CHAPTER 3 EXAMINATION OF THE MARROW
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
Morphologic Interpretation of Marrow Preparations
Adequacy of the Marrow Sample
Bone Marrow Cellularity
Infiltrative Diseases of the Marrow
Differentiation of the Hematopoietic Lineages
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
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.
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.
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.
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
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 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
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 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.
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
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.
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 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.
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
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 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.
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.
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.
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn