, Souza L: Kinetics and mechanisms of recombinant human granulocyte-colony stimulating factor-induced neutrophilia. Am J Pathol 133:630, 1988.

598.
DiPersio JF, Abboud CN: Activation of neutrophils by granulocyte-macrophage colony-stimulating factor, in Granulocyte Responses to Cytokines: Basic and Clinical Research, edited by RG Coffey, pp 457–484. Immunology Series, vol 57, Marcel Dekker, New York, 1992.

599.
Ghebrehiwet B, Muller-Eberhard HJ: C3e: an acidic fragment of human C3 with leukocytosis-inducing activity. J Immunol 123:616, 1979.

600.
Kubo H, Graham L, Doyle NA, et al: Complement fragment-induced release of neutrophils from bone marrow and sequestration within pulmonary capillaries in rabbits. Blood 92:283, 1998.

601.
Deinard AS, Page AR: A study of steroid-induced granulocytosis. Br J Haematol 28:333, 1974.

602.
Vogel MJ, Yankee RA, Kimball HR, et al: The effect of etiocholanolone on granulocyte kinetics. Blood 30:474, 1967.

603.
Cybulsky MI, McCoumb DJ, Movat HZ: Neutrophil leukocyte emigration induced by endotoxin: Mediator roles of interleukin-1 and tumor necrosis factor alpha. J Immunol 140:3144, 1988.

604.
Terashima T, English D, Hogg JC, van Eeden SF: Release of polymorphonuclear leukocytes from the bone marrow by interleukin-8. Blood 92:1062, 1998.

605.
Wang JM, Rambaldi A, Biondi A, et al: Recombinant human interleukin 5 is a selective eosinophil chemoattractant. Eur J Immunol 19:701, 1989.

606.
Palframan RT, Collins PD, Williams TJ, Rankin SM: Eotaxin induces a rapid release of eosinophils and their progenitors from the bone marrow. Blood 91:2240, 1998.

607.
Ebisawa M, Yamada T, Bickel C, et al: Eosinophil transendothelial migration induced by cytokines: III. Effect of the chemokine RANTES. J Immunol 153:2153, 1994.

608.
Lundahl J, Moshfegh A, Gronneberg R, Hallden G: Eotaxin increases the expression of CD11b/CD18 and adhesion properties in IL-5, but not fMLP-prestimulated human peripheral blood eosinophils. Inflammation 22:123, 1998.

609.
Dutt P, Wang JF, Groopman JE: Stromal cell-derived factor-1 alpha and stem cell factor/kit ligand share signaling pathways in hemopoietic progenitors: a potential mechanism for cooperative induction of chemotaxis. J Immunol 161:3652, 1998.

610.
Hedrick JA, Helms A, Vicari A, Zlotnik A: Characterization of a novel chemokine, HCC-4, whose expression is increased by interleukin-10. Blood 91:4242, 1998.

611.
Kim CH, Broxmeyer HE: Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol 65:6, 1999.

612.
Kim CH, Broxmeyer HE: SLC/exodus2/6Ckine/TCA4 induces chemotaxis of hematopoietic progenitor cells: differential activity of ligands of CCR7, CXCR3, or CXCR4 in chemotaxis vs. suppression of progenitor proliferation. J Leukoc Biol 66:455, 1999.

613.
Morales J, Homey B, Vicari AP, et al: CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells. Proc Natl Acad Sci USA 96:14470, 1999.

614.
Zlotnik A, Yoshie O: Chemokines: a new classification system and their role in immunity. Immunity 12:121, 2000.

615.
Tanaka H, Tatsumi N, Kan E, et al: EPO test in hemodialysis patients. Biomater Artif Cells Immobil Biotechnol 21:221, 1993.

616.
Chamberlain JK, Weiss L, Weed RI: Bone marrow sinus cell packing: a determinant of cell release. Blood 46:91, 1975.

617.
Waugh RE, Sassi M: An in vitro model of erythroid egress in bone marrow. Blood 68:250, 1986.

618.
Dabrowski A, Szygula Z, Miszta H: Do changes in bone marrow pressure contribute to the egress of cells from the bone marrow? Acta Physiol Pol 32:729, 1981.

619.
Iversen PO, Nicolaysen G, Benestad HB: Blood flow to bone marrow during development of anemia or polycythemia in the rat. Blood 79:594, 1992.

620.
Iversen PO, Nicolaysen G, Benestad HB: The leukopoietic cytokine granulocyte colony-stimulating factor increases blood flow to rat bone marrow. Exp Hematol 21:231, 1993.

621.
Lichtman MA, Chamberlain JK, Santillo PA: Factors thought to contribute to the regulation of egress of cells from marrow, in The Year in Hematology 1978, edited by R Silber, J LoBue, A Gordon, pp 243–279. Plenum, New York, 1978.

622.
Van Eeden SF, Miyagashima R, Haley L, Hogg JC: A possible role for L-selectin in the release of polymorphonuclear leukocytes from bone marrow. Am J Physiol 272:H1717, 1997.

623.
LeMarer N, Skacel PO: Up-regulation of alpha2,6 sialylation during myeloid maturation: a potential role in myeloid cell release from the bone marrow. J Cell Physiol 179:315, 1999.

624.
Reinhardt PH, Elliott JF, Kubes P: Neutrophils can adhere via alpha2beta1-integrin under flow conditions. Blood 89:3837, 1997.

625.
Burns AR, Bowden RA, Abe Y, et al: P-selectin mediates neutrophil adhesion to endothelial cell borders. J Leukoc Biol 65:299, 1999

626.
Jagels MA, Chambers JD, Arfors KE, Hugli TE: C5a- and tumor necrosis factor-alpha-induced leukocytosis occurs independently of beta 2 integrins and L-selectin: differential effects on neutrophil adhesion molecule expression in vivo. Blood 85:2900, 1995.

627.
Stroncek DF, Kaszcz W, Herr GP, et al: Expression of neutrophil antigens after 10 days of granulocyte-colony-stimulating factor. Transfusion 38:663, 1998.

628.
Jung U, Ley K: Mice lacking two or all three selectins demonstrate overlapping and distinct functions for each selectin. J Immunol 162:6755, 1999.

629.
Scurfield G, Radley JM: Aspects of platelet formation and release. Am J Hematol 10:285, 1981.

630.
Radley JM, Haller CJ: Fate of senescent megakaryocytes in bone marrow. Br J Haematol 53:277, 1983.

631.
Efrati P, Rozenszajn L: The morphology of buffy coats in normal human adults. Blood 16:1012, 1960.

632.
Tinggaard-Pedersen N, Laursen B: Megakaryocytes in cubital venous blood in patients with chronic myeloproliferative diseases. Scand J Haematol 30:50, 1983.

633.
Mantovani A, Vecchi A, Sozzani S, et al: Tumors as a paradigm for the in vivo role of chemokines in leukocyte recruitment, in Chemokines and Cancer, Contemporary Cancer Research, edited by BJ Rollins, pp 35–49. Humana Press Inc, NJ, 1999.

634.
Kim CH, Broxmeyer HE: In vitro behavior of hematopoietic progenitor cells under the influence of chemoattractants: stromal cell-derived factor-1, steel factor, and the bone marrow environment. Blood 91:100, 1998.

635.
Aiuti A, Webb IJ, Bleul C, et al: The chemokine SDF-1 is a chemoattractant for human hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med 185:111, 1997.

636.
Aiuti A, Tavian M, Cipponi A, et al: Expression of CXCR4, the receptor for stromal cell-derived factor-1 on fetal and adult human lympho-hematopoietic progenitors. Eur J Immunol 29:1823, 1999.

637.
Roberts MM, Swart BW, Simmons PJ, et al: Prolonged release and c-kit expression of haemopoietic precursor cells mobilized by stem cell factor and granulocyte colony stimulating factor. Br J Haematol 104:778, 1999.

638.
Wang JB, Mukaida N, Zhang Y, et al: Enhanced mobilization of hematopoietic progenitor cells by mouse MIP-2 and granulocyte colony-stimulating factor. J Leuk Biol 62:503, 1997.

639.
Vermeulen M, Le Pesteur F, Gagnerault MC, et al: Role of adhesion molecules in the homing and mobilization of murine hematopoietic stem and progenitor cells. Blood 92:894, 1998.

640.
Craddock CF, Nakamoto B, Elices M, Papayannopoulou TH: The role of CS1 moiety of fibronectin in VLA mediated haemopoietic progenitor trafficking. Br J Haematol 98:828, 1997.

641.
Craddock CF, Nakamoto B, Andrews RG, et al: Antibodies to VLA4 integrin mobilize long-term repopulating cells and augment cytokine-induced mobilization in primates and mice. Blood 90:4779, 1997.

642.
Papayannopoulou T, Priestley GV, Nakamoto B: Anti-VLA4/VCAM-1-induced mobilization requires cooperative signaling through the kit/mkit ligand pathway. Blood 91:2231, 1998.

643.
Papayannopoulou T: Hematopoietic stem/progenitor cell mobilization. A continuing quest for etiologic mechanisms. Ann NY Acad Sci 872:187, 1999.

644.
Maloney MA, Patt HM: Migration of cells from shielded to irradiated marrow. Blood 39:804, 1972.

645.
Link H, Arseniev L, Bahre O, et al: Combined transplantation of allogeneic bone marrow and CD34+ blood cells. Blood 86:2500, 1995.

646.
Dercksen MW, Gerristen WR, Rodenhuis S, et al: Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after blood stem cell transplantation. Blood 85:3313, 1995.

647.
Watanabe T, Dave B, Heiman DG, et al: Cell adhesion molecule expression on CD34+ cells in grafts and time to myeloid and platelet recovery after autologous stem cell transplantation. Exp Hematol 26:10, 1998.

648.
Timeus F, Crescenzio N, Marranca D, et al: Cell adhesion molecules in cord blood hematopoietic progenitors. Bone Marrow Transplant 22(suppl 1):S61, 1998.

649.
Katayama Y, Mahmut N, Takimoto H, et al: Hematopoietic progenitor cells from allogeneic bone marrow transplant donors circulate in the very early post-transplant period. Bone Marrow Transplant 23:659, 1999.

650.
Hardy CL: The homing of hematopoietic stem cells to the bone marrow. Am J Med Sci 309:260, 1995.

651.
Pipia GG, Long MW: Human hematopoietic progenitor cell isolation based on galactose-specific cell surface binding. Nat Biotechnol 15:1007, 1997.

652.
Shirota T, Tavassoli M: Alterations of bone marrow sinus endothelium induced by ionizing irradiation: implications in homing of intravenously transplanted marrow cells. Blood Cells 18:197, 1992.

653.
Yamazaki K, Allen TD: The structure and function of the blood-marrow barrier: early ultrastructural changes in irradiated bone marrow sinus endothelial cells detected by vascular perfusion fixation (comment). Blood Cells 18:215, 1992.

654.
Bolante-Cervantes R, Li S, Sahota A, et al: Pattern of localization of primitive hematopoietic cells in vivo using a novel mouse model. Exp Hematol 27:1346, 1999.

655.
Cui J, Wahl RL, Shen T, et al: Bone marrow cell trafficking following intravenous administration. Br J Haematol 107:895, 1999.

656.
Imai K, Kobayashi M, Wang J, et al: Selective secretion of chemoattractants for haemopoietic progenitor cells by bone marrow endothelial cells: a possible role in homing of haemopoietic progenitor cells to bone marrow. Br J Haematol 106:905, 1999.

657.
Jo DY, Rafii S, Hamada T, Moore MA: Chemotaxis of primitive hematopoietic cells is in response to stromal cell-derived factor-1. J Clin Invest 105:101, 2000.

658.
Lanzkron SM, Collector MI, Sharkis SJ: Hematopoietic stem cell trafficking in vivo: a comparison of short-term and long-term repopulating cells. Blood 93:1916, 1999.

659.
Kessinger A: Collection of autologous peripheral blood stem cells in steady state, in Peripheral Stem Cells in Bone Marrow Transplantation, edited by NC Gorin, pp 19–26, Baillieres Best Practice and Research Clinical Haematology, vol 12, #1/2, 1999.

660.
Shpall EJ: The utilization of cytokines in stem cell mobilization strategies. Bone Marrow Transplant 23(suppl 2):S13, 1999.

661.
Facon T, Harousseau JL, Maloisel F, et al: Stem cell factor in combination with filgastrim after chemotherapy improves peripheral blood progenitor cell yield and reduces apheresis requirements in multiple myeloma patients: a randomized, controlled trial. Blood 94:1218, 1999.

662.
Lyman SD: Biologic effects and potential clinical applications of Flt3 ligand. Curr Opin Hematol 5:192, 1998.

663.
Fischmeister G, Kurz M, Haas OA, et al: G-CSF versus GM-CSF for stimulation of peripheral blood progenitor cells (PBPC) and leukocytes in healthy volunteers: comparison of efficacy and tolerability. Ann Hematol 78:117, 1999.

664.
Chao NJ, Schriber JR, Grimes K, et al: Granulocyte colony-stimulating factor “mobilized” peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy. Blood 81:2031, 1993.

665.
Damia G, Komschlies KL, Faltynek CR, et al: Administration of recombinant human interleukin-7 alters the frequency and number of myeloid progenitor cells in the bone marrow and spleen of mice. Blood 79:1121, 1992.

666.
Somlo G, Sniecinski I, ter Veer A, et al: Recombinant human thrombopoietin in combination with granulocyte colony-stimulating factor enhances mobilization of peripheral blood progenitor cells, increases peripheral blood platelet concentration, and accelerates hematopoietic recovery following high-dose chemotherapy. Blood 93:2798, 1999.

667.
MacVittie TJ, Farese AM, Davis TA, et al: Myelopoietin, a chimeric agonist of human interleukin 3 and granulocyte colony-stimulating factor receptors, mobilizes CD34+ cells that rapidly engraft lethally x-irradiated nonhuman primates. Exp Hematol 27:1557, 1999.

668.
Welham MJ, Schrader JW: Modulation of c-kit mRNA and protein by hemopoietic growth factors. Mol Cell Biol 11:2901, 1991.

669.
Pilarski LM, Pruski E, Wizniak J, et al: Potential role for hyaluronan and the hyaluronan receptor RHAMM in mobilization and trafficking of hematopoietic progenitor cells. Blood 93:2918, 1999.

670.
Chamberlain JK, Leblond PF, Weed RI: Reduction of adventitial cell cover: an early direct effect of erythropoietin on bone marrow ultrastructure. Blood Cells 1:655, 1975.

671.
Pruijt JF, Williamze R, Fibbe WE: Mechanisms underlying hematopoietic stem cell mobilization induced by the CXC chemokine interleukin-8. Curr Opin Hematol 6:152, 1999.

672.
Pruijt JF, Fibbe WE, Laterveer L, et al: Prevention of interleukin-8-induced mobilization of hematopoietic progenitor cells in rhesus monkeys by inhibitory antibodies against the metalloproteinase gelatinase B (MMP-9). Proc Natl Acad Sci USA 96:10863, 1999.

673.
Watanabe T, Kawano Y, Kanamaru S, et al: Endogenous interleukin-8 (IL-8) surge in granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization. Blood 93:1157, 1999.

674.
Pruijt JF, van Kooyk Y, Figdor CG, et al: Murine hematopoietic progenitor cells with colony-forming or radioprotective capacity lack expression of the beta 2-integrin LFA-1. Blood 93:107, 1999.

675.
Pruijt JF, van Kooyk Y, Figdor CG, et al: Anti-LFA-1 blocking antibodies prevent mobilization of hematopoietic progenitor cells induced by interleukin-8. Blood 91:4099, 1998.

676.
Semerad, CL, Poursine-Laurent J, Liu F, et al: A role for G-CSF receptor signaling in the regulation of hematopoietic cell function but not lineage commitment or differentiation. Immunity 11:153, 1999.

677.
Liu F, Poursine-Laurent J, Link D: The granulocyte colony-stimulating factor receptor is required for the mobilization of murine hematopoietic progenitors into peripheral blood by cyclophosphamide or interleukin-8 but not Flt-3 ligand. Blood 90:2522, 1997.

678.
Betsuyaku T, Liu F, Senior RM, et al: A functional granulocyte-macrophage colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation. J Clin Invest 103:825, 1999.

679.
Murphy G, Gavrilovic J: Proteolysis and cell migration: creating a path? Curr Opin Cell Biol 11:614, 1999.

680.
Webb LM, Ehrengruber MU, Clark-Lewis I, et al: Binding of heparan sulfate or heparin enhances neutrophil responses to interleukin-8. Proc Natl Acad Sci USA 90:7158, 1993.

681.
Dias Baruffi M, Pereira-da-Silva G, Jamur MC, Roque-Barreira MC: Heparin potentiates in vivo neutrophil migration induced by IL-8. Glyconj J 15:523, 1998.

682.
Rainger GE, Rowley AF, Nash GB: Adhesion-dependent release of elastase from human neutrophils in a novel, flow-based model: specificity of different chemotactic agents. Blood 92:4819, 1998.

683.
Wize J, Sopata I, Smerdel A, Maslinski S: Ligation of selectin L and integrin CD11b/CD18 (Mac-1) induces release of gelatinase B (MMP-9) from human neutrophils. Inflamm Res 47:325, 1998.

684.
Janowska-Wieczorek A, Marquez LA, Nabholtz J-M, et al: Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34+ cells and their transmigration through reconstituted basement membrane. Blood 93:3379, 1999.

685.
Gallacher L, Murdoch B, Wu DM, Karanu FN, et al: Isolation and characterization of human CD34(–)Lin(–) and CD34(+)Lin(–) hematopoietic stem cells using cell surface markers AC133 and CD7. Blood 95:2813, 2000.

686.
Gehling UM, Ergun S, Schumacher U, Wagener C, et al: In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood 95:3106, 2000.

687.
Peichev M, Naiyer AJ, Pereira D, Zhu Z, et al: Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood 95:952, 2000.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

CHAPTER 3 EXAMINATION OF THE MARROW

CHAPTER 3 EXAMINATION OF THE MARROW
Williams Hematology

CHAPTER 3 EXAMINATION OF THE MARROW

DANIEL H. RYAN

Indications for Marrow Aspirate or Biopsy
Marrow Aspiration Technique
Needle Biopsy Technique
Preparation of Marrow Specimens for Study

Films

Touch Preparations

Special Studies

Histologic Sections
Morphologic Interpretation of Marrow Preparations

Overview

Adequacy of the Marrow Sample

Bone Marrow Cellularity

Infiltrative Diseases of the Marrow

Differentiation of the Hematopoietic Lineages
Chapter References

The examination of the marrow in concert with the prior examination of the blood remains the dyad required for the diagnosis of many hematologic diseases. The marrow examination provides a semi-quantitative and qualitative assessment of the state of hematopoiesis and the normalcy of the blood cell precursors of all lineages. It can provide the diagnosis of several hereditary and acquired benign and malignant diseases. The marrow is a source of cells for clonal hematopoietic cell assays, cells for histocytologic, immunocytologic, cytogenetic, and molecular analysis. It is an easy, safe, and inexpensive means to arrive at the diagnosis of important abnormalities of the hematopoietic system. It is an important test to assess the response to treatment of the leukemias and some lymphomas. It can be useful in assessing the state of iron stores and of metabolic diseases that affect macrophages, such as Gaucher disease. It represents the cornerstone of hematologic diagnosis, even as hematology moves to a more molecularly- and genetically-based discipline.

Acronyms and abbreviations that appear in this chapter include: ALIP, abnormal localization of immature precursor cells; M/E, myeloid/erythroid.

Marrow progenitors give rise to all hematopoietic lineages in the adult. Therefore, direct visual examination of the marrow has long been a mainstay of hematologic diagnosis. Even with the advent of specialized biochemical and molecular assays that capitalize on advances in understanding of the biology of hematopoiesis, the primary diagnosis of hematologic malignancies and many nonneoplastic hematologic disorders relies on visual examination of the marrow. Marrow may be obtained without significant risk and with only minor discomfort and is quickly and easily processed for examination.
At birth all bones contain hematopoietic marrow. Fat cells begin to replace hematopoietic marrow in the extremities in the fifth to seventh year, and by adulthood the hematopoietic marrow is limited to the axial skeleton and the proximal portions of the extremities.1,2 The structure and function of the marrow and the distribution of marrow in the skeleton are discussed in Chap. 4. Fatty marrow appears yellow, while hematopoietic marrow is red. Red marrow does contain fat, however, and fat droplets are visible grossly in aspirated marrow specimens. Histologically, yellow marrow consists almost entirely of fat cells and supporting connective tissue, while red marrow contains an abundance of hematopoietic cells along with fat cells and connective tissue. The marrow fills the spaces between the trabeculae of bone in the marrow cavity. It is soft and friable and can be readily aspirated or biopsied with a needle.
INDICATIONS FOR MARROW ASPIRATE OR BIOPSY
The marrow should be examined when the clinical history, laboratory test results, or blood film suggests the possibility of a primary or secondary hematologic disorder for which morphologic analysis or special studies of the marrow would aid in the diagnosis. Although marrow aspiration and biopsy techniques are safe, they should be performed with a clear idea as to how the results will aid in distinguishing the differential diagnoses under consideration or provide assessment of treatment. In some hematologic disorders, such as most cases of iron deficiency anemia, thalassemia, pernicious anemia, and Gaucher disease, examination of the blood and specialized laboratory tests may be sufficient to make the diagnosis without the need for a marrow examination.
When examination of the marrow is indicated, it should be decided whether an aspirate only or aspirate plus biopsy is desired. The aspirate is always performed, because of the superior morphology offered by examination of the marrow aspirate film. However, a marrow biopsy is superior to the aspirate in quantitating marrow cellularity and diagnosing infiltrative diseases of the marrow and should be performed when these conditions are part of the differential diagnosis.3,4,5,6 and 7 In low-grade lymphoma the marrow is frequently involved at the time of diagnosis, and this involvement is most sensitively detected by marrow biopsy.8 Marrow biopsy is also useful in diagnosing and following the course of disorders that are commonly associated with fibrosis, such as megakaryoblastic leukemia, hairy-cell leukemia, and the chronic myeloproliferative disorders.9,10 In myelodysplastic syndromes, marrow biopsy is useful in evaluating the abnormal localization of immature precursor cells (ALIP) as well as evaluating abnormal megakaryocytes.9 Marrow necrosis and gelatinous transformation are more readily detected in marrow sections than in aspirate films. In some clinical settings where the diagnostic question is very targeted, such as diagnosis of childhood ITP or surveillance follow-up of leukemia patients, marrow aspirate alone may be appropriate. It is important to anticipate whether additional sample volume is required for cytogenetic or molecular studies.
MARROW ASPIRATION TECHNIQUE
The posterior iliac crest (Fig. 3-1) is the preferred site for both marrow aspiration and biopsy. In adults, the sternum and the anterior iliac crest can also be utilized (Fig. 3-2). The sternum should be used for aspiration only, and the anterior iliac crest is less preferred than the posterior crest in adults due to its thick cortical bone. The anteromedial surface of the tibia is an option for infants less than 1 year old (particularly newborns), but the posterior iliac crest is still the preferred site. The spinous processes of the vertebrae, the ribs, or other marrow-containing bones are rarely used. The hazards of marrow aspiration include hemorrhage, infection, and reactions to anesthetic agents, but these are very rare when the procedure is carefully performed. Penetration of the bone with damage to the underlying structures is possible with all marrow aspirations, but the hazard is greatest in sternal aspirations, since the sternum at the second interspace is only about 1 cm thick in the adult.

FIGURE 3-1 (a) Jamshidi biopsy instrument. (b) Site of marrow biopsy. [(a) From Jamshidi and Swaim13 by permission; (b) from Ellis, Jensen, and Westerman3 by permission.]

FIGURE 3-2 Sites used for marrow aspiration. (Modified from SO Schwartz, WH Hartz Jr, and JH Robbins, Hematology in Practice, part 1, p 36. McGraw-Hill, New York, 1961.)

For either marrow biopsy or aspirate, conscious sedation minimizes anxiety and pain,11 particularly in children, but must be performed with proper patient monitoring to minimize risk. Marrow biopsies and aspirates performed for staging purposes can often be done while a patient is under anesthesia for other reasons (insertion of central line). Several different types of needles, most of which are satisfactory, are available for marrow aspiration. For adults an 18-gauge needle is sufficiently large to permit aspiration of adequate specimens; larger needles are unnecessary. The patient is prone or in the left or right lateral decubitus position. Sterile precautions must be observed. The skin over the puncture site is shaved if necessary and cleansed with a disinfectant solution, and the skin, subcutaneous tissues, and periosteum are infiltrated with a local anesthetic solution such as 1% lidocaine. Adequate infiltration of the anesthetic at the periosteal surface is important, but no more than 20 ml of 1% lidocaine should be used in an adult.12 An air gun may be used to anesthetize the skin surface prior to application of anesthetic to the periosteal surface by injection. After the anesthesia has taken effect, the marrow needle is inserted through the skin, subcutaneous tissue, and cortex of the bone with a slight twisting motion. In obese patients, the length of the needle must be sufficient to reach the iliac crest. The stylet should be locked into place on the hub of the needle to prevent plugging of the needle with tissue prior to entry into the marrow cavity. Penetration of the cortex can be sensed by a slight, rapid forward movement accompanied by a sudden increase in the ease of advancing the needle. The stylet of the needle is removed promptly, the hub is attached to a 10- or 20-ml syringe, and about 0.2 to 0.5 ml of fluid is aspirated. The actual aspiration of the marrow causes a transient painful sensation for most patients. If additional specimen volume is required, another syringe is fitted on the marrow needle and marrow is aspirated. The stylet may be reinserted and the marrow needle slightly repositioned between aspirations. When aspiration is complete, the stylet is reinserted and the needle removed from the bone immediately. Pressure is applied to the skin over the aspiration site for at least 5 min to minimize bruising at the site. In a thrombocytopenic patient, firm pressure should be applied for at least 10 to 15 min.
The bloody fluid that is aspirated contains light-colored particles of marrow from 0.5 to 1 mm in diameter. They are often readily visible in the syringe but may not be detected until the syringe contents are discharged on a glass slide to prepare films.
Occasionally nothing enters the syringe when aspiration is performed. This may mean that the needle has not been properly placed in the marrow cavity. It may be cautiously advanced 1 to 2 mm after reinsertion of the stylet and aspiration may be attempted again, or it may be more desirable to remove the needle from the bone and reinsert it in a nearby site in the anesthetized area. The thickness of the bone must be kept in mind when one is attempting to adjust the needle in the bone. Occasionally the needle must be rotated on its longitudinal axis, or in a larger orbit, in order to loosen the marrow mechanically before it can be aspirated. If a small amount of blood has been aspirated, it is wise to use a new needle because of the probability of clotting of the aspirate when it is finally obtained. Aspiration with a 50-ml syringe may succeed when failure has been encountered with a smaller syringe. Leukemic marrow may be so densely packed in the bone as to resist all attempts at aspiration, in which case biopsy is necessary. The marrow in myelofibrosis also may be impossible to aspirate. The commonest cause of failure to obtain marrow is faulty positioning of the needle, and a second attempt at aspiration will usually succeed.
NEEDLE BIOPSY TECHNIQUE
Needle biopsy is usually performed with the Jamshidi needle,13 using the same preparation as described above. The Jamshidi instrument (Fig. 3-1) consists of a cylindrical needle of constant bore except for a concentrically tapered distal portion ending in a sharp, beveled cutting tip. The stylet fits precisely inside the opening at the tapered tip, interlocks at the hub of the needle, and extends 1 to 2 mm beyond the end of the needle. An 11-gauge needle is most commonly used in the United States. After one has anesthetized the skin and the periosteum of the biopsy site, a 3-mm incision is made in the skin, and the needle, with obturator in place, is inserted into the skin incision and through the subcutaneous tissue to the cortex of the bone. The needle is directed toward the posterior iliac spine and advanced with a twisting motion. Penetration of the cortex is sensed by a decreased resistance to forward movement of the needle. The obturator is then removed, and the needle is slowly advanced with reciprocal clockwise-counterclockwise twisting motions around the long axis. After sufficient penetration of the bone (up to about 3 cm), the needle is rotated several times on its axis and withdrawn about 2 to 3 mm. The needle is then reinserted to the original depth at a slightly different angle, with care taken not to bend the needle, and rotated several times in order to free the specimen from attachments in the marrow cavity. Next the needle is slowly withdrawn, using the same twisting motion employed during insertion. The core of marrow inside the needle is removed by inserting the probe through the cutting tip and extruding the specimen through the hub of the needle. The smaller size of the cutting aperture relative to the bore of the shaft of the Jamshidi instrument yields a specimen which fits loosely inside the needle and is therefore less subject to compression, distortion, or fragmentation. This technique reliably produces biopsy specimens of good quality. Marrow biopsy should be performed before marrow aspiration is attempted (or in a slightly different site on the iliac crest) to avoid hemorrhage and distorted marrow architecture in the biopsy core.
With the availability of the biopsy needles described above, open (surgical) biopsies are rarely necessary but may be performed, for example, for the diagnosis of deeply situated bone lesions.
PREPARATION OF MARROW SPECIMENS FOR STUDY
Several types of preparations can be made from the marrow aspirate to make maximal use of the diagnostic material. Most important is the direct film, which is made immediately from the unmanipulated aspirate. This is the best preparation for evaluation of cellular morphology and differential counts of the marrow. The particle film is best for estimation of marrow cellularity and megakaryocyte abundance, but morphology is obscured in the thicker parts of the film. A concentrate film, prepared from a buffy coat of the marrow, is useful to detect low abundance cells, such as megakaryoctyes and metastatic tumor, or when the marrow is hypocellular. However, the relative proportions of cell lineages are not reliably maintained in this preparation (often erythroid precursors are relatively enriched). This preparation is also subject to anticoagulant-induced changes in nuclear morphology or cytoplasmic vacuolation. The touch imprint from the biopsy is essential to evaluate cellular morphology in case of a “dry tap”14 and provides cytologic detail of cells that may not appear in the aspirate specimen.15
FILMS
After aspiration, about 0.5 ml of marrow is placed on a glass slide, and the rest mixed into an EDTA-anticoagulated tube. The marrow specimen is examined to be sure that “spicules” or particles of marrow containing bony or fatty pieces are present, indicating successful aspiration of the marrow cavity. Direct marrow films are immediately prepared by transferring drops of the unanticoagulated marrow pool to fresh slides and making push films with coverslips. Sufficient slides should be made for special stains. Heparinization of the aspirate is not necessary if the operator works rapidly and should be avoided as it may introduce artifacts.
It is useful to prepare a film enriched in marrow particles (“particle film”) by picking up with a pipette several spicules from the pool of marrow, discharging a drop or two on a slide, covering the particles with a second slide, pressing these gently together to express most of the blood into a gauze sponge, and then pulling the slides apart longitudinally. Such preparations may contain an increased number of broken cells if too much pressure is applied, but they provide a large number of particles from which cellularity of the marrow may be estimated and which are useful for estimation of the amount of hemosiderin present.
An aliquot of the EDTA-anticoagulated sample is centrifuged (1500 g for 10 min) in a Wintrobe tube to concentrate the cellular elements of the marrow. After centrifugation, the fatty layer and plasma are removed, and the “buffy coat” is mixed with an equal amount of plasma; then multiple films are made of this preparation (“bone marrow concentrate”). These slides should be air dried, labeled, and retained as unstained preparations in case special stains are required.
TOUCH PREPARATIONS
After one has obtained a biopsy with a Jamshidi needle, the biopsy specimen should be extruded through the hub of the needle and then gently rolled across a glass slide (using forceps to move the specimen) before it is placed in fixative, taking care to avoid crushing. The touch preparations are allowed to dry and are stained in the same manner as films.
SPECIAL STUDIES
One of the reasons that it is essential to formulate the diagnostic question before performing a marrow aspiration is to be sure that adequate sample is obtained for all the special studies that may be needed to make the correct diagnosis. A sterile anticoagulated sample containing viable unfixed cells in single-cell suspension is the best substrate for nearly all special studies that are likely to be required on a marrow sample. Specifically, flow cytometry is best performed on an EDTA- or heparin-anticoagulated aspirate specimen, which is stable for at least 24 h at room temperature. For cytogenetic or cell culture analysis, anticoagulated marrow should be added to tissue culture medium and analyzed as soon as possible to maintain optimal cell viability. Cytogenetic samples are generally not adversely affected by overnight incubation.16
For molecular analysis of genomic DNA, sample preparation and storage as described for cell marker studies is adequate, since DNA is relatively stable. DNA can be extracted and analyzed even from paraffin-embedded tissue sections. However, RT-PCR assays, involving amplification of cDNA prepared from cellular messenger RNA, are often needed for molecular diagnosis of translocations associated with leukemia and lymphoma. Messenger RNA has a variable half-life in an intact cell and is degraded rapidly (on the order of seconds to minutes) in a cell lysate by ubiquitous RNAses. For maximal mRNA recovery, cell suspensions (typically buffy coat or mononuclear cell preparations) should be lysed in an appropriate RNAse-inhibitor containing buffer as soon as possible after sampling. Air-dried films may retain varying amounts of detectable mRNA.17 EDTA is the preferred anticoagulant, as heparin can interfere with some molecular assays.
Archival storage of marrow specimens is of increasing interest in light of advances in molecular diagnosis that may necessitate validation studies using samples of known origin or testing of diagnostic material from a patient now in remission. Isolated DNA or RNA can be stored for long periods at –70°C (–158°F), while viable, intact cells are most reliably preserved by controlled rate freezing in DMSO and storage in liquid nitrogen.
HISTOLOGIC SECTIONS
A variety of techniques have been advocated for preparing aspirated material for histologic study. All are designed to collect a sufficient number of marrow particles in a small volume so that adequate sections may be prepared. This may be accomplished by discharging the marrow aspirate onto a glass slide, allowing the particles to settle for a few seconds, and then gently tilting the slide so that the excess blood runs off. The particles are then pushed together with an applicator stick, and the remaining blood is allowed to clot. The clot is then promptly fixed in Zenker solution, B5, or buffered formalin18 for tissue processing and sectioning. An alternative method employing filtration of anticoagulated aspirate specimen has been described.19
The core marrow biopsy specimen is processed for histologic examination by fixation in Zenker solution, B5 fixative, or neutral buffered formalin, followed by decalcification and embedding in paraffin. Sections of high quality cut at 4 µm and stained with hematoxylin and eosin or Giemsa stain are eminently satisfactory for routine work. Refinements in fixation and embedding techniques have made it possible to use most immunologic markers in decalcified paraffin-embedded marrow biopsy specimens.19 Fixation in neutral buffered formalin and embedding in plastic has the advantage of superior morphology20 and suitability for most immunochemical procedures,21 but is technically more demanding and expensive.22
MORPHOLOGIC INTERPRETATION OF MARROW PREPARATIONS
OVERVIEW
The Wright-Giemsa-stained direct marrow aspirate film should be examined as quickly as possible to provide a preliminary assessment of the marrow morphology and allow specialized testing based on this preliminary evaluation to be set up while the sample is still fresh. The final interpretation of the marrow biopsy and aspirate should be integrated with results from the blood film, cell counts, laboratory data, clinical history, cell marker studies, and molecular or cytogenetic data. There is no other histologic specimen in which a state-of-the-art interpretation is dependent on such an array of supportive data. This is a result of the wealth of basic biologic information gained from in vitro studies of blood cells which has been translated into useful diagnostic tests. The challenge for the hematopathologist and hematologist is to understand the advantages and limitations of each diagnostic approach, so that apparently conflicting results can be reconciled and put into perspective.
ADEQUACY OF THE MARROW SAMPLE
The first question in interpreting the marrow is whether the sample is adequate for diagnosis. The best indicator at the time of the procedure that the needle entered the medullary cavity and marrow was successfully withdrawn is the presence of marrow particles in the aspirate. Marrow particles are bony with a glistening appearance caused by fat in the particles. A biopsy specimen should minimally contain at least a 0.5-cm length of marrow cavity. Correlation of biopsy specimen length with positivity rate for metastatic neoplasia suggests that a length of at least 1.2 cm is preferred for this purpose.23 Specimens containing cortical bone, muscle, or other tissue with little or no medullary bone are inadequate for marrow interpretation (although they may provide other information). Also inadequate are samples with extensive crush artifact or hemorrhage, underscoring the importance of proper technique in obtaining a readable sample.
The marrow cavity was entered if the aspirate contains marrow particles or hematopoietic precursors (e.g., megakaryocytes, nucleated red cells) not found in the blood film. This does not ensure, however, that the specimen is adequate for diagnosis, since the amount of marrow actually aspirated can vary significantly in disease states, as discussed below.24 Also, some cell types, notably fibroblasts and metastatic tumor cells, are not as readily removed from the marrow space by aspiration as are normal precursors. Lack of particles or precursor cells does not prove that the marrow cavity was not entered, as marrow packed with leukemic cells or infiltrated with fibroblasts may yield a “dry tap.”14 Marrow aspirations resulting in a dry tap are usually due to significant pathology (only 7 percent show normal histology on biopsy14), indicating the necessity of examining a biopsy specimen in these cases.
An unspoken assumption is that the piece of marrow provided for diagnostic evaluation is representative of the marrow as a whole. This is generally a reasonable assumption, but studies employing bilateral biopsies,25 comparison with radiologic studies,26 or immunologic markers27 indicate that the focal nature of tumor deposits contributes to false-negative results of marrow biopsy staging.
BONE MARROW CELLULARITY
The “gold standard” for overall marrow cellularity is examination of an adequate marrow biopsy specimen.28,29 The normal cellularity (percent of the nonbony marrow space occupied by hematopoietic cells as opposed to fatty and nonhematopoietic tissue) of iliac crest marrow decreases from a mean of 80 percent in early childhood to 50 percent by age 30, with further decreases after age 70.30 Marrow cellularity should therefore be evaluated with reference to normal individuals of the same age as the patient.31 The normal range of iliac crest marrow cellularity is broader than one might expect.30 In evaluating cellularity, it must be remembered that marrow spaces directly adjacent to cortical bone are frequently fatty in the elderly and are not representative of the cellularity of the deeper marrow spaces.32
Cellularity assessment by examination of the direct marrow aspirate film is more difficult because of loss of histologic structure and mixture with blood. The aspirate may suggest that the marrow is more hypocellular than indicated by the biopsy.29 Marrow particles (seen in the direct film or a particle preparation) are the best indicators of cellularity. These particles are like “minibiopsies” and contain sufficient hematopoietic and fatty elements to give some idea of the cellularity of the marrow. Cellularity estimates from careful examination of particles in the aspirate preparation agree well with cellularity estimated from the marrow biopsy.31
The degree of dilution of marrow aspirate specimens with blood during the aspiration is quite variable and may affect interpretation of marrow cellularity. Adult marrows with over 30 percent lymphocytes plus monocytes are likely to be substantially admixed with blood, as shown by cytokinetic studies of paired marrow aspirate and biopsy preparations.33 Radiolabeled erythrocytes and serum albumin have been used to estimate the admixture of nucleated cells from blood with those from marrow in sternal marrow aspirates.24 In patients with hematologic disease, from 6 to 93 percent of the nucleated cells were derived from the blood.24 The greatest admixture occurred in patients with leukemia. Substantial dilution with blood may occur in difficult aspirates or when multiple draws are taken from the same puncture site. Based on cell markers and progenitor assays, the first 1.0 ml of marrow aspirated from healthy donors was found to be only 8 percent contaminated with peripheral blood nucleated cells, while subsequent aspirates performed for marrow harvesting were 20 percent contaminated with nucleated blood cells.34 The bulk volume of the “marrow” aspirate (i.e., plasma, red cells) is almost completely derived from blood, even if the nucleated cells are mostly marrow derived.34 Assessment of marrow cellularity by measuring the buffy coat observed after centrifugation of the aspirate specimen is unreliable.29
Cellularity of individual lineages is also best assessed by examination of the biopsy. Erythroid cells are typically arranged in clusters, while megakaryocytes are scattered throughout the biopsy. Erythroid and megakaryoctic cellularity is best appreciated at low power. In the aspirate, a myeloid/erythroid (M/E) ratio is frequently calculated to give some impression of the relative cellularity of these two major lineages. The rule of thumb is that this value should normally lie between 2:1 and 4:1 (for normal ranges in men and women, see Table 3-1). The relative proportions of cell types should be assessed only on the direct marrow film or particle preparation, not a concentrate film, which has been manipulated by centrifugation. A decreased M/E ratio could be interpreted as either myeloid hypocellularity or erythroid hyperplasia, depending on the overall marrow cellularity. Megakaryoctye numbers can be assessed in the direct marrow aspirate film, where there should be at least 5 megakaryocytes in the optimal portion of the smear. In the particle preparation, most large particles should contain one or more megakaryocytes. Megakaryocyte number varies markedly in direct marrow aspirate films of normal subjects35 (Table 3-1) and is dependent on the degree of admixture of the specimen with blood. Megakaryocytes are variably enriched in the feathered edge of concentrate films.

TABLE 3-1 NORMAL VALUES FOR MARROW DIFFERENTIAL CELL COUNT AT DIFFERENT AGES (PERCENT OF CELLS)**

INFILTRATIVE DISEASES OF THE MARROW
METASTATIC TUMOR
Metastatic nonhematopoietic tumor in the marrow biopsy is characterized by disruption of the marrow architecture with groups of cytologically abnormal cells. Assessment of the tissue of origin is primarily based on morphology, clinical history, and immunocytochemical staining. The tendency of carcinoma cells to form tightly adherent clusters is frequently helpful in recognizing these neoplasms. Such clumps can also appear on the marrow aspirate, but the aspirate is less sensitive than the biopsy for detection of metastatic tumor. Tumor clumps may be infrequent in the aspirate, often appearing only on side or feathered edges of the film or only in the concentrate preparation. These tumor clumps must be distinguished from clumps of damaged hematopoietic cells which commonly appear in aspirate preparations, especially the concentrate. This is best accomplished by examining cells at the periphery of the clumps to determine if they show the morphology of hematopoietic precursors or cytologically atypical cells. Isolated nonhematopoietic tumor cells are infrequent in aspirate preparations, even when tumor is obvious in the biopsy, due to the adherent nature of most nonhematopoietic tumors. Examination of multiple films may be necessary to find isolated tumor cell clumps.36
Myeloma37 and lymphomas8 are also more reliably detected in the biopsy preparation. Lymphoma cells frequently form abnormal lymphoid aggregates which must, however, be distinguished from lymphoid aggregates found in reactive conditions or in older patients.38 Neoplastic aggregates are more likely to show cytologic atypia, monomorphous cellular population, and are often adjacent to bony trabeculae, but the distinction can be difficult in some cases. The cellular morphology can often be appreciated better on the marrow aspirate, but the key histologic features are lost. Lymphoma cells do not form the tight clusters seen in nonhematopoietic tumors on the marrow aspirate smear. In hairy cell leukemia, however, the hematopoietic cells are sufficiently adherent to each other and the marrow matrix that aspirate specimens are often “dry,” while the biopsy shows extensive infiltration with tumor. Special studies such as in situ hybridization for kappa versus lambda light-chain mRNA39 or immunohistochemistry/flow cytometry to determine cell lineage and demonstrate surface light-chain restriction may be necessary to distinguish a reactive process from malignant lymphoma. Flow cytometry and morphologic examination of the bone marrow are complementary and can improve detection of lymphomatous involvement when used together.56
FIBROSIS
Bone marrow fibrosis is typically recognizable only on a marrow biopsy specimen, with the aspirate merely showing reduced or absent recovery of hematopoietic cells. Early stages of fibrosis are characterized by increased stainable marrow reticulin fibers. Fibrosis may accompany either primary hematopoietic disorders (e.g., myelofibrosis) or infiltrative diseases such as metastatic tumor.
STORAGE DISEASES
Storage disorders, such as Gaucher and Niemann-Pick diseases (described in Chap. 79), are characterized by abnormal macrophages containing stored material in various forms. These cells can be appreciated on both the biopsy and aspirate. In the latter preparation, they are typically more common in the feathered edge of the films. Reactive cells, such as the histiocytes with “sea-blue” inclusion granules or pseudo-Gaucher cells,40 which are seen in chronic myelogenous leukemia can resemble those seen in storage disorders.
INFECTIONS
Infectious organisms with an intracellular location, such as Leishmania,41 Histoplasma,45 and Toxoplasma,42 can be visualized in monocytic cells by morphologic examination of the marrow. Identification of mycobacterial organisms in the marrow by acid-fast staining lacks sensitivity but allows early diagnosis in one-third of cases of HIV-related Mycobacterium avium complex infection.43 Morphologic examination and culture of the marrow is the most sensitive diagnostic test for disseminated leishmaniasis, a troublesome problem in HIV-infected patients who are exposed to this organism.44 Marrow morphology is also a sensitive diagnostic tool for detection of disseminated histoplasmosis in patients with AIDS.45 The presence of marrow granulomas, recognizable only on biopsy specimens, necessitates examination by special stains for fungal and mycobacterial organisms, but the differential diagnosis is extensive.46
NECROSIS AND GELATINOUS TRANSFORMATION
Marrow necrosis may occur in a variety of disorders, particularly sickle cell disease and neoplastic processes involving the marrow.47 Aspirates of necrotic marrow stained with polychrome stains contain cells with indistinct margins and smudged basophilic nuclei surrounded by acidophilic material. In advanced necrosis all nuclei become acidophilic, with blurred outlines. Sections of marrow stained with hematoxylin and eosin or with polychrome stains show loss of normal marrow architecture, indistinct cellular margins, and a background of amorphous eosinophilic material. Patients with severe weight loss may develop gelatinous transformation of the marrow, characterized by amorphous extracellular material (proteoglycans), fat atrophy, and marrow hypoplasia.48 The findings of gelatinous transformation are reversible.49
DIFFERENTIATION OF THE HEMATOPOIETIC LINEAGES
OVERVIEW
The marrow aspirate films should be examined under low-power magnification to assess the relative amounts of fat and hematopoietic cells in particles and the number of megakaryocytes, plasma cells, and mast cells present. Low-power examination will also permit detection of osteoclasts or osteoblasts, groups of malignant cells, Gaucher cells, lymphoid follicles, and granulomas. The entire film should be examined, including the particles, and higher magnification should be employed to study any abnormalities discovered. Similarly, biopsy sections are examined at low power to assess adequacy, cellularity, presence of infiltrative disease, and cellularity of the major hematopoietic lineages.
After the low-power survey, the films should be examined under oil-immersion magnification to determine the various hematopoietic cell types present and assess adequacy of differentiation in each hematopoietic lineage. For most diagnostic questions, a careful and systematic visual examination of the marrow is sufficient to assess differentiation, but a marrow differential count can be performed to quantify the status of hematopoietic differentiation, particularly in the granulocytic lineages. Because a large variety of cell types are normally present in the marrow and their distribution is irregular, an accurate marrow differential count requires examination of 300 to 500 nucleated cells. Normal values for these determinations are presented in Table 3-1, including data for infants from birth to 18 months of age.50 Between birth and age 1 month there is an increase in lymphocytes and a decrease in erythroid and granulocytic precursors. After 1 month the marrow differential count varies little to age 18 months, the duration of the study.50 The proportion of polymorphonuclear neutrophils is increased with large volumes of aspirate, probably because of dilution of marrow cells by mature granulocytes in the blood.51 The range of normal for all cell types is broad, and differential counts and M/E ratios are to be considered rough guides to the character of the marrow as a whole.
Morphologically recognizable cells in the normal marrow include mature granulocytes and their precursors, erythroid precursors, lymphocytes in varying stages of development, plasma cells, monocytes, macrophages (histiocytes), stromal cells, megakaryocytes, osteoblasts, osteoclasts, and mast cells. It should be recognized that typically only the later stages of differentiation, in which progenitors become fully committed to a given lineage, are morphologically recognizable. The earliest committed progenitors of all lineages are typically rather unremarkable cells without distinctive morphologic attributes.
The morphologic characteristics of each cell type are briefly described below. Detailed descriptions of the normal development and differentiation in the major hematopoietic lineages are found in the specific chapters related to the erythroid (Chap. 22 and Chap. 29), granulocytic (Chap. 64, Chap. 65, Chap. 68, and Chap. 69), monocytic (Chap. 73 and Chap. 75), megakaryocytic (Chap. 110), and lymphoid (Chap. 80) series.
GRANULOCYTES
The term granulocytes is used to refer to the precursors and mature forms of leukocytes characterized by neutrophilic, eosinophilic, or basophilic granules in their cytoplasm in the more mature stages of development. This series is sometimes referred to as the myeloid series. The overall trend is a gradual decrease in nuclear size and enhanced clumping of nuclear chromatin as cells lose proliferative capacity, while granules of varying types progressively appear in the cytoplasm.
The myeloblast (Plate X-1) is round and large, about 14 to 18 µm in diameter on a dried film. The nucleus occupies most of the cell. The nuclear chromatin is very fine, and two to five nucleoli are present. The cytoplasm is basophilic but less so than that of the erythroid series. No granules are present.
The promyelocyte (progranulocyte) (Plate X-2) is larger than the myeloblast. The chromatin pattern is coarser than that of the myeloblast, but nucleoli are usually present. The cytoplasm is basophilic with a clear Golgi area and is characterized by a small number of prominent, large red granules. These are called primary, nonspecific, or azurophilic granules, and in the marrow they usually mark the cell as a granulocyte precursor, although similar-appearing granules (with different enzymatic composition, however) may occur in large lymphocytes.
The myelocyte (Plate X-7) is slightly smaller than the promyelocyte. This is the most mature dividing cell in the granulocytic lineage. Its nucleus is round or oval and is often located eccentrically. The chromatin pattern is coarser than that of the promyelocyte, and nucleoli are usually not visible. The defining feature is the presence of specific (secondary) granules in the cytoplasm, which identify the cell lineage. These may be neutrophilic (fine, variable size, lilac color), eosinophilic (larger, round, orange-red), or basophilic (larger still, irregular in size, deep blue). Based on the type of specific granules present, myelocytes, metamyelocytes, and bands are described as being either neutrophilic, eosinophilic, or basophilic. These granules first appear in the perinuclear area. The cytoplasm is only slightly basophilic.
The metamyelocyte (Plate X-8) is about the same size as the myelocyte and resembles it closely, except that the nucleus is indented, the chromatin is more coarse, and the cytoplasm is less basophilic.
The band cell (Plate X-6) is characterized by a nucleus which is horseshoe-shaped or lobulated but not segmented in that the rudimentary lobes are connected by a thick band of chromatin rather than the thin thread or filament which characterizes the mature polymorphonuclear leukocyte. The cytoplasm is yellowish pink or nearly colorless. Fine neutrophilic granules are abundant in the cytoplasm. Nuclear chromatin is dense, but less so than the segmented granulocyte.
Polymorphonuclear (segmented) granulocytes (Plate X-6) differ from the band cell by the multilobed character of the nucleus. At least two separate lobes are defined by a complete rounded shape, whether or not the thin filament joining them is seen. Nuclear chromatin is very dense. The mature eosinophil typically has only two lobes, while the neutrophil averages three to four lobes. Basophil nuclei are often obscured by the abundant basophilic granules.
MONOCYTES
Monocytes in normal marrow are identical morphologically to those in the blood (Plate VII). Promonocytes have more delicate chromatin, visible nucleoli, often a few fine granules, and somewhat more basophilic cytoplasm.
MACROPHAGES (HISTIOCYTES)
These cells are derived from monocytes but are larger, reaching 20 to 30 µm in the longest dimension. The nucleus is oval with delicate reticular chromatin and one or two small nucleoli. The cytoplasm ranges from blue-gray to pale and colorless and often contains phagocytosed cells, degenerating cell debris, and vacuoles. Normally, intact red cells are rarely visible inside marrow histiocytes. However, uncontrolled activation of histiocytic cells leads to a “hemophagocytic syndrome,” which is associated with a variety of neoplastic, viral, and reactive conditions.52
ERYTHROID CELLS
During erythroid differentiation, the nucleus progressively becomes smaller and nuclear chromatin more condensed, as the cell’s proliferative capacity decreases, while cytoplasm gradually loses the bluish color imparted by mRNA, replacing it with the pink-staining hemoglobin. Cells in the erythroid series are termed “erythroblasts” or “normoblasts.” The latter term was used to distinguish the normal sequence from that observed in megaloblastic anemia, in which the erythroid precursors are called “megaloblasts” because of their large size. It should be recognized that these stages are arbitrary divisions within a continuum of differentiation.
The proerythroblast (Plate V-1) is a large round cell measuring from 15 to 20 µm in diameter. The nucleus occupies most of the cell. The chromatin is present in a fine reticular or stippled pattern, but is more densely stained than the chromatin of the myeloblast. Nucleoli are present and are often bluish. The cytoplasm is typically more basophilic than the myeloblast.
The basophilic erythroblast (Plate V-2) is smaller than the proerythroblast, and the nucleus occupies less of the cell. The chromatin pattern is stippled, and the small, condensed masses of chromatin are sharply defined and separated by pale parachromatin. The cytoplasm is deeply basophilic.
The polychromatophilic erythroblast (Plate V-3) is smaller than the basophilic erythroblast. The nucleus occupies even less of the cell, and the chromatin pattern is more condensed, with larger masses of chromatin sharply defined by pale parachromatin. The cytoplasm is gray or grayish-pink due to the increasing amounts of hemoglobin.
The orthochromatic erythroblast (Plate V-4) is only slightly larger than the mature erythrocyte. The nucleus is small and pyknotic. The cytoplasm is red, like that of the mature erythrocyte.
The erythrocyte (Plate I-1) is the mature anucleate red cell. Polychromatophilic erythrocytes are mature anucleate red cells that are just released from the marrow and still have sufficient residual mRNA to impart a slight grayish tinge to the cytoplasm. The gray color of the cytoplasm is due to a combination of cytoplasmic RNA and hemoglobin.
MEGAKARYOCYTES
Megakaryocytes are large cells (30 to 150 µm) with darkly stained, irregularly lobed nuclei (Plate XI). The cytoplasm is blue cotton-candy-textured, and the more mature cells contain many purple-red granules. About half the megakaryocytes should have platelets adjacent to their periphery.
LYMPHOCYTES
In normal marrow lymphocytes similar to those found in the blood occur in variable numbers dependent on the degree of peripheral blood contamination of the marrow. Immature lymphoid cells with very high nuclear/cytoplasmic ratio and moderately dense but finely distributed chromatin are often seen in pediatric marrow aspirates and may cause diagnostic difficulty in some clinical settings, such as the “rebound” lymphocytosis that occurs after cessation of maintenance chemotherapy for acute lymphoblastic leukemia.53 These mostly represent varying stages of B-cell precursor development.54 Mature lymphocytes and smaller numbers of immature lymphoid forms are prominent in infant marrows but diminish in number with age.
PLASMA CELLS
Normal plasma cells vary somewhat in size but are usually 12 to 16 µm in diameter. They are round or oval. The nucleus is small, round, eccentrically placed, and stained densely purple. The chromatin is coarse and clumped. Nucleoli are not visible. The cytoplasm is deep blue, often with a paranuclear clear zone. Binucleate forms may be found in normal marrow (Plate XVI-1, Plate XVI-2, and Plate XVI-3).
OTHER CELL TYPES
Mast cells are readily recognized by their content of dark-blue granules, which usually completely fill the cytoplasm and may obscure the nucleus. The cells are round or spindle-shaped and are often located deep in the particles, frequently lying along blood vessels. The nucleus is often not visible, but, when seen, it is round or oval with a vesicular chromatin pattern (Plate VII-5).
Osteoclasts and osteoblasts are uncommon and are seen more frequently in marrow obtained from children and from adults with hyperparathyroidism or osteoblastic reactions to tumors (Plate XV). Osteoclasts are large cells and may be more than 100 µm in diameter. They superficially resemble megakaryocytes but contain multiple separated nuclei which have a moderately fine chromatin pattern with nucleoli. The cytoplasm varies from slightly basophilic to intensely eosinophilic due to the content of eosinophilic granules. Osteoclasts may contain coarse basophilic debris.
Osteoblasts are usually oval cells up to 30 µm in the longest diameter. They often occur in groups. The nucleus is usually eccentric and is relatively small. The chromatin pattern is uniform, and there are one to three nucleoli. The cytoplasm is light blue and may contain a few red granules. Osteoblasts may be mistaken for plasma cells. In osteoblasts the pale centrosomal region of the cytoplasm is separated from the nucleus, in contrast to that of the plasma cell, in which it abuts the nucleus directly.
EVALUATION OF IRON STORES
Marrow examination should include evaluation of the iron stores, especially if the patient is anemic. This is accomplished by staining a marrow film or section by the Prussian blue technique. Marrow macrophages are evaluated for storage iron, and erythroblasts are examined for the presence of iron granules in the cytoplasm (sideroblasts). In order to sensitively detect presence of iron stores in macrophages, a film containing marrow particles should be examined. Late erythroblasts are readily identified by their small size and the size, shape, and chromatin pattern of the nucleus. The proportion of late erythroblasts in normal subjects which contain one or more Prussian blue granules is extremely variable (3 to 69 percent).35 Abnormal sideroblasts are characterized by increased number (>5) or size of iron granules, particularly if these are arranged in a ring around the nucleus, reflecting accumulation of iron in mitochondria.
CHAPTER REFERENCES

1.
Piney A: Anatomy of bone marrow. Br Med 2:792, 1922.

2.
Custer RP, Ahlfeldt FE: Studies on the structure and function of bone marrow: II. Variations in cellularity in various bones with advancing years of life and their relative response to stimuli. J Lab Clin Med 17:960, 1932.

3.
Ellis LD, Jensen WN, Westerman MP: Needle biopsy of bone marrow: an experience with 1,445 biopsies. Arch Intern Med 114:213, 1964.

4.
Sabharwal BD, Malhotra V, Aruna S, Grewal R: Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections. J Postgrad Med 36:194, 1990.

5.
Bearden JD, Ratkin GA, Coltman CA: Comparison of the diagnostic value of bone marrow biopsy and bone marrow aspiration in neoplastic disease. J Clin Pathol 27:738, 1974.

6.
Pasquale D, Chikkappa G: Comparative evaluation of bone marrow aspirate particle smears, biopsy imprints, and biopsy sections. Am J Hematol 22:381, 1986.

7.
Kidd PG, Saminathan T, Drachtman RA, Ettinger LJ: Comparison of the cellularity and presence of residual leukemia in bone marrow aspirate and biopsy specimens in pediatric patients with acute lymphoblastic leukemia (ALL) at day 7–14 of chemotherapy. Med Pediatr Oncol 29:541, 1997.

8.
Montserrat E, Villamor N, Reverter JC, et al: Bone marrow assessment in B-cell chronic lymphocytic leukaemia: aspirate or biopsy? A comparative study in 258 patients. Br J Haematol 93:111, 1996.

9.
Winfield DA, Polacarz SU: Bone marrow histology 3: value of bone marrow core biopsies in acute leulemia, myelodysplastic syndromes, and chronic myeloid leukemia. J Clin Pathol 45:855, 1992.

10.
Bartl R, Frisch B, Wilmanns W: Potential of bone marrow biopsy in chronic myeloproliferative disorders (MPD). Eur J Haematol 50:41, 1993.

11.
Dunlop TJ, Deen C, Lind S, et al: Use of combined oral narcotic and benzodiazepine for control of pain associated with bone marrow examination. South Med J 92:477, 1999.

12.
Cannell H: Evidence for safety margins of lignocaine local anaesthetics for peri-oral use. Br Den J 181:243, 1996.

13.
Jamshidi K, Swaim WR: Bone marrow biopsy with unaltered architecture: a new biopsy device. J Lab Clin Med 77:335, 1971.

14.
Humphries J: Dry tap bone marrow aspiration: clinical significance. Am J Hematol 247, 1990.

15.
James L, Stass S, Schumacher H: Value of imprint preparation of bone marrow biopsies in hematologic diagnosis. Cancer 46:173, 1980.

16.
Dewald G, Allen JE, Strutzenberg DK: A cytogenetic method for mailed in bone marrow specimens for the study of hematologic disorders. Lab Med 13:225, 1982.

17.
Akoury DA, Seo JJ, James CD, Zaki SR: RT-PCR detection of mRNA recovered from archival glass slide smears. Mod Pathol 6:195, 1993.

18.
Lillie RD, Fullmer HM: Histopathologic Technique and Practical Histochemistry. McGraw-Hill, New York, 1976.

19.
Hyun BH, Stevenson AJ, Hanau CA: Fundamentals of bone marrow examination. Hematol Oncol Clin North Am 8:651, 1994.

20.
Moosavi H, Lichtman MA, Donnelly JA, Churukian CJ: Plastic-embedded human marrow biopsy specimens: improved histochemical methods. Arch Pathol Lab Med 105:269, 1981.

21.
Blythe D, Hand NM, Jackson P, et al: Use of methyl methacrylate resin for embedding bone marrow trephine biopsy specimens. J Clin Pathol 50:45, 1997.

22.
Brown DC, Gatter KC: The bone marrow trephine biopsy. A review of normal histology. Histopathology 22:411, 1992.

23.
Bishop PW, McNally K, Harris M: Audit of bone marrow trephines. J Clin Pathol 45:1105, 1992.

24.
Holdrinet RSG, Egmond J, Wessels JMC, Haanen C: A method for quantification of peripheral blood admixture in bone marrow aspirates. Exp Hematol 8:103, 1980.

25.
Juneja SK, Wolf MM, Cooper IA: Value of bilateral bone marrow biopsy specimens in non-Hodgkin’s lymphoma. J Clin Pathol 43:630, 1990.

26.
Perrin-Resche I, Bizais Y, Buhe T, Fiche M: How does iliac crest bone marrow biopsy compare with imaging in the detection of bone metastases in small cell lung cancer? EurJ Nucl Med 20:420, 1993.

27.
Cheung NK, Heller G, Kushner BH, et al: Detection of metastatic neuroblastoma in bone marrow: when is routine marrow histology insensitive? J Clin Oncol 15:2807, 1997.

28.
Ozkaynak MF, Scribano P, Gomperts E, et al: Comparative evaluation of the bone marrow by the volumetric method, particle smears, and biopsies in pediatric disorders. Am J Hematol 29:144, 1988.

29.
Gruppo RA, Lampkin BC, Granger S: Bone marrow cellularity determination: comparison of the biopsy, aspirate, and buffy coat. Blood 49:29, 1977.

30.
Hartsock RJ, Smith EB, Petty CS: Normal variations with aging of the amount of hemopoietic tissue in bone marrow from the anterior iliac crest. Am J Clin Path 43:326, 1965.

31.
Tuzuner N, Cox C, Rowe JM, Bennett JM: Bone marrow cellularity in myeloid stem cell disorders: impact of age correction. Leuk Res 18:559, 1994.

32.
Wilkins BS: Histology of normal haemopoiesis: bone marrow histology I. J Clin Pathol 45:645, 1992.

33.
Abrahamsen JF, Lund-Johansen F, Laerum OD, et al: Flow cytometric assessment of peripheral blood contamination and proliferative activity of human bone marrow cell populations. Cytometry 19:77, 1995.

34.
Batinic D, Marusic M, Pavletic Z, et al: Relationship between differing volumes of bone marrow aspirates and their cellular composition. Bone Marrow Transplant 6:103, 1990.

35.
Bain BJ: The bone marrow aspirate of healthy subjects. Br J Haematol 94:206, 1996.

36.
Atac B, Lawrence C, Goldberg S: Metastatic tumor: the complementary role of the marrow aspirate and biopsy. Am J Med Sci 302:211, 1991.

37.
Terpstra W, Lokhorst H, Blomjous F: Comparison of plasma cell infiltration in bone marrow biopsies and aspirates in patients with multiple myeloma. Br J Haematol 82:46, 1992.

38.
Navone R, Valpreda M, Pich A: Lymphoid nodules and nodular lymphoid hyperplasia in bone marrow biopsies. Acta Haematol 74:19, 1985.

39.
Erber WN, Asbahr HD, Phelps PN: In situ hybridization of immunoglobulin light chain mRNA on bone marrow trephines using biotinylated probes and the APAAP method. Pathology 25:63, 1993.

40.
Anastasi J, Musvee T, Roulston D, et al: Pseudo-Gaucher histiocytes identified up to 1 year after transplantation for CML are BCR/ABL-positive. Leukemia 12:233, 1998.

41.
Magill AJ, Grogl M, Gasser RA Jr, et al: Visceral infection caused by Leishmania tropica in veterans of Operation Desert Storm. N Engl J Med 328:1383, 1993.

42.
Brouland JP, Audouin J, Hofman P, et al: Bone marrow involvement by disseminated toxoplasmosis in acquired immunodeficiency syndrome: the value of bone marrow trephine biopsy and immunohistochemistry for the diagnosis. Hum Pathol 27:302, 1996.

43.
Hussong J, Peterson LR, Warren JR, Peterson LC: Detecting disseminated Mycobacterium avium complex infections in HIV- positive patients. The usefulness of bone marrow trephine biopsy specimens, aspirate cultures, and blood cultures. Am J Clin Pathol 110:806, 1998.

44.
Agostoni C, Dorigoni N, Malfitano A, et al: Mediterranean leishmaniasis in HIV-infected patients: epidemiological, clinical, and diagnostic features of 22 cases. Infection 26:93, 1998.

45.
Neubauer MA, Bodensteiner DC: Disseminated histoplasmosis in patients with AIDS. South Med J 85:1166, 1992.

46.
Eid A, Carion W, Nystrom JS: Differential diagnoses of bone marrow granuloma. West J Med 164:510, 1996.

47.
Norgard MJ, Carpenter JTJ, Conrad ME: Bone marrow necrosis and degeneration. Arch Intern Med 139:905, 1979.

48.
Seaman JP, Kjeldsberg CR, Linker A: Gelatinous transformation of the bone marrow. Hum Pathol 9:685, 1978.

49.
Tavassoli M, Eastlund DT, Yam LT, et al: Gelatinous transformation of bone marrow in prolonged self-induced starvation. Scand J Haematol 16:311, 1976.

50.
Rosse C, Krauner MJ, Dillon TL, et al: Bone marrow cell populations of normal infants: the predominance of lymphocytes. J Lab Clin Med 89:1225, 1977.

51.
Dresch C, Faille A, Poirier O, Kadouche J: The cellular composition of the granylocyte series in the normal human bone marrow according to the volume of the sample. J Clin Pathol 27:106, 1974.

52.
Janka G, Imashuku S, Elinder G, et al: Infection- and malignancy-associated hemophagocytic syndromes. Secondary hemophagocytic lymphohistiocytosis. Hematol Oncol Clin North Am 12:435, 1998.

53.
Pritchard-Jones K, Toogood IR, Rice MS: The significance of an M2 bone marrow at cessation of chemotherapy in childhood acute lymphoblastic leukemia. Am J Pediatr Hematol Oncol 10:292, 1988.

54.
Longacre TA, Foucar K, Crago S, et al: Hematogones: a multiparameter analysis of bone marrow precursor cells. Blood 73:543, 1989.

55.
Glaser K, Limarzi LR, Poncher HG: Cellular composition of the bone marrow in normal infants and children. Pediatrics 6:789, 1950.

56.
Duggan PR, Easton D, Linder J, Auer IA: Bone marrow staging of patients with non-Hodgkin lymphoma by flow cytometry: correlation with morphology. Cancer 88:894, 2000.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

CHAPTER 2 EXAMINATION OF THE BLOOD

CHAPTER 2 EXAMINATION OF THE BLOOD
Williams Hematology

CHAPTER 2 EXAMINATION OF THE BLOOD

DANIEL H. RYAN

Quantitative Measures of Hematopoietic Elements in the Blood

Red Cells

Leukocytes

Platelets
Morphologic Examination of the Blood

Red Cell Morphology

Platelet Morphology

Leukocyte Morphology

Leukocyte Inclusions

Leukocyte Artifacts

The Need for Examination of the Blood Film
Chapter References

Examination of the blood is central to the diagnosis and management of hematologic diseases. In few other disciplines can the physician make a specific diagnosis and monitor therapy with easily accessible tissue samples and readily available methodologies, many of which can be performed in a physician’s office. Assessment of the prevalence of red cells, of the several types of leukocytes, and of platelets, usually from automated particle counters, and examination of the blood film for qualitative changes in the appearance of red cells, leukocytes, and platelets, and the presence of marrow precursors, malignant cells, and intracellular parasites can be used to diagnose specific diseases, gain insight into pathophysiology, and measure the response to treatment.

Acronyms and abbreviations that appear in this chapter include: MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MPV, mean platelet volume; PDW, platelet volume distribution width; RBCs, red blood cells; RDW, red cell distribution width.

The blood is examined in order to answer two principal questions: Is the marrow producing sufficient numbers of mature cells in the hematopoietic lineages? Is the development of each hematopoietic lineage qualitatively normal? Quantitative measures routinely available from automated cell counters are generally reliable and provide a rapid and cost-effective way to screen for major disturbances of hematopoiesis. Morphologic observation of the blood film is essential to confirm certain quantitative results and to investigate qualitatively abnormal differentiation of the hematopoeitic lineages. Based on examination of the blood, the physician is directed toward a more focused assessment of the marrow or to systemic disorders which secondarily involve the hematopoietic system. Table 2-1 lists blood cell values in a normal population.

TABLE 2-1 BLOOD CELL VALUES IN A NORMAL POPULATION*

The complete blood count is a necessary part of the diagnostic workup in a broad variety of clinical conditions. Similarly, the leukocyte differential count and examination of the blood film, in spite of limitations as a screening test for occult disease,1 is important in sorting out the differential diagnosis in most ill patients. Quantitative and morphologic examination of the formed elements of the peripheral blood are for convenience considered separately in this chapter, but it should be understood that the distinction between these two is not absolute, and measures once strictly confined to the “qualitative” realm can become quantifiable and routinely measurable as technology advances.
QUANTITATIVE MEASURES OF HEMATOPOIETIC ELEMENTS IN THE BLOOD
In a typical automated blood cell counter, the aspirated blood sample is separated into two portions, one of which is lysed and diluted to permit measurement of hemoglobin concentration and leukocyte enumeration, and the other which is diluted without lysis to enable counting and sizing of red cells and platelets. Some recently developed instruments offer automated reticulocyte counting as well.
RED CELLS
Most automated blood cell counters measure the red cell count, MCV (mean corpuscular volume), and hemoglobin concentration directly. All other red cell parameters, including the hematocrit, are derived from these primary values. The red cell count is most commonly measured by passing a well-mixed sample of blood diluted in an electrolyte solution through a small orifice through which the electrical impedance can be measured.2 Each cell causes a jump in impedance as it passes through the opening, since it cannot readily conduct an electrical signal through its lipid membrane. Red cells are distinguished from platelets by the magnitude of the impedance signal, which is proportional to cell size. Alternatively, red cells and other hematopoietic elements can be counted and sized by measuring the intensity and angle at which laser light is scattered as the cells pass by.
MEASUREMENT OF THE RED CELL COUNT AND HEMATOCRIT
In electronic instruments the hematocrit (proportion of blood volume occupied by erythrocytes) is calculated from direct measurements of the erythrocyte count and the mean corpuscular volume: (Hct (µl/100 µl) = [RBC in millions per µl × MCV in fl] ¸ 10). Falsely elevated MCV and decreased red cell counts can be observed when red cell autoantibodies are present and retain binding capability at room temperature, particularly cold agglutinins and in some cases of autoimmune hemolytic anemia.3 This causes red cells to clump and, by affecting the accuracy of both RBC count and MCV, also affects the derived hematocrit.
The hematocrit may also be determined by subjecting the blood to sufficient centrifugal force to pack the cells into as small a volume as possible.4 Before standardized methods for hemoglobin quantitation were available, the hematocrit was the best method of determining adequacy of red cell production. However, the “spun” hematocrit is a manual procedure not well adapted to routine processing in a high-volume clinical laboratory. The microhematocrit includes plasma trapped between red cells in the packed cell volume,5 which is a source of systematic bias between the “spun” and automated hematocrit. The amount of plasma remaining in the packed cells is typically about 2 to 3 percent.6,7 Microhematocrits from blood containing abnormal erythrocytes (sickle cell anemia, thalassemia, iron deficiency, spherocytosis, macrocytosis) are relatively increased because of enhanced plasma trapping that generally is due to increased red cell rigidity.6,7 Fully oxygenated blood also has about a 2 percent lower hematocrit than deoxygenated blood.8 In polycythemic samples (Hct > 55), plasma trapping is commonly increased. Therefore, although automated hematocrit values are adjusted to be equivalent to spun hematocrit for normal samples, in abnormal samples, the spun hematocrit may be artifactually elevated (up to 6% in microcytosis9). In general, the automated hematocrit is more accurate and easier to routinely obtain than the spun hematocrit, although the hemoglobin determination is preferred to either, as it is directly measured.
MEASUREMENT OF HEMOGLOBIN
Hemoglobin is intensely colored, and this property has been utilized in methods for estimating its concentration in blood. Erythrocytes contain a mixture of hemoglobin, oxyhemoglobin, carboxyhemoglobin, methemoglobin, and minor amounts of other forms of hemoglobin. To determine hemoglobin concentration in the peripheral blood, red cells are lysed and hemoglobin variants are converted to the stable compound cyanmethemoglobin for quantitation by absorption at 540 nm.10 All forms of hemoglobin are readily converted to cyanmethemoglobin except sulfhemoglobin, which is rarely present in significant amounts. In automated blood cell counters, hemoglobin is accurately and directly measured, and hence this determination is preferable to the hematocrit for the diagnosis of anemia. In practice, the major interference with this measurement is chylomicronemia.11 Methodological improvements in recent instrumentation may minimize this interference.12
The hemoglobin level varies with age (see Table 2-2). Changes in hemoglobin in the neonatal period are discussed in Chap. 7. After the first week or two of extrauterine life, the hemoglobin falls from levels of about 17 g/dl to levels of about 12 g/dl by 2 months of age. Thereafter the levels remain relatively constant throughout the first year of life. Any child with a hemoglobin level below 11 g/dl should be considered to be anemic.13,14 Changes in hemoglobin levels in the elderly are discussed in Chap. 8.

TABLE 2-2 NORMAL LEUKOCYTE COUNT, DIFFERENTIAL COUNT, AND HEMOGLOBIN CONCENTRATION AT VARIOUS AGES*

DETERMINATION OF SIZE AND HEMOGLOBIN CONTENT OF ERYTHROCYTES (RED CELL INDICES)
The size and hemoglobin content of erythrocytes (red cell indices) have traditionally been used to assist in the differential diagnosis of anemia.15 In current practice, the most useful parameter is the MCV.16,17
Automated blood counters measure the MCV directly using the Coulter principle,2 in which the cross-sectional area of a nonconducting particle (i.e., any cell) in an electrolyte solution is proportional to the increase in electrical impedance as the particle passes through a narrow orifice.18 The MCV has been used to guide the diagnostic workup in patients with anemia, for example, testing patients with microcytic anemia for iron deficiency or thalassemia,17 and those with macrocytic anemia for folate or B12 deficiency.19 This assumption has practical value, but its limitations should be recognized,20 for instance, in elderly patients with megaloblastic anemia, who may have an MCV in the normal range.21 In about one-third of elderly patients, the cause of an elevated MCV is not evident.22 Numerous mathematical manipulations of the red cell indices, particularly the MCV and red cell count, have been devised to assist in the differential diagnosis of iron deficiency anemia and thalassemia,23 but their utility has been questioned24 due to significant overlap and the availability of more definitive tests for these conditions.
The other red cell indices are less useful in clinical decision-making. The MCH (mean corpuscular hemoglobin, or the amount of hemoglobin per red cell) is calculated by the formula MCH (pg/cell) = [hemoglobin in g/dl ¸ red cell count in millions/µl] × 10. Changes in MCH accompany similar alterations in the MCV and generally provide little additional diagnostic information. The MCHC (mean corpuscular hemoglobin concentration, or the concentration of hemoglobin in the red cell volume) is calculated by the formula MCHC (g/dl) = [hemoglobin in g/dl ¸ hematocrit in µl/100 µl] × 100. An MCHC greater than 35 has been associated with hereditary spherocytosis,25 and low MCHC is typical of iron deficiency,26 but its diagnostic usefulness is limited.27 The dynamic range of the MCHC measurement in most automated instruments is limited by technical considerations,28 but technical improvements in newer instruments may improve the usefulness of this parameter.12 In the clinical laboratory, the MCHC is useful as a warning of potential interferences with the measurement of MCV or RBC count. For instance, an abnormally low MCHC suggests the possibility of artifactually high MCV due to osmotic shifts that occur when red cells from patients with severe hyperglycemia are diluted in saline prior to analysis.29
The MCV, MCH, and MCHC are average quantities and therefore may not detect abnormalities in blood with mixed-cell populations. For example, patients with sideroblastic anemia usually have a dimorphic blood picture, with both hypochromic and normochromic cells. The indices may be in the normal range, and the important finding of the mixed-cell population would not be detected. It is possible to identify mixed populations by direct examination of a histogram of MCV (or red cell hemoglobin concentration, in instruments that measure this parameter on individual cells30) values for individual cells that is printed out by the instrument but typically not included in the laboratory report. Another index, the red cell distribution width (RDW), is specifically designed to reflect the variability of red cell size. It is based on the width of the red blood cell volume distribution curve, with larger values indicating greater variability. An elevated RDW may be an early sign of iron deficiency anemia,31,32 and, although proposed as an aid in distinguishing iron deficiency from other causes of microcytic anemia,33 the RDW is not sufficiently diagnostic to obviate the need for more specific tests.34 The RDW can be used in the laboratory as a flag to select which samples submitted for automated blood count should have manual review of the blood film for red cell morphology.
As with any laboratory test, the clinical use of these red cell parameters depends on the prevalence of disease and the clinical setting. For instance, the Centers for Disease Control recommends routine hemoglobin screening and a 1-month therapeutic trial of oral iron for those with anemia in populations at particularly high risk of iron-deficiency anemia (9- to 18-month-old infants, pregnant women). In the absence of clinical evidence for other causes of anemia, a further workup beyond hemoglobin measurement is recommended only if the hemoglobin is not increased by at least 1 g/dl during the therapeutic trial.35 In contrast, for other populations, anemia detected during routine medical examinations should be fully evaluated for its cause.35
LEUKOCYTES
LEUKOCYTE COUNT
Leukocyte counts are performed by automated blood counters on blood samples appropriately diluted with a solution that lyses the erythrocytes (e.g., acid or a detergent) but preserves leukocyte integrity. Manual counting of leukocytes is used only when the instrument reports a potential interference or the count is beyond instrument linearity limits. Manual counts are subject to much greater technical variation than automated counts due to technical and statistical factors. Leukocyte counts may be falsely elevated due to cryoglobulins or cryofibrinogen,36 clumped platelets or fibrin from an inadequately anticoagulated or mixed sample,37 EDTA-induced platelet aggregation,38 nucleated red blood cells (RBCs),37 or nonlysed RBCs. These interferences cause a population of small-sized particles to appear in the leukocyte volume histogram and trigger a flag for manual review.39
LEUKOCYTE DIFFERENTIAL
Leukocytes in the peripheral blood serve different functions and arise from different hematopoietic lineages, so it is important to separately evaluate each of the major leukocyte types. The size differences among lymphocytes, monocytes, and neutrophils were initially used to produce a “three-part” leukocyte differential. Modern automated instruments use additional parameters (typically light scatter at different angles or electrical conductivity) to identify and enumerate the five major morphologic leukocyte types in peripheral blood. Complex algorithms flag samples likely to contain abnormal cells (or variants such as immature granulocytes and reactive lymphocytes) for manual review.40 “Band neutrophils” cannot be specifically identified by any current automated cell counter but usually trigger a manual review flag if present in increased numbers. Current instruments can perform an accurate automated “five-part” differential without need for manual review in about 50 to 80 percent of samples from medical center patient populations.40,41 It should be recognized, however, that small numbers of abnormal cells can escape detection by either automated or manual methods. The false negative rate for detection of abnormal cells varies from 1 to 20 percent, depending on the instrument and the detection limit desired (1–5% abnormal cells).42,43 and 44 Lymphoma cells and reactive lymphocytes are the most problematic41 for both automated instruments and the human observer. If one needs to search for infrequent abnormal cells or evaluate leukocyte morphology, there is still no substitute for examination of a properly stained blood film by a trained observer. In spite of instrumentation that permits automated analysis of a majority of clinical samples, the test is still quite labor-intensive relative to other high-volume laboratory tests, and its value as a case-finding tool in screening of asymptomatic patients has been questioned.1,45
The normal differential leukocyte count varies with age (see Table 2-2). As described in Chap. 7, in the first few days after birth polymorphonuclear neutrophils are predominant, but thereafter lymphocytes account for the majority of leukocytes. This persists up to about 4 to 5 years of age, when the polymorphonuclear leukocyte again becomes the predominant cell and remains so throughout the rest of childhood and adult life. Changes in the leukocyte count in the elderly are discussed in Chap. 8. The leukocyte count may decrease slightly in older subjects because of a fall in the lymphocyte count. The reference range for neutrophil counts is lower in African Americans, Africans, and some Middle Eastern populations than caucasians (Table 2-3).46,47,48 and 49

TABLE 2-3 ETHNIC DIFFERENCES IN NORMAL BLOOD CELL VALUES

PLATELETS
Platelets are usually counted electronically by enumerating particles in the unlysed sample within a specified volume window (e.g., 2–20 fl). The platelet count was more difficult to automate than the red cell count, because of the small size, tendency to aggregate, and potential overlap of platelets with more numerous red cells. Current instruments typically construct a platelet volume histogram based on measured platelet size within the platelet volume window and mathematically extrapolate this histogram to account for platelets whose size overlaps with debris or small red cells. The normal platelet count is lower in individuals of African ethnic origin49 (Table 2-3).
Since platelet volumes in health or disease follow a log-normal distribution,50 volume histograms not consistent with such a distribution are flagged for manual review. Automated platelet counting by current instrumentation is accurate and reliable, even in the thrombocytopenic range,51 and far more precise than manual methods.51 Platelet counts by either manual or automated methods may be falsely decreased if the sample is incompletely anticoagulated (often indicated by small clots in the specimen or fibrin strands on the stained film). Infrequently, it may be necessary to confirm automated results by a manual (phase contrast) platelet count or platelet estimate from the blood film when potential interferences are present. These include severe microcytosis and leukocyte fragmentation (falsely elevated count) or platelet clumping/“satellitism” (falsely decreased count). Current instruments are able to identify and flag samples when these interferences are present. Some newer automated cell counters incorporate novel approaches, such as staining with antiglycoprotein IIIa antibody or volume/refractive index two-parameter measurement, to minimize the need for manual review of the platelet count.12 Platelet clumping, or platelet satellitism (adherence of platelets to neutrophils), may occur due to platelet reactive antibodies,52 which cause no clinical symptoms. Paradoxically, these antibodies recognize epitopes on adhesion molecules which are exposed in the absence of divalent cations, and so become activated in EDTA- or citrate-anticoagulated blood specimens.52 This condition occurs in about 0.1 percent of hospitalized patients, and the origin of the thrombocytopenia in such cases can be suspected by the appearance of small particles (representing the platelet clumps) on the leukocyte volume histogram.39 Platelet counting under these conditions is difficult but can be minimized by collecting blood in citrate39 or estimating platelet count from a freshly prepared fingerstick blood smear.
Platelet volume is measured in the same fashion as red cell size, and the mean platelet volume (MPV) has been proposed as a clinically useful tool in the differential diagnosis of thrombocytopenias53 and as a risk factor for thrombotic disease.54,55 Increased MPV may be related in a complex way to thrombopoietic stimulus56 and not platelet age per se.57 However, in spite of the known association of increased platelet size on blood films with consumptive thrombocytopenias, platelet size is a difficult parameter to accurately quantitate and use diagnostically, because of a wide physiologic variation of the MPV in normal subjects (i.e., Mediterranean macrothrombocytopenia58,59) and susceptibility of anticoagulated platelets to time-dependent swelling in vitro.60 A platelet volume distribution width (PDW) can be calculated just as the RDW and is correlated with platelet count and MPV.61 This measurement has yet to find an established clinical use, although a higher-than-expected PDW has been observed in thrombocytoses due to myeloproliferative disease.61
MORPHOLOGIC EXAMINATION OF THE BLOOD
Microscopic examination of the blood spread on a glass slide or coverslip yields useful information regarding all the formed elements of the blood. The process of preparing a thin blood film causes mechanical trauma to the cells. Also, the cells flatten on the glass during drying, and the fixation and staining involve exposure to methanol and water. Some artifacts are inevitably introduced, but these can be minimized by good technique. The optimal part of the stained blood film to use for morphologic examination of the formed blood elements should be sufficiently thin that only a few erythrocytes in a 100× field touch each other but not so thin that no red cells are touching. Selection of a portion of the blood film for analysis that is too thick or too thin for proper morphologic evaluation is by far the most common error in blood film interpretation. For example, leukemic blasts may appear dense and rounded and lose their characteristic features when viewed in the thick part of the film. For specific purposes, the thick portion or side and “feathered” edges of the film are of interest (for instance, to detect microfilariae and malarial parasites or to search for large abnormal cells and platelet clumps). It is sometimes advantageous to examine fresh blood diluted in saline under the microscope to avoid artifacts of fixation or staining which may mimic spherocytosis or acanthocytosis.
The blood film is first scanned at medium power (×20) to confirm reasonably even distribution of leukocytes and check for abnormally large or immature cells in the side and feathered edges of the film. The feathered edge is examined for platelet clumps. Abnormal cells, red cell aggregation or rouleaux, background bluish staining consistent with paraproteinemia, and parasites are all findings that can be suggested by medium-power examination. The optimal portion of the film is then examined at high power (50–100×, oil immersion) to systematically assess the size, shape, and morphology of the major hematopoietic lineages.
RED CELL MORPHOLOGY
Erythrocytes should be examined for size, shape, hemoglobin concentration and distribution, staining properties, distribution on the film, and inclusions (see Plate I, Plate II, Plate III and Plate IV).
Normal erythrocytes on dried films are nearly uniform in size, with a normal distribution about a mean of 7.2 to 7.9 µm. Erythrocyte diameter can be evaluated by the use of a micrometer disc inserted into the microscope, although experienced morphologists usually evaluate erythrocyte size without this aid. It is helpful to compare erythrocyte size with the similar diameter of small lymphocyte nuclei. Note that the MCV is a more sensitive measure of red cell volume than the red cell diameter. However, an experienced observer should be able to recognize abnormalities in average red cell size when the MCV is markedly elevated or decreased. Anisocytosis is used to describe variation in erythrocyte size and is the morphologic correlate of the RDW. Macrocytes may be seen in a number of disease states. Cells are considered to be macrocytes if they are well hemoglobinized and their diameters exceed 9 µm. Early (“shift”) reticulocytes (i.e., those with the most residual RNA) appear in stained films as large, bluish cells, often referred to as polychromatophilic cells. Microcyte is the term used to describe a cell less than 6 µm in diameter.
The normal erythrocyte on a dried film is round with central pallor. Poikilocytosis is a term used to describe variations in the shape of erythrocytes. The predominant appearance of a specific abnormality in red cell shape can be an important diagnostic clue in patients with anemia. These are described in detail in Chap. 22 and Plate I, Plate II, Plate III and Plate IV. Erythrocytes with evenly spaced spikes (crenated cells) can be an artifact caused by prolonged storage or may reflect metabolic erythrocyte abnormalities.
The normal erythrocyte appears as a disc with a rim of hemoglobin and a clear central area. The central pallor normally occupies less than one-half the diameter of the cells. Increased central pallor (hypochromia) is associated with disorders characterized by diminished hemoglobin synthesis. Evaluation of red cell hemoglobinization as well as red cell size is complelety dependent on examining the proper part of the blood film. Cells at the far feathered edge will always be large and lack central pallor, while cells in the thick part of the film will look small and rounded and will also lack central pallor. A sharp refractile border demarcating the central area of pallor is an artifact secondary to inadequate drying of the film before staining (due to high humidity, and more common in anemic samples). Spherocytes are more densely stained and appear smaller because of their rounded shape and will show decreased or absent central pallor. Some automated blood counters produce a histogram of red cell hemoglobin concentration that identifies hypochromic, normochromic, and hyperchromic populations.62 The hemoglobin may appear to be abnormally distributed in erythrocytes, particularly in a form of cell in which there is a spot or disc of hemoglobin in the center surrounded by a clear area which is in turn surrounded by a rim of hemoglobin at the outer edge of the cell, giving the appearance of a target—a target cell. This is in reality a cup-shaped cell which is distorted as it is flattened on the glass slide. These cells are typically found in disorders of hemoglobin synthesis (e.g., thalassemia, iron deficiency), where the cell surface to cell volume ratio is high.
Erythrocytes are usually distributed evenly throughout the film. In some films the cells become aligned in aggregates (rouleaux) resembling stacks of coins. Such rouleaux formation is normal in the thicker part of the film; when found in the optimal viewing portion of the film, it may be due to the presence of a paraprotein and suggests the diagnosis of plasma cell myeloma or macroglobulinemia.
Inclusions that may be observed in erythrocytes on films stained with Wright stain are described in Chap. 22.
PLATELET MORPHOLOGY
Platelets appear in normal stained blood as small blue or colorless bodies with red or purple granules (see Plate XII and Plate XIII). Normal platelets average about 1 to 2 µm in diameter but show wide variation in shape, from round to elongated, cigar-shaped forms. A rough estimate of the platelet count can be made by observation of the stained blood film. If the platelet count is normal, approximately 8 to 15 platelets (individually or in small clumps) should be visible in each 100× oil-immersion field. There should be one platelet present for every 10 to 30 erythrocytes. This is a valuable check when the automated platelet count is in question or an unexpected result is obtained.
In improperly prepared films, platelets may form large aggregates in some areas and appear to be diminished or absent in others. The occurrence of giant platelets or platelet masses may indicate a myeloproliferative disorder (see Chap. 118) or improper collection of the blood specimen. The latter circumstance can occur when venipuncture technique is faulty and platelets become activated before the blood sample is thoroughly mixed with anticoagulant. These platelet masses are readily recognized (typically in the feathered edge of the film), but this maldistribution may create a mistaken impression of thrombocytopenia if the aggregates are not detected. Platelet clumping throughout the blood film, or platelet satellitism (adherence of platelets to neutrophils), may be due to platelet agglutinins as previously discussed (see Plate XII and Plate XIII).
A platelet will occasionally overlie an erythrocyte, where it may be mistaken for an inclusion body or a parasite. The differentiation depends on the observation of a halo around the platelet, determination that it lies above the plane of the erythrocyte, and observation of the characteristics of a normal platelet in the “inclusion.”
LEUKOCYTE MORPHOLOGY
The distribution of leukocytes on glass slides is not uniform, and the larger cells, such as monocytes and polymorphonuclear leukocytes, tend to be concentrated on the side and feathered edges of the film. The cells that are normally found in blood are polymorphonuclear leukocytes of the neutrophilic, eosinophilic, and basophilic types; lymphocytes; and monocytes (see Plate VII and Plate VIII). These cell types are described below, and normal values for the differential count are presented in Table 2-2.
Neutrophils are round cells ranging from 10 to 14 µm in diameter (see Plate VII). The nucleus is lobulated, with two to five lobes connected by a thin chromatin thread. The defining feature of the mature neutrophil is the round lobes with condensed chromatin, since the chromatin thread may overlie the nucleus and not be visible. The chromatin stains purple and is coarse and arranged in clumps. The nucleus of 1 to 16 percent of the neutrophils from females may have an appendage that is shaped like a drumstick and is attached to one lobe by a strand of chromatin (see Plate VII). Nuclear spicules or appendages attached by a broad base occur in normal individuals but may be increased in number in chronic illnesses or after cytotoxic or radiation therapy.63 The cytoplasm is clear and contains many small tan to pink granules distributed evenly throughout the cell, although they may not be apparent when they lie over the nucleus.
Bands are identical to mature polymorphonuclear leukocytes except that the nucleus is U-shaped or has rudimentary lobes connected by a band containing chromatin rather than by a thin thread (see Plate VII). The nuclear chromatin is slightly less condensed than the mature neutrophil.
Eosinophils are on the average slightly larger than neutrophils. The nucleus usually has only two lobes. The chromatin pattern is the same as that in the neutrophil, but the nucleus tends to be more lightly stained. The differentiating characteristic of these cells is the presence of many refractile, orange-red granules that are distributed evenly throughout the cell and may be visible overlying the nucleus (see Plate VII). These granules are larger than those in the neutrophil and are more uniform in size. Occasionally some of the granules in eosinophils stain light blue rather than orange-red.
Basophils are similar to the other polymorphonuclear cells and are slightly smaller than neutrophils. The nucleus may stain more faintly and usually is less segmented and has less distinct chromatin condensation than is the case in neutrophils. The large deeply basophilic granules are fewer in number and less regular in size and shape than in the eosinophil. The granules are visible overlying the nucleus and, in some cells, almost completely obscure the lightly stained nuclear chromatin. Because the granular constituents are water-soluble, some granules may stain only faintly or not at all (see Plate V).
Lymphocytes on blood films are usually small, about 10 µm in diameter, but larger forms up to 20 µm in diameter are seen. The small lymphocyte, the predominant type in normal blood, is round and contains a relatively large, round, densely stained nucleus in which the chromatin is distributed in coarse masses (see Plate V). The cytoplasm is scanty and stains pale to dark blue. In the large lymphocytes the nuclear/cytoplasmic ratio is lower and the chromatin is less condensed than in the small lymphocytes. The nucleus is usually round but may be oval or indented. The cytoplasm is abundant and may contain a few azurophilic granules. Large lymphocytes containing azurophilic granules and relatively abundant cytoplasm are designated large granular lymphocytes and often represent cytotoxic T cells or NK cells. Reactive lymphocytes, as seen in viral infections caused by EBV, CMV, adenovirus, or other organisms, are large with indented nuclei and abundant blue cytoplasm. Nuclear chromatin condensation is variable, and nucleoli may be evident. A low nuclear/cytoplasmic ratio distinguishes these reactive T lymphocytes from neoplastic cells.
Monocytes are the largest normal cells in the blood, usually measuring from 15 to 22 µm in diameter. The nucleus is of various shapes—round, kidney-shaped, oval, or lobulated—and frequently appears to be folded (see Plate VII). The chromatin is arranged in fine strands with sharply defined margins. The cytoplasm is light blue or gray, contains numerous fine lilac or purple granules, and is frequently vacuolated, especially in films made from blood anticoagulated with EDTA. The gray (as opposed to blue) color of monocyte cytoplasm is due to fine granules (staining pink) seen on the background of RNA-containing cytoplasm (staining blue) and helps to distinguish between monocytes and reactive lymphocytes. The monocyte nuclear chromatin contains a fine stringlike structure as opposed to the coarser clumps of the lymphoid chromatin. Nuclear shape and cytoplasmic vacuolation are less reliable distinguishing features between monocytic and lymphoid cells.
LEUKOCYTE INCLUSIONS
Leukocytes may contain abnormal inclusions as a result of genetic or acquired disorders.
ABNORMAL GRANULES
In patients with conditions associated with a systemic inflammatory reaction, neutrophil granules may appear larger than normal and stain more darkly, often assuming a dark blue-black color. This has been called toxic granulation. These granules can be confused with the larger granules of basophils. In mucopolysaccharidoses, coarse, dark granules may be found in the neutrophils (the Alder-Reilly anomaly), and large azurophilic granules are often found in some lymphocytes (Gasser cells) and monocytes. Huge misshapen granules are found in the polymorphonuclear leukocytes, and giant azurophilic granules are present in the lymphocytes of patients exhibiting the Chédiak-Higashi anomaly (see Chap. 72).64 Auer rods are sharply outlined, red-staining rods found in the cytoplasm in blast cells, and occasionally in more mature leukemic cells, in the blood of some patients with acute myelogenous leukemia (see Plate XVI).
ABNORMAL RNA AGGREGATIONS
Light blue round or oval bodies about 1 to 2 µm in diameter may be seen in the cytoplasm of neutrophils of patients with infections, burns, and other inflammatory states. These have been named Döhle bodies. The blue staining is due to RNA, since it is blocked by treating the slide with ribonuclease prior to staining. Ultrastructurally, Döhle bodies contain rough-surfaced endoplasmic reticulum. Similar blue inclusions are seen in patients with the May-Hegglin anomaly. The staining of May-Hegglin inclusions is also attributable to RNA, but ultrastructurally they differ from Döhle bodies, suggesting alterations in the RNA.65
LEUKOCYTE ARTIFACTS
CRUSHED (“SMUDGE,” “BASKET”) CELLS
During the process of preparing the film, leukocytes may be damaged, with consequent alteration in their appearance and staining. In some damaged leukocytes the nucleus appears enlarged, with alteration of the chromatin so that the strands appear more homogeneous, stain with a distinct reddish hue, and are more widely separated; the cytoplasm may or may not appear intact. Such cells may appear to have a large blue nucleolus. There is no specific association with disease other than chronic lymphocytic leukemia, where the neoplastic lymphocytes are fragile and smudge cells are frequent.
RADIAL NUCLEAR SEGMENTATION
This refers to abnormal segmentation of the nuclei of leukocytes on the blood film, in which the lobes appear to radiate from a single point, giving a cloverleaf or cartwheel picture. This change is common in cytocentrifuged preparations (i.e., from a body fluid), EDTA anticoagulated blood after excessive storage, or samples collected in oxalate.
VACUOLATION
Vacuoles may develop in the nucleus and cytoplasm of leukocytes, especially monocytes and neutrophils, with prolonged storage in EDTA anticoagulated blood. Vacuoles may be associated with swelling of the nuclei and loss of granules from the cytoplasm. In blood films prepared without anticoagulation, vacuoles in neutrophils suggest sepsis.
ENDOTHELIAL CELLS
If the blood film is prepared from the first drop of blood issuing from the microsampling wound, endothelial cells may be present singly or in clumps. Such cells are illustrated in Fig. 2-1. These cells appear quite immature and may be misinterpreted as blasts or metastatic tumor cells.

FIGURE 2-1 Endothelial cells in blood film. (Courtesy of Dr. HA Wurzel.)

THE NEED FOR EXAMINATION OF THE BLOOD FILM
The quantitative determinations discussed earlier in this chapter—hemoglobin level, hematocrit, and erythrocyte, platelet, and leukocyte counts—describe the blood in sufficient detail that the physician will often recognize the need for further laboratory and clinical study. Quantitative analysis of the peripheral blood may suggest diseases involving erythrocytes, leukocytes, and/or platelets that can then be confirmed by examination of a stained blood film. A number of diseases in which the blood counts may be relatively unremarkable but in which examination of the blood film will suggest the disorder are listed in Table 2-4. Based on the quantitative and morphologic examination of the peripheral blood, the physician can assess the need for direct examination of the marrow, as described in Chap. 3.

TABLE 2-4 CONDITIONS IN WHICH THE BLOOD COUNT MAY BE RELATIVELY UNREMARKABLE BUT EXAMINATION OF THE BLOOD FILM WILL SUGGEST OR CONFIRM THE DISORDER

CHAPTER REFERENCES

1.
Shapiro MF, Hatch RL, Greenfield S: Cost containment and labor-intensive tests. The case of the leukocyte differential count. JAMA 252:231, 1984.

2.
Coulter WH: High speed automatic blood cell counter and cell size analyzer. Proc Natl Elect Conf 12:1034, 1956.

3.
Bessman JD, Banks D: Spurious macrocytosis, a common clue to erythrocyte cold agglutinins. Am J Clin Pathol 74:797, 1980.

4.
Wintrobe MM: Macroscopic examination of the blood. Am J Med Sci 185:58, 1933.

5.
England JM, Walford DM, Waters DA: Re-assessment of the reliability of the haematocrit. Br J Haematol 23:247, 1972.

6.
Pearson TC, Guthrie DL: Trapped plasma in the microhematocrit. Am J Clin Pathol 78:770, 1982.

7.
Fairbanks VF: Nonequivalence of automated and manual hematocrit and erythrocyte indices. Am J Clin Pathol 73:55, 1980.

8.
Dacie JV, Lewis SL: Practical Hematology. Churchill Livingstone, Edinburgh, 1991.

9.
England JM: Blood Cell Sizing. Churchill Livingstone, New York, 1991.

10.
International Committee for Standardization in Haematology; Expert Panel on Haemoglobinometry: Recommendations for reference method for haemoglobinometry in human blood (ICSH standard 1986) and specifications for international haemoglobincyanide reference preparation (3rd edition). Clin Lab Haematol 9:73, 1987.

11.
Gagne C, Auger PL, Moorjani S, et al: Effect of hyperchylomicronemia on the measurement of hemoglobin. Am J Clin Pathol 68:584, 1977.

12.
Kickler TS: Clinical analyzers. Advances in automated cell counting. Anal Chem 71:363R, 1999.

13.
Dallman PR, Bart GD, Allen CM, et al: Hemoglobin concentration in white, black and Oriental children: Is there a need for separate criteria in screening for anemia? Am J Clin Nutr 31:377, 1978.

14.
Dallman PR, Siimes MA: Percentile curves for hemoglobin and red cell volume in infancy and childhood. J Pediatr 94:26, 1979.

15.
Wintrobe MM: Anemia: Classification and treatment on the basis of differences in the average volume and hemoglobin content of the red corpuscles. Arch Intern Med 54:256, 1934.

16.
Hillman RS: After sixty years: the MCV is still alive and well. J Gen Intern Med 5:264, 1990.

17.
Rund D, Filon D, Strauss N et al: Mean corpuscular volume of heterozygotes for beta-thalassemia correlates with the severity of mutations. Blood 79:238, 1992.

18.
Segel GB, Cokelet GR, Lichtman MA: The measurement of lymphocyte volume: Importance of reference particle deformability and counting solution tonicity. Blood 57:894, 1981.

19.
Griner PF, Oranburg PR: Predictive values of erythrocyte indices for tests of iron, folic acid, and vitamin B12 deficiency. Am J Clin Pathol 70:748, 1978.

20.
Seward SJ, Safran C, Marton KI, Robinson SH: Does the mean corpuscular volume help physicians evaluate hospitalized patients with anemia? J Gen Intern Med 5:187, 1990.

21.
Carmel R: Pernicious anemia. The expected findings of very low serum cobalamin levels, anemia, and macrocytosis are often lacking. Arch Intern Med 148:1712, 1988.

22.
Mahmoud MY, Lugon M, Anderson CC: Unexplained macrocytosis in elderly patients. Age Ageing 25:10, 1996.

23.
Eldibany MM, Totonchi KF, Joseph NJ, Rhone D: Usefulness of certain red blood cell indices in diagnosing and differentiating thalassemia trait from iron-deficiency anemia. Am J Clin Pathol 111:676, 1999.

24.
Lafferty JD, Crowther MA, Ali MA, Levine M: The evaluation of various mathematical RBC indices and their efficacy in discriminating between thalassemic and non-thalassemic microcytosis. Am J Clin Pathol 106:201, 1996.

25.
Michaels LA, Cohen AR, Zhao H, Raphael RI, Manno CS: Screening for hereditary spherocytosis by use of automated erythrocyte indexes. J Pediatr 130:957, 1997.

26.
Bentley SA, Ayscue LH, Watson JM, Ross DW: The clinical utility of discriminant functions for the differential diagnosis of microcytic anemias. Blood Cells 15:575, 1989.

27.
Mahu JL, Leclercq C, Suquet JP: Usefulness of red cell distribution width in association with biological parameters in an epidemiological survey of iron deficiency in children. Int J Epidemiol 19:646, 1990.

28.
Rose MS: Epitaph for the M.C.H.C. Br Med J 4:169, 1971.

29.
Holt JT, DeWandler MJ, Arvan DA: Spurious elevation of the electronically determined mean corpuscular volume and hematocrit caused by hyperglycemia. Am J Clin Pathol 77:561, 1982.

30.
Liu TC, Seong PS, Lin TK: The erythrocyte cell hemoglobin distribution width segregates thalassemia traits from other nonthalassemic conditions with microcytosis. Am J Clin Pathol 107:601, 1997.

31.
McClure S, Custer E, Bessman JD: Improved detection of early iron deficiency in nonanemic subjects. JAMA 253:1021, 1985.

32.
Patton WN, Cave RJ, Harris RI: A study of changes in red cell volume and haemoglobin concentration during phlebotomy induced iron deficiency and iron repletion using the Technicon H1. Clin Lab Haematol 13:153, 1991.

33.
Bessman JD, Gilmer PR Jr, Gardner FH: Improved classification of anemias by MCV and RDW. Am J Clin Pathol 80:322, 1983.

34.
Flynn MM, Reppun TS, Bhagavan NV: Limitations of red blood cell distribution width (RDW) in evaluation of microcytosis. Am J Clin Pathol 85:445, 1986.

35.
Anonymous: Recommendations to prevent and control iron deficiency in the United States. Centers for Disease Control and Prevention. MMWR 47:1, 1998.

36.
Gulati GL, Hyun BH, Gagaldon H: Falsely elevated automated leukocyte count on cryoglobulinemic and/or cryofibrinogenic blood samples. Lab Med 8:14, 1977.

37.
Williams LJ: Cell histograms: New trends in data interpretation and classification. J Med Technol 1:189, 1984.

38.
Lombarts AJ, deKieviet W: Recognition and prevention of pseudothrombocytopenia and consomitant pseudoleukocytosis. Am J Clin Pathol 89:534, 1988.

39.
Bartels PC, Schoorl M, Lombarts AJ: Screening for EDTA-dependent deviations in platelet counts and abnormalities in platelet distribution histograms in pseudothrombocytopenia. Scand J Clin Lab Invest 57:629, 1997.

40.
Ferrero-Vacher C, Sudaka I, Jambou D, et al: Evaluation of the ABX Cobas Vega automated hematology analyzer and comparison with the Coulter STKS. Hematol Cell Ther 39:149, 1997.

41.
Cornbleet PJ, Myrick D, Levy R: Evaluation of the Coulter STKS five-part differential. Am J Clin Pathol 99:72, 1993.

42.
Thalhammer-Scherrer R, Knobl P, Korninger L, Schwarzinger I: Automated five-part white blood cell differential counts. Efficiency of software-generated white blood cell suspect flags of the hematology analyzers Sysmex SE-9000, Sysmex NE-8000, and Coulter STKS. Arch Pathol Lab Med 121:573, 1997.

43.
Warner BA, Reardon DM, Marshall DP: Automated haematology analysers: a four-way comparison. Med Lab Sci 47:285, 1990.

44.
Zaccaria A, Celso B, Raspadori D, et al: Comparative evaluation of differential leukocyte counts by Coulter VCS cytometer and direct microscopic observation. Haematologica 75:412, 1990.

45.
Atwater S, Corash L: Advances in leukocyte differential and peripheral blood stem cell enumeration. Cur Opin Hematol 3:71, 1996.

46.
Reed WW, Diehl F: Leukopenia, neutropenia, and reduced hemoglobin levels in healthy American blacks. Arch Intern Med 151:501, 1991.

47.
Haddy TB, Rana SR, Castro O: Benign ethnic neutropenia: what is a normal absolute neutrophil count? J Lab Clin Med 133:15, 1999.

48.
Caramihai E, Karayalcin G, Aballi AJ, et al: Leukocyte count differences in healthy white and black children 1 to 5 years of age. J Pediatr 86:252, 1975.

49.
Bain BJ: Ethnic and sex differences in the total and differential white cell count and platelet count. J Clin Pathol 49:664, 1996.

50.
Paulus JM: Platelet size in man. Blood 46:321, 1975.

51.
Lawrence JB, Yomtovian RA, Dillman C, et al: Reliability of automated platelet counts: comparison with manual method and utility for prediction of clinical bleeding. Am J Hematol 48:244, 1995.

52.
Fiorin F, Steffan A, Pradella P, et al: IgG platelet antibodies in EDTA-dependent pseudothrombocytopenia bind to platelet membrane glycoprotein IIb. Am J Clin Pathol 110:178, 1998.

53.
Levin J, Bessman JD: The inverse relation between platelet volume and platelet number. Abnormalities in hematologic disease and evidence that platelet size does not correlate with platelet age. J Lab Clin Med 101:295, 1983.

54.
Bath PM, Butterworth RJ: Platelet size: measurement, physiology and vascular disease. Blood Coagul Fibronol 7:157, 1996.

55.
van der Loo B, Martin JF: A role for changes in platelet production in the cause of acute coronary syndromes. Arterioscler Thromb Vasc Biol 19:672, 1999.

56.
Bessman JD: The relation of megakaryocyte ploidy to platelet volume. Am J Hematol 16:161, 1984.

57.
Thompson CB, Love DG, Quinn PG, Valeri CR: Platelet size does not correlate with platelet age. Blood 62:487, 1983.

58.
Behrens WE: Mediterranean macrothrombocytopenia. Blood 46:199, 1975.

59.
Altes A, Pujol-Moix N, Muniz-Diaz E, et al: Hereditary macrothrombocytopenia and pregnancy. Thrombosis and Haemostasis 76:29, 1996.

60.
O’Malley T, Ludlam CA, Fox KA, Elton RA: Measurement of platelet volume using a variety of different anticoagulant and antiplatelet mixtures. Blood Coagul Fibronol 7:431, 1996.

61.
Osselaer JC, Jamart J, Scheiff JM: Platelet distribution width for differential diagnosis of thrombocytosis. Clin Chem 43:1072, 1997.

62.
Ross DW, Bentley SA: Evaluation of an automated hematology system (Technicon H*1). Arch Pathol Lab Med 10:803, 1986.

63.
Bessis M: Living Blood Cells and Their Ultrastructure, Springer-Verlag, New York, 1973.

64.
Brunning RD: Morphologic alterations in nucleated blood and marrow cells in genetic disorders. Hum Pathol 1:99, 1970.

65.
Jenis EH, Takeuchi A, Dillon DE, et al: The May-Hegglin anomaly: Ultrastructure of the granulocytic inclusion. Am J Clin Pathol 55:187, 1971.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

CHAPTER 1 APPROACH TO THE PATIENT

CHAPTER 1 APPROACH TO THE PATIENT
Williams Hematology

CHAPTER 1 APPROACH TO THE PATIENT

ERNEST BEUTLER
MARSHALL A. LICHTMAN
BARRY S. COLLER
THOMAS J. KIPPS
URI SELIGSOHN
WILLIAM J. WILLIAMS

History

Drugs and Chemicals

General Symptoms

Specific Symptoms

Family History

Sexual History
Physical Examination

Skin
Chapter References

Ideally, the physician’s goal is to prevent illness, and many opportunities exist for hematologists to prevent the development of hematologic disorders. These opportunities include identification of individual genetic risk factors and either avoidance of situations that may make a latent disorder manifest or actual prophylactic therapy, as for example in avoiding venous stasis in patients heterozygous for protein C deficiency or administering prophylactic subcutaneous heparin after major surgery in such patients. Hematologists may also prevent disease by participating in public health and community medicine efforts, such as eliminating sources of environmental lead that may result in childhood anemia. Prenatal diagnosis of hematologic disorders can provide information to families in which a fetus is affected with a hematologic disorder.
When preventive opportunities are not available or fail, the care of a patient begins with a systematic attempt to determine the nature of the illness by eliciting an in-depth medical history and performing a physical examination. The physician should identify the important symptoms and obtain as much relevant information as possible about their origin and evolution and about the general health of the patient by appropriate questions designed to explore the patient’s recent and remote experience. Reviewing previous records may add important data for understanding the onset or progression of illness. Hereditary and environmental factors should be carefully sought and evaluated. The physician follows the medical history with a physical examination to obtain data on the patient’s general health and to permit a careful search for signs of the illnesses suggested by the history. Additional history is obtained during the physical examination, as findings suggest an additional or alternative diagnosis. Thus, the history and physical examination should be considered as a unit, providing the basic information with which further diagnostic information is integrated.
Primary hematologic diseases are uncommon, while hematologic manifestations secondary to other diseases occur frequently. For example, the signs and symptoms of anemia and the presence of enlarged lymph nodes are common clinical findings that may be related to hematologic disease but occur even more frequently as secondary manifestations of disorders not considered primarily hematologic. A wide variety of diseases may produce signs or symptoms of hematologic illness. Thus, in patients with metastatic carcinoma, all the signs and symptoms of anemia may be elicited and lymphadenopathy may be pronounced, but additional findings are usually present that indicate primary involvement of some system besides the blood and lymph nodes. In this discussion, therefore, emphasis is placed on the clinical findings resulting from either primary hematologic disease or the complications of hematologic disorders in order to avoid presenting an extensive catalog of signs and symptoms encountered in general clinical medicine.
In each discussion of specific diseases in subsequent chapters, the signs and symptoms that accompany the particular disorder are presented, and the clinical findings are covered in detail. In this chapter a more general systematic approach is taken.

Acronyms and abbreviations that appear in this chapter include: PS, performance status.

HISTORY1,2,3 and 4
DRUGS AND CHEMICALS
DRUGS
Drug therapy, either self-prescribed or ordered by a physician, is extremely common in our society. Drugs often induce or aggravate hematologic disease, and it is therefore essential that a careful history of drug ingestion, including beneficial and adverse reactions, be obtained from all patients. Drugs taken regularly often become a part of the patient’s way of life and are often forgotten or are not recognized as “drugs.” Agents such as aspirin, laxatives, tranquilizers, medicinal iron, vitamins, other nutritional supplements, and sedatives belong to this category. Further, drugs may be ingested in unrecognized form, such as antibiotics in food or quinine in tonic water. Specific, persistent questioning, often on several occasions, may be necessary before a complete history of drug use is obtained. It is very important to obtain detailed information on alcohol consumption from every patient. The four “CAGE” questions—about cutting down, being annoyed by criticism, having guilt feelings, and needing an eye-opener—provide an effective approach to the history of alcohol use. Patients should also be asked about the use of recreational drugs. The use of “alternative medicines” and herbal medicines are common, and many patients will not consider these medications or may actively withhold information about their use. Nonjudgmental questioning may be successful in identifying agents in this category that the patient is taking.
CHEMICALS
In addition to drugs, most people are exposed regularly to a variety of chemicals in the environment, some of which may be potentially harmful agents in hematologic disease. Similarly, occupational exposure to chemicals must be considered. When a toxin is suspected, the patient’s daily activities and environment must be carefully reviewed, since significant exposure to toxic chemicals may occur incidentally.
VACCINATION
Vaccinations can be potent triggers of exacerbations of immune thrombocytopenia.
NUTRITION
Nutrition information can be useful in deducing the possible role of dietary deficiency in anemia. The avoidance of certain food groups, as might be the case with vegans, or the ingestion of uncooked fish or meat can be clues to the pathogenesis of anemia.
GENERAL SYMPTOMS
Performance status (PS) is a useful concept in establishing the seriousness of the patient’s disability at the outset and in evaluating the effects of therapy.2 A well-founded set of criteria for evaluating performance status is presented in Table 1-1.

TABLE 1-1 CRITERIA OF PERFORMANCE STATUS

Weight loss is a frequent accompaniment of many serious diseases, including primary hematologic entities, but it is not a prominent accompaniment of most hematologic disease. Many “wasting” diseases, such as disseminated carcinoma or tuberculosis, cause anemia, and pronounced emaciation should suggest one of these diseases rather than anemia as the primary disorder.
Fever is a common manifestation of the lymphomas or leukemias, usually because of secondary infection but sometimes as a result of the disease itself. In patients with “fever of unknown origin,” leukemia or lymphoma, and particularly Hodgkin’s disease, should be considered. Myelofibrosis and chronic lymphocytic leukemia may also cause fever. In rare patients with severe pernicious anemia or hemolytic anemia, fever may be pronounced. Chills may accompany severe hemolytic processes and the bacteremia that may complicate the immunocompromised or neutropenic patient. Night sweats suggest the presence of low-grade fever and may occur in patients with lymphoma or leukemia.
Fatigue, malaise, and lassitude are such common accompaniments of both physical and emotional disorders that their evaluation is complex and often difficult. In patients with serious disease, these symptoms may be readily explained by fever, muscle wasting, or other associated findings. Patients with anemia frequently complain of fatigue, malaise, or lassitude, and these symptoms may accompany the hematologic malignancies. Fatigue or lassitude may occur also with iron deficiency even in the absence of sufficient anemia to account for the symptom. In slowly developing chronic anemias, the patient may not recognize reduced exercise tolerance, etc., except in retrospect, after a remission has been induced by appropriate therapy. Anemia may be responsible for more symptoms than has been traditionally recognized, as suggested by the remarkable improvement in quality of life of most uremic patients treated with erythropoietin.3
Weakness may accompany anemia or the wasting of malignant processes, in which cases it is manifest as a general loss of strength or reduced capacity for exercise. The weakness may be localized as a result of neurologic complications of hematologic disease. In pernicious anemia there may be weakness of the lower extremities, accompanied by numbness, tingling, and unsteadiness of gait. Peripheral neuropathy also occurs with dysproteinemias. Weakness of one or more extremities in patients with leukemia, myeloma, or lymphoma may signify central or peripheral nervous system invasion or compression. Myopathy secondary to malignancy occurs with the hematologic malignancies and is usually manifest as weakness of proximal muscle groups. Foot drop or wrist drop may occur in lead poisoning, amyloidosis, systemic autoimmune diseases, or as a complication of vincristine therapy. Paralysis may occur in acute intermittent porphyria.
SPECIFIC SYMPTOMS
NERVOUS SYSTEM
Headache may be due to a number of causes related to hematologic diseases. Anemia or polycythemia may cause mild to severe headache. Invasion or compression of the brain by leukemia or lymphoma, or infection of the central nervous system by Cryptococcus or tuberculosis, may also cause headache in patients with hematologic malignancies. Hemorrhage into the brain or subarachnoid space in patients with thrombocytopenia or other bleeding disorders may cause sudden, severe headache.
Paresthesias may occur because of peripheral neuropathy in pernicious anemia or secondary to hematologic malignancy or amyloidosis. They may also result from therapy with vincristine.
Confusion may accompany malignant or infectious processes involving the brain, sometimes as a result of the accompanying fever. Confusion may also occur with severe anemia, hypercalcemia, or glucocorticoid therapy. Confusion or apparent senility may be a manifestation of pernicious anemia. Frank psychosis may develop in acute intermittent porphyria or with glucocorticoid therapy.
Impairment of consciousness may be due to increased intracranial pressure secondary to hemorrhage or tumor in the central nervous system. It may also accompany severe anemia or polycythemia, or it may be due to hyperviscosity secondary to a paraprotein in the plasma.
EYES
Visual disturbances may be manifestations of anemia, polycythemia, leukemia, or macroglobulinemia. Occasionally blindness may result from retinal hemorrhages secondary to anemia and thrombocytopenia or severe hyperviscosity. Diplopia or disturbances of ocular movement may occur with orbital tumors or paralysis of the third, fourth, or sixth cranial nerves because of compression by tumor.
EARS
Vertigo, tinnitus, and “roaring” in the ears may occur with marked anemia, polycythemia, or macroglobulinemia-induced hyperviscosity.
NASOPHARYNX AND MOUTH
Epistaxis may occur with any bleeding disorder. Anosmia or olfactory hallucinations occur in pernicious anemia. The nasopharynx may be invaded by a malignant tumor, with the symptoms dependent on the structures invaded. Sore tongue occurs in pernicious anemia and may accompany iron deficiency or vitamin deficiencies. Macroglossia occurs in amyloidosis. Bleeding gums may occur with bleeding disorders. Infiltration of the gingiva with leukemic cells occurs in acute monocytic leukemia. Ulceration of the tongue or oral mucosa may be severe in the leukemias or in patients with neutropenia. Dryness of the mouth may be due to hypercalcemia, secondary, for example, to plasma cell myeloma. Dysphagia may be seen in patients with severe mucous membrane atrophy associated with chronic iron-deficiency anemia.
NECK
Painless swelling in the neck is characteristic of lymphoma but may be due to a number of other diseases as well. Occasionally, the enlarged lymph nodes of lymphomas may be tender or painful because of secondary infection or rapid growth. Diffuse swelling of the neck and face may occur with obstruction of the superior vena cava due to lymphoma.
CHEST AND HEART
Both dyspnea and palpitations, usually on effort but occasionally at rest, may occur because of anemia. Congestive heart failure may supervene, and angina pectoris may become manifest in anemic patients. The impact of anemia on the circulatory system depends in part on the rapidity with which it develops, and chronic anemia may become severe without producing major symptoms; with acute blood loss, the patient may develop shock with a nearly normal hemoglobin level. Cough may result from enlarged mediastinal nodes. Chest pain may arise from involvement of the ribs or sternum with lymphoma or multiple myeloma, nerve-root invasion or compression, or herpes zoster; the pain of herpes zoster usually precedes the skin lesions by several days. Tenderness of the sternum may be quite pronounced in chronic myelogenous or acute leukemia, in myelofibrosis, or if the sternal marrow is invaded by lymphoma or myeloma.
GASTROINTESTINAL SYSTEM
Dysphagia has already been mentioned under “Nasopharynx.” Anorexia frequently occurs but usually has no specific diagnostic significance. Hypercalcemia and azotemia cause anorexia, nausea, and vomiting. A variety of ill-defined gastrointestinal complaints grouped under the heading “indigestion” may occur with hematologic diseases. Abdominal fullness, premature satiety, belching, or discomfort may occur because of a greatly enlarged spleen, but such splenomegaly may also be entirely asymptomatic. Abdominal pain may arise from intestinal obstruction by lymphoma, retroperitoneal bleeding, lead poisoning, ileus secondary to therapy with the Vinca alkaloids, acute hemolysis, allergic purpura, the abdominal crises of sickle cell disease, or acute intermittent porphyria. Diarrhea may occur in pernicious anemia. It also may be prominent in the various forms of intestinal malabsorption, although significant malabsorption may occur without diarrhea. Malabsorption may be a manifestation of small-bowel lymphoma. Gastrointestinal bleeding related to thrombocytopenia or other bleeding disorder may be entirely occult but often is manifest as hematemesis or melena. Constipation may occur in the patient with hypercalcemia or in one receiving treatment with the Vinca alkaloids.
GENITOURINARY AND REPRODUCTIVE SYSTEMS
Impotence or bladder dysfunction may occur with spinal cord or peripheral nerve damage due to one of the hematologic malignancies or with pernicious anemia. Priapism may occur in leukemia or sickle cell disease. Hematuria may be a manifestation of any of the bleeding disorders. Red urine may also occur with intravascular hemolysis (hemoglobinuria), myoglobinuria, or porphyrinuria. Injection of anthracycline drugs or ingestion of drugs such as pyridium regularly causes the urine to turn red. Beeturia also occurs as a benign genetic trait. Amenorrhea may accompany any serious disease. It may also be induced by certain drugs, such as antimetabolites or alkylating agents. Menorrhagia is a common cause of iron deficiency, and care must be taken to obtain an accurate history of the extent of menstrual blood loss. Semiquantification can be obtained from estimates of the number of days of heavy bleeding (usually 1 to 2), the number of days of any bleeding (usually 5 to 7), number of tampons or pads used, degree of blood soaking, and clots formed. Menorrhagia may occur in patients with bleeding disorders.
BACK AND EXTREMITIES
Back pain may accompany acute hemolytic reactions or be due to involvement of bone or the nervous system in malignant disease. It is one of the commonest manifestations of myeloma.
Arthritis or arthralgia may occur with gout secondary to increased uric acid production in patients with hematologic malignancies, myelofibrosis, or hemolytic anemia. They also occur in the plasma cell dyscrasias, acute leukemias, and sickle cell disease without evidence of gout, and in allergic purpura. Arthritis may accompany hemochromatosis. Hemarthroses in patients with severe bleeding disorders cause marked joint pain. Autoimmune diseases may present as anemia and/or thrombocytopenia, and arthritis appears as a later manifestation. Shoulder pain on the left may be due to infarction of the spleen and on the right from gall bladder disease associated with chronic hemolytic anemia such as hereditary spherocytosis. Bone pain may occur with bone involvement by the hematologic malignancies or metastatic tumor; it is common in the congenital hemolytic anemias, such as sickle cell anemia, and may occur in myelofibrosis. In patients with Hodgkin’s disease, ingestion of alcohol may induce pain at the site of any lesion, including those in bone. Edema of the lower extremities, sometimes unilateral, may occur because of obstruction to veins or lymphatics by enlarged lymph nodes. Leg ulcers are a common complaint in sickle cell anemia and occur rarely in other hereditary anemias.
SKIN
Skin manifestations of hematologic disease may be of great importance; they include changes in texture or color, itching, and the presence of specific or nonspecific lesions. The skin in iron-deficient patients may become dry, the hair dry and fine, and the nails brittle. In hypothyroidism, which may cause anemia, the skin is dry, coarse, and scaly. Jaundice may be apparent with pernicious anemia or congenital or acquired hemolytic anemia. The skin of patients with pernicious anemia is said to be “lemon yellow” because of the simultaneous appearance of jaundice and pallor. Jaundice may also occur in patients with hematologic diseases as a result of liver involvement or biliary tract obstruction. Pallor is a common accompaniment of anemia, although some severely anemic patients may not appear pale. Widespread erythroderma occurs in cutaneous T-cell lymphoma and in some cases of chronic lymphocytic leukemia or lymphocytic lymphoma. The skin is often involved, sometimes severely, in graft-versus-host disease following marrow transplantation. Patients with hemachromatosis may have bronze or grayish pigmentation of the skin. Cyanosis occurs with methemoglobinemia, either hereditary or acquired; sulfhemoglobinemia; abnormal hemoglobins with low oxygen affinity; and primary and secondary polycythemia. Cyanosis of the ears or the fingertips may occur after exposure to cold in individuals with cryoglobulins or cold agglutinins.
Itching may occur in the absence of any visible skin lesions in Hodgkin’s disease and may be extreme. Mycosis fungoides or other lymphomas with skin involvement may also present as itching. A significant number of patients with polycythemia vera will complain of itching after bathing.
Petechiae and ecchymoses are most often seen in the extremities in patients with thrombocytopenia, nonthrombocytopenic purpura, or nonthrombocytopenic bleeding disorders. Unless secondary to trauma, these lesions usually are painless, although the lesions of psychogenic purpura and erythema nodosum are painful. Easy bruising is a common complaint, especially among women, and when no other hemorrhagic symptoms are present, usually no abnormalities are found after detailed study. This symptom may, however, indicate a mild hereditary bleeding disorder, such as von Willebrand’s disease or one of the platelet disorders.
Infiltrative lesions may occur in the leukemias and lymphomas and are sometimes the presenting complaint. Necrotic lesions may occur with intravascular coagulation, purpura fulminans, warfarin-induced skin necrosis, or rarely with exposure to cold in patients with circulating cryoproteins or cold agglutinins.
FAMILY HISTORY
A carefully obtained family history may be of great importance in the study of patients with hematologic disease. In the case of hemolytic disorders, questions should be asked regarding jaundice, anemia, and gallstones in relatives. In patients with disorders of hemostasis or venous thrombosis, particular attention must be given to bleeding manifestations, and clots in family members. In the case of autosomal recessive disorders such as pyruvate kinase deficiency the parents are usually not affected, but a similar clinical syndrome may have occurred in siblings. It is particularly important to inquire about siblings who may have died in infancy, since these may be forgotten, especially by older patients. When sex-linked inheritance is suspected, it is necessary to inquire about symptoms in the maternal grandfather, maternal uncles, male siblings, and nephews. In patients with disorders with dominant inheritance, such as hereditary spherocytosis, one may expect to find that one of the parents and possibly siblings and children of the patient have stigmata of the disease. Ethnic background may be important in the consideration of certain diseases such as thalassemia, sickle cell anemia, glucose-6-phosphate dehydrogenase deficiency, or other inherited disorders that are concentrated in geographic areas.
SEXUAL HISTORY
Because of the epidemic of infections with the human immunodeficiency viruses, it is important to ascertain the sexual preferences and risk factors of patients.
PHYSICAL EXAMINATION
A detailed physical examination should be performed on every patient, with sufficient attention paid to all systems to obtain a full evaluation of the general health of the individual. Certain body areas are especially pertinent to hematologic disease and therefore deserve special attention. These are the skin, eyes, tongue, lymph nodes, skeleton, spleen and liver, and nervous system.
SKIN
PALLOR AND FLUSHING
The color of the skin is due to the pigment contained therein and to the blood flowing through the skin capillaries. The component of skin color related to the blood may be a useful guide to anemia or polycythemia, since pallor may result when the hemoglobin level is reduced and redness when the hemoglobin level is increased. The amount of pigment in the skin will modify skin color and may mislead the clinician, as in individuals with pallor due to decreased pigment, or make skin color useless as a guide because of the intense pigmentation present.
Alterations in blood flow and in hemoglobin content may change skin color; this too may mislead the clinician. Thus emotion may cause either pallor or blushing. Exposure of the skin to cold or heat may similarly cause pallor or blushing. Chronic exposure to wind or sun may lead to permanent redness of the skin, and chronic ingestion of alcohol to a flushed face. The degree of erythema of the skin can be evaluated by pressing the thumb firmly against the skin, as on the forehead, so that the capillaries are emptied, and then comparing the color of the compressed spot with the surrounding skin immediately after the thumb is removed.
The mucous membranes and nail beds are usually more reliable guides to anemia or polycythemia than the skin. The conjunctivae and gums may be inflamed, however, and therefore not reflect the hemoglobin level, or the gums may appear pale because of pressure from the lips. The gums and the nail beds may also be pigmented and the capillaries correspondingly obscured. In some individuals, the color of the capillaries does not become fully visible through the nails unless pressure is applied to the fingertip, either laterally or on the end of the nail.
The palmar creases are useful guides to the hemoglobin level and appear pink in the fully opened hand unless the hemoglobin is 7 g/dl or less. Liver disease may induce flushing of the thenar and hypothenar eminences of the palm, even in patients with anemia.
CYANOSIS
The detection of cyanosis, like the detection of pallor, may be made difficult by skin pigmentation. Cyanosis is a function of the total amount of reduced hemoglobin, methemoglobin, or sulfhemoglobin present. The minimum amounts of these pigments that cause detectable cyanosis are about 5 g of reduced hemoglobin, 1.5 to 2.0 g of methemoglobin, and 0.5 g of sulfhemoglobin per deciliter of blood.
JAUNDICE
Jaundice may be observed in the skin of individuals who are not otherwise deeply pigmented or in the conjunctivae or the mucous membranes. The patient should be examined in daylight rather than under incandescent or fluorescent light, because the yellow color of the latter masks the yellow color of the patient. Jaundice is due to actual staining of the skin by bile pigment, and bilirubin glucuronide (direct-reacting or conjugated bilirubin) stains the skin more readily than the unconjugated form. Jaundice of the skin may not be visible if the bilirubin level is below 2 to 3 mg/dl. Yellow pigmentation of the skin may also occur with carotenemia, especially in young children.
PETECHIAE AND ECCHYMOSES
Petechiae are small (1 to 3 mm), round, red or brown lesions resulting from hemorrhage into the skin and are present primarily in areas with high venous pressure, such as the lower legs. These lesions do not blanch on pressure, and this can be demonstrated most readily by compressing the skin with a glass microscope slide or magnifying lens. Petechiae may occasionally be elevated slightly, i.e., palpable; this finding suggests vasculitis. Ecchymoses may be of various sizes and shapes and may be red, purple, blue, or yellowish green, depending on the intensity of the skin hemorrhage and its age. They may be flat or elevated; some are painful and tender. The lesions of hereditary hemorrhagic telangiectasia are small, flat, nonpulsatile, and violaceous. They blanch with pressure.
EXCORIATION
Itching may be intense in some hematologic disorders such as Hodgkin’s disease, even in the absence of skin lesions. Excoriation of the skin from scratching is the only physical manifestation of this severe symptom.
LEG ULCERS
Open ulcers or scars from healed ulcers are often found in the region of the internal or external malleoli in patients with sickle cell anemia and, rarely, in other hereditary anemias.
NAILS
Detection of pallor or rubor by examining the nails was discussed earlier. The fingernails in chronic, severe iron-deficiency anemia may be ridged longitudinally and flattened or concave rather than convex. The latter change is referred to as koilonychia and is uncommon in present practice.
EYES
Jaundice, pallor, or plethora may be detected from examination of the eyes. Jaundice is usually more readily detected in the sclerae than in the skin. Ophthalmoscopic examination is also essential in patients with hematologic disease. Retinal hemorrhages and exudates occur in patients with severe anemia and thrombocytopenia. These hemorrhages are usually the typical “flame-shaped” hemorrhages, but they may be quite large and elevate the retina so that they may appear as a darkly colored tumor. Round hemorrhages with white centers are also often seen. Dilatation of the veins may be seen in polycythemia; in patients with macroglobulinemia, the veins are engorged and segmented, resembling link sausages.
MOUTH
Pallor of the mucosa has already been discussed. Ulceration of the oral mucosa occurs commonly in neutropenic patients. In leukemia there may also be infiltration of the gums with swelling, redness, and bleeding. Bleeding from the mucosa may occur with a hemorrhagic disease. A dark line of lead sulfide may be deposited in the gums at the base of the teeth in lead poisoning. The tongue may be completely smooth in pernicious anemia and iron-deficiency anemia. Patients with an upper dental prosthesis may also have papillary atrophy, presumably on a mechanical basis. The tongue may be smooth and red in patients with nutritional deficiencies. This may be accompanied by fissuring at the corners of the mouth, but fissuring may also be due to ill-fitting dentures.
LYMPH NODES
Lymph nodes are widely distributed in the body, and in disease any node or group of nodes may be involved. The major concern on physical examination is the detection of enlarged or tender nodes in the cervical, supraclavicular, axillary, epitrochlear, inguinal, or femoral regions. Under normal conditions in adults, the only readily palpable lymph nodes are in the inguinal region, where several firm nodes 0.5 to 2.0 cm long are normally attached to the dense fascia below the inguinal ligament and in the femoral triangle. In children, multiple small (0.5 to 1.0 cm) nodes may be palpated in the cervical region as well. Supraclavicular nodes may sometimes be palpable only when the patient performs the Valsalva maneuver.
Enlarged lymph nodes are ordinarily detected in the superficial areas by palpation, although they are sometimes large enough to be seen. Palpation should be gentle and is best performed with a circular motion of the fingertips, using slowly increasing pressure.
Nodes too deep to palpate may be detected by radiologic examination, including computerized tomography, magnetic resonance imaging, radiographic lymphangiography, isotopic lymphangiography, or by ultrasound.
CHEST
Increased rib or sternal tenderness is an important physical sign often ignored. Increased bone pain may be generalized, as in leukemia, or spotty, as in plasma cell myeloma or in metastatic tumors. The superficial surfaces of all bones should be examined thoroughly by applying intermittent firm pressure with the fingertips to locate potential areas of disease.
SPLEEN5,6,7 and 8
The normal adult spleen is usually not palpable on physical examination but occasionally may be felt. Palpability of the normal spleen may be related to body habitus, but there is disagreement on this point. Enlarged spleens may be detected by percussion, palpation, or a combination of these two methods. Some enlarged spleens may be visible through the abdominal wall.
The normal spleen weighs about 150 g and lies in the peritoneal cavity against the diaphragm and the posterolateral abdominal wall at the level of the lower three ribs. As it enlarges it remains close to the abdominal wall, while the lower pole moves downward, anteriorly, and to the right. Spleens enlarged only 40 percent above normal may be palpable, but significant splenic enlargement may occur and the organ still not be felt on physical examination. A good but imperfect correlation has been reported between spleen size estimated from radioisotope scanning or ultrasonography and spleen weight determined after splenectomy or at autopsy. Although it is common to fail to palpate an enlarged spleen on physical examination, palpation of a normal-sized spleen is unusual, and therefore a palpable spleen is usually a significant physical finding.
In examining for an enlarged spleen, it should be remembered that the organ lies just beneath the abdominal wall and that it is identified by its movement during respiration. The splenic notch may be evident if the organ is moderately enlarged. During the examination the patient lies in a relaxed, supine position. The examiner, standing on the patient’s right, gently palpates the left upper abdomen with the right hand while exerting pressure forward with the palm of the left hand placed over the lower ribs posterolaterally. If nothing is felt, the palpation should be performed repeatedly, moving the examining hand about 2 cm toward the inguinal ligament each time. It is often advantageous to carry out the examination initially with the patient lying on the right side with left knee flexed and to repeat it with the patient supine.
It is not always possible to be sure that a left upper quadrant mass is spleen; masses in the stomach, colon, kidney, or pancreas may mimic splenomegaly on physical examination. When there is uncertainty regarding the nature of a mass in the left upper quadrant, imaging procedures will usually permit accurate diagnosis.
LIVER7,8,9,10 and 11
Palpation of the edge of the liver in the right upper quadrant of the abdomen is commonly used to detect hepatic enlargement, although the inaccuracies of this method have been demonstrated. It is necessary to determine both the upper and lower borders of the liver by percussion in order to properly assess liver size. The normal liver may be palpable as much as 4 to 5 cm below the right costal margin but is usually not palpable in the epigastrium. The height of liver dullness is best measured in a specific line 8, 10, or 12 cm to the right of the midline. Techniques should be standardized so that serial measurements can be made. The vertical span of the normal liver determined in this manner will range about 10 cm in an average-size man and about 2 cm smaller in women. Because of variations introduced by technique, each physician should determine the normal area of liver dullness by his or her own procedure. Correlation of radioisotope imaging data with results from routine physical examinations indicates that often a liver of normal size is considered enlarged on physical examination and an enlarged liver is considered normal. Imaging procedures are useful in demonstrating localized infiltrative lesions.
NERVOUS SYSTEM
A thorough evaluation of neurologic function is necessary in many patients with hematologic disease. Vitamin B12 deficiency impairs cerebral, olfactory, spinal cord, and peripheral nerve function, and severe chronic deficiency may lead to irreversible neurologic degeneration. Leukemic meningitis is often manifested by headache, visual impairment, or cranial nerve dysfunction. Tumor growth in the brain or spinal cord compression may be due to malignant lymphoma or plasma cell myeloma. A variety of neurologic abnormalities may develop in patients with various leukemias and lymphomas as a consequence of infiltration, bleeding, or infection.
JOINTS
Deformities of the knees, elbows, ankles, shoulders, wrists, or hips may be the result of repeated hemorrhage in patients with hemophilia A, hemophilia B, or severe factor VII deficiency. Often, a target joint is prominently affected.
CHAPTER REFERENCES

1.
Bates B: A Guide to Physical Examination and History Taking, 6th ed. Lippincott, Philadelphia, 1995.

2.
Mor V, Laliberte L, Morris JN, Wiemann M: The Karnovsky Performance Status Scale: an examination of its reliability and validity in a research setting. Cancer 53:2002, 1984.

3.
Sackett DL: A primer on the precision and accuracy of the clinical examination. JAMA 267:2638, 1992.

4.
Enelow AJ, Forde DL, Brummel-Smith K: Interviewing and Patient Care, 4th ed. Oxford University Press, Oxford, 1996.

5.
Arkles LB, Gill GD, Nolan MP: A palpable spleen is not necessarily enlarged or pathological. Med J Aust 145:15, 1986.

6.
Barkun AN, Camus M, Green L, et al: The bedside assessment of splenic enlargement. Am J Med 91:512, 1991.

7.
Halpern S, Coel M, Ashburn W, et al: Correlation of liver and spleen size: determinations by nuclear medicine studies and physical examination. Arch Intern Med 134:123, 1974.

8.
Downey MT: Estimation of splenic weight from ultrasonographic measurements. Can Assn Rad J 43:273, 1992.

9.
Castell DO, O’Brien KD, Muench H, Chalmers TC: Estimation of liver size by percussion in normal individuals. Ann Intern Med 70:1183, 1969.

10.
Bennett WF, Dova JG: Review of hepatic imaging and a problem-oriented approach to liver masses. Hepatology 12:761, 1990.

11.
Barloon TJ, Brown BP, Abu-Yousef MM, et al: Teaching physical examination of the adult liver with the use of real-time sonography. Acad Radiol 5:101, 1998.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology