CHAPTER 66 PRODUCTION, DISTRIBUTION, AND FATE OF NEUTROPHILS
CHAPTER 66 PRODUCTION, DISTRIBUTION, AND FATE OF NEUTROPHILS
BERNARD M. BABIOR
DAVID W. GOLDE
Regulation of Neutrophilic Granulopoiesis
Evaluation of Adequacy of Neutrophil Production
Dna Measurement: Tritiated Thymidine and Microfluorimetry
Tracer Techniques for Studying Neutrophil Kinetics in Humans
Lysozyme and Transcobalamin I
Accumulation in Inflammatory Sites
In Vitro Marrow Culture
The neutrophil count in the blood is maintained in a normal steady state by the balance among neutrophilopoiesis in the marrow, the distribution of neutrophils between the marginated pool in the microvasculature and the freely circulating pool in the blood, and the rate of egress from blood to tissues. Marrow production is regulated by three principal glycoprotein hormones, or cytokines: interleukin-3, granulocyte-monocyte, and granulocyte colony-stimulating factors. The latter two cytokines are available as recombinant pharmaceutical products that can be administered therapeutically to ameliorate certain causes of neutropenia. Neutrophil interaction with endothelium is mediated by selectins, polypeptides that contain sugar-binding sites and enter tissues in response to inflammatory mediators by the up-regulation and exposure of integrins on the neutrophil and endothelial cell, which permits firm attachment to endothelium and emigration into tissues through intercellular junctions under the influence of chemoattractant chemicals. Neutrophils migrate from blood to tissues in an age-independent (random) manner, with a half-disappearance time of about 7 h. This process can be accelerated when inflammation is present and highlights the need for a sustained rate of production to maintain a normal blood neutrophil count. The pathogenesis of neutropenia is more complex to analyze kinetically than anemia or thrombocytopenia because at least four compartments are involved: marrow storage pool, circulating pool, marginated pool, and tissue pool. The latter is particularly difficult to assay. Measurements can be further complicated in the nonsteady state, when dramatic increases in turnover rates and distribution among the four principal pools are in disequilibrium, such as during acute inflammatory states.
Acronyms and abbreviations that appear in this chapter include: CFU, colony-forming unit; CFU-GM, colony forming unit–granulocyte-monocyte; CSF, colony-stimulating factor; DF32P, diisopropyl fluorophosphate; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-monocyte colony stimulating factor; [3H]TdR, tirtiated thymidine; ICAM-1, intracellular adhesion molecule-1; IL, interleukin; Mr, relative molecular mass; NTR, neutrophil turnover rate; PECAM-1, platelet–endothelial cell adhesion molecule-1; PMN, polymorphonuclear neutrophil; T1/2, half-time; TBNP, total blood neutrophil pool.
Neutrophils are produced in the marrow, where they arise from progenitor and precursor cells by a process of cellular proliferation and maturation. They differentiate from the pluripotential stem cell1,2 through a series of more and more narrowly committed progenitor, or colony-forming, units (CFU), including the CFU for granulocytes and monocytes (CFU-GM) and the CFU for granulocytes (CFU-G), which give rise to neutrophils.3 These early progenitor cells cannot be recognized under the microscope but can be identified by marrow culture (see Chap. 14). The earliest microscopically recognizable neutrophil precursor is the myeloblast. From there, the formal sequence of precursor development is myeloblast ® promyelocyte ® myelo-cyte ® metamyelocyte ® band neutrophil ® segmented neutrophil (see Chap. 64). The term granulocyte is often loosely used to refer to neutrophils but strictly speaking includes eosinophils and basophils as well. Eosinophilic and basophilic granulocytes develop from progenitors in a manner analogous to the neutrophils, although commitment to neutrophilic, eosinophilic, or basophilic development is probably established at an early progenitor stage.
The normal human neutrophil production rate is 0.85 to 1.6 × 109 cells per kilogram per day. The mature neutrophils are stored in the marrow before release into the blood. They leave the circulation randomly, with a half-disappearance time of about 6 h. These cells then enter the tissues and probably function for a day or two before their death or loss into the gastrointestinal tract through mucosal surfaces.
The neutropoietic system has a high production volume, and yet it is finely modulated in the steady state and has a great capacity to increase production in response to inflammatory stimuli. This chapter outlines current concepts of neutrophil production, distribution, and survival. For detailed data and methods, the reader is referred to primary articles and reviews on neutropoiesis and neutrophil kinetics.4,5,6,7,8,9,10,11,12,13 and 14
REGULATION OF NEUTROPHILIC GRANULOPOIESIS
Although the primary cellular manifestation of commitment is the expression of receptors for lineage-specific hematopoietins, the “decision” for a stem cell to self-renew or differentiate may in part be a random or stochastic event.1,15 On the other hand, stromal elements, collectively referred to as the hematopoietic microenvironment, release short-range signals that regulate the process of commitment from multipotential stem cell pools. Although the details of hematopoietic stem cell regulation are poorly understood, much is now known regarding the interaction of hematopoietic hormones with committed granulocyte progenitor cells and their mature progeny.16,17,18,19 and 20
The humoral regulators involved in granulopoiesis have been defined by in vitro culture systems.16,17 Originally identified by their ability to stimulate colony formation from marrow progenitor cells, the hemopoietins came to be called colony-stimulating factors (CSF). With regard to neutrophil production, at least four human CSF have been defined. GM-CSF is a 22,000-Mr glycoprotein that stimulates the production of neutrophils, monocytes, and eosinophils. G-CSF has an Mr of 20,000 and stimulates only the production of neutrophils. Interleukin-3 (IL-3), or multi-CSF, also has an Mr of 20,000 and acts relatively early in hemopoiesis, affecting multipotential stem cells. Finally, stem cell factor (also known as c-kit ligand or steel factor), with an Mr of 28,000, acts in combination with IL-3 or GM-CSF to stimulate the proliferation of the early hematopoietic precursor cells. In addition to their effects on neutrophil precursors, both G-CSF and GM-CSF act directly on the neutrophil to enhance its function. These hemopoietins therefore are important in regulating the production, survival, and functional activity of neutrophils.17,18,21,22 and 23 The mature neutrophil lacks IL-3 receptors and thus is not affected by IL-3. IL-3 receptors are present, however, on mature eosinophils and monocytes. IL-3 is produced by activated T lymphocytes and thus would be expected to have a physiologic role in circumstances of cell-mediated immunity. GM-CSF is also produced by activated lymphocytes, but, like G-CSF, it is also elaborated by mononuclear phagocytes and endothelial and mesenchymal cells when these cell types are stimulated by certain cytokines, including IL-1 and tumor necrosis factor, or bacterial products, such as endotoxin.24,25 and 26 Stem cell factor is secreted by a variety of cells, including marrow stromal cells,27 and affects the development of several kinds of tissues.28,29 and 30 (see Chap. 4 and Chap. 14).
The activities of exogenously administered biosynthetic (recombinant) human G-CSF and GM-CSF in humans are well documented.18,31 G-CSF administration rapidly induces neutrophilia, whereas GM-CSF causes an increase in neutrophils, eosinophils, and monocytes. GM-CSF cannot be detected easily in normal plasma, and thus its role as a day-to-day, long-range modulator of neutrophil production is uncertain. Mice in whom the GM-CSF gene is “knocked out” have generally normal hematopoiesis but show macrophage abnormalities, pulmonary alveolar proteinosis, and decreased resistance to microbial challenge.32,33,34 and 35
G-CSF, however, appears to be a critical regulator of neutrophilopoiesis, since giving an animal an antibody to G-CSF leads to profound neutropenia.36 The G-CSF knockout mouse shows severe neutropenia.37 Also, neutropenia that results from a production disturbance, such as exposure to cytotoxic drugs, is associated with high circulating serum concentrations of G-CSF.38 As part of an inflammatory response, macrophages and T lymphocytes are activated. They release CSF and also produce cytokines that cause endothelial and mesenchymal cells to release CSF. These CSF stimulate marrow neutrophil production. When the microorganism is contained and eliminated, the stimulus for CSF gene expression is removed and neutrophil production returns to baseline.
Many biologic systems employ negative feedback mechanisms whereby the end product of a process has an inhibitory effect on its further production. Tissue-specific inhibitors are referred to as chalones, and a granulocytic chalone elaborated by mature neutrophils has been reported.39,40 The role of other inhibitors, such as lactoferrin and acidic isoferritins, is uncertain.41,42
Methods used to study granulocyte kinetics may be listed under the following categories: (1) neutrophil depletion or destruction to determine the size and rate of mobilization of reserves and the level of compensatory neutropoiesis; (2) the use of radioactive tracers to study neutrophil distribution, production rates, and survival times; (3) mitotic indices of marrow granulocytic cells to assess proliferative activity and cell cycle times; and (4) induced inflammatory lesions to study cell movement into the tissues. Of these categories, the most popular has been the use of radioactive tracers.
Neutrophilopoiesis and neutrophil kinetics are usually analyzed by describing neutrophil movement through a number of interconnected compartments. These compartments may be arranged in three major groups: the marrow, the blood, and the tissue (Fig. 66-1).
FIGURE 66-1 A scheme of maturation of neutrophil precursor cells. The myeloblast (MB) is the first recognizable precursor of neutrophils. Myeloblasts undergo division and maturation into promyelocytes (Prom) and thereafter into neutrophilic myelocytes (Myelocyte), after which stage mitotic capability is lost. The major compartments of precursor proliferation and distribution are indicated across the top of the figure: marrow, blood, and tissues. The marrow precursor compartment is made up of the proliferating compartment (myeloblasts through myelocytes) and the maturation and storage compartment [metamyelocytes (Meta) to mature polymorphonuclear neutrophils (PMN)]. Under normal conditions, there is no return of cells from the tissue compartment to the blood or marrow.
Marrow neutrophils may be divided into the mitotic, or proliferative, compartment and the maturation-storage compartment (see Fig. 66-1). Myeloblasts, promyelocytes, and myelocytes are capable of replication and constitute the mitotic compartment. Earlier progenitor cells are few in number, not morphologically identifiable, and usually neglected in kinetic studies. Metamyelocytes, bands, and mature neutrophils, none of which replicate, constitute the maturation-storage compartment (see Color Plate VII).
The number of cell divisions from the myeloblast to the myelocyte stage in the proliferative compartment has been estimated at between four and five.43 Data obtained using radioactive diisopropyl fluorophosphate (DF32P) suggest that there are three divisions at the myelocyte stage, but the number of cell divisions at each step may not be constant. The major increase in neutrophil number probably occurs at the myelocyte level, since the myelocyte pool is at least four times the size of the promyelocyte pool. Because of the difficulties in measuring human intramarrow neutrophil kinetics, a precise model of the dynamics of the mitotic compartment is not available. Estimates of the sizes of the marrow neutrophil compartments and the transit times and cell cycle stages of the cells in the various compartments are given in Table 66-1. Precise studies have measured a postmitotic pool of (5.59 ± 0.9) × 109 cells per kilogram and a mitotic pool (promyelocytes and myelocytes) of (2.11 ± 0.36) × 109 cells per kilogram. These studies have led to a calculated normal marrow neutrophil production of 0.85 × 109 cells per kilogram per day. Radioautographic studies with [3H]thymidine support the concept of an orderly progression from metamyelocytes to mature PMN within the maturation-storage compartment. These studies also suggest a “first in, first out” pattern for cells leaving this compartment and entering the blood. Several labeling techniques indicate that the myelocyte-to-blood transit time is 5 to 7 days.11,44 Previous studies with DF32P gave a range of 8 to 14 days.8,43 During infections, however, the myelocyte-to-blood transit time may be as short as 48 h.45
TABLE 66-1 MARROW NEUTROPHIL KINETICS
It is not known with certainty whether the production of neutrophils in the mitotic compartment exactly equals the neutrophil turnover rate. Studies in dogs have suggested that some immature neutrophils die in the marrow (“ineffective neutrophilopoiesis”).46 Ineffective neutrophilopoiesis has not been shown in normal humans, however,13,47 although ineffective neutrophilopoiesis occurs in some pathologic states. In the preleukemic syndromes48 there is probably substantial intramedullary cell death, as may occur also in myelofibrosis and perhaps some of the idiopathic neutropenic disorders. At present, however, there is no convenient means to quantitate ineffective neutrophilopoiesis.
On completion of maturation, the neutrophils are stored in the marrow and are referred to as the mature neutrophil reserve. This reserve contains many more cells than are normally circulating in the blood. Comparative data on the characteristics of the maturation-storage compartment are given in Table 66-2. Under stress, maturation time may be shortened, divisions may be skipped, and release into the blood may occur prematurely.
TABLE 66-2 COMPARATIVE DATA ON MARROW MATURATION-STORAGE COMPARTMENT
Neutrophils leave the marrow storage compartment and enter the blood without significant reentry into the marrow. The total blood neutrophil pool consists of all the neutrophils in the vascular spaces. Some of these neutrophils are free in the circulation (the circulating pool), while others roll along the endothelium of small vessels (the marginated pool). Cells in the two pools are freely exchangeable. When neutrophils labeled with DF32P are injected into normal subjects, approximately half can be accounted for in the circulating pool; the remainder enter the marginated pool.4,5 and 6 Neutrophils shift from the marginated to the circulating pool with exercise, epinephrine injection, or stress but eventually leave the blood and enter the tissues. Once they have entered the tissues, they do not normally return to the blood; the flow of cells is unidirectional.
The behavior of neutrophils in the blood appears to be controlled by two classes of membrane-bound adhesion proteins: selectins and integrins. Selectins are polypeptides containing a sugar-binding site, while integrins are heterodimers composed of a large a subunit (Mr » 150,000) and a smaller b subunit (Mr » 95,000).49,50 and 51 Two se-lectins have been found to participate in the interaction between neutrophils and endothelial cells: L-selectin, a protein also found on lym-hocytes and monocytes, and E-selectin, also found on endothelial cells. The ligand for L-selectin has not been identified, although heparan sulfates on the luminal surface of endothelial cells are candidates.52 For E-selectin, ligands include certain sialylated fucosylated glycolipids that are expressed by neutrophils. Through interactions between these selectins and their ligands, circulating neutrophils attach reversibly to the endothelium, where they retain their spherical shape and roll with the flowing blood.53,54 These endothelium-associated neutrophils exchange freely with circulating neutrophils and probably constitute the marginated pool.
Exposure to inflammatory mediators causes L-selectin to be shed and neutrophil integrins to be activated.55,56 and 57 The principal neutrophil integrins are aLb2 (CD11a/CD18) and aMb2 (CD11b/CD18). These interact with ICAM-1, an integrin counterligand that is displayed on the luminal surfaces of endothelial cells exposed to inflammatory mediators.58,59 [A third neutrophil integrin, aXb2 (CD11c/CD18), interacts with complement-coated particles but has little to do with the binding of neutrophils to endothelial cells.] As a result of the interaction between the neutrophil integrins and ICAM-1, the neutrophils flatten onto the endothelial surface, to which they are now attached irreversibly.60 Migration of the neutrophils into the tissues now begins. Neutrophils leave the blood vessels by crawling between the endothelial cells. This process depends on the phosphorylation of an endothelial cell protein called platelet–endothelial cell adhesion molecule (PECAM-1), also known as CD31. PECAM-1 phosphorylation occurs in response to molecules such as endotoxin61 and platelet-activating factor,62 which are known to stimulate the egress of neutrophils from the circulation into the tissues.
DF32P-labeled neutrophils disappear from the circulation with a half-time (T1/2) of 6.7 h.6,63,64 These data are supported by the finding that over one-half of Pelger-Huët cells infused into a normal individual disappeared after 6 to 8 h.65 (Data obtained with 51Cr-labeled neutrophils give substantially longer half-times.66) The exponential disappearance of cells from the blood suggests that they leave in a random manner. Thus, neutrophils newly released from the marrow are as likely to leave the blood as are neutrophils that have been circulating for several hours. Certain senescent neutrophils, however, may be eliminated in a nonrandom fashion, perhaps by programmed cell death induced by growth factor deficiency,67 and are probably disposed of by the macrophage system.45
Assuming a random loss of neutrophils from the blood, the neutrophil turnover rate (NTR) can be calculated from the half-time and the total blood neutrophil pool (TBNP): NTR = 0.693 × TBNP/T1/2. In the steady state, the neutrophil turnover rate measures the rate of effective neutrophilopoiesis. Definitions and calculations related to blood neutrophil kinetics are given in Table 66-3 and data for normal humans in Table 66-4. The high production rate of neutrophils under normal conditions is remarkable, especially since it may increase severalfold in response to inflammatory stimuli.
TABLE 66-3 DEFINITIONS AND CALCULATIONS RELATING TO BLOOD NEUTROPHIL KINETICS
TABLE 66-4 DATA FOR HUMAN BLOOD NEUTROPHIL KINETICS
Glucocorticoids increase the total blood neutrophil pool by increasing influx from the marrow and decreasing efflux from the circulation. Five hours after a pharmacologic dose of glucocorticoid, the neutrophil count increases by about 4000/µl due to release from the marrow, demargination, and prolongation of the T1/2 to approximately 10 h.68,69 and 70 Consistent with the increase in the T1/2, prednisone reduces the accumulation of neutrophils at induced sites of skin inflammation.71 (Dexamethasone has been reported to produce a contrary effect.72) With alternate-day, single-dose prednisone, neutrophil counts and kinetics are normal 24 h after administration and during the day off.71 Endotoxin causes a prompt neutropenia as a result of cell margination and sequestration, followed in 2 to 4 h by a rebound neutrophilia as a result of cell release from the marrow. The size of the neutrophilic response correlates with the functional marrow reserves.73,74,75 and 76 After administration of epinephrine, a peak leukocytosis occurs in 5 to 10 min and rarely lasts more than 20 min. This reflects a shift of cells from the marginated to the circulating pool.
The migration of neutrophils into areas of inflammation has been widely studied, but little is known of the fate of these cells in normal tissues. Neutrophils normally migrate into the lung, oral cavity, gastrointestinal tract, liver, and spleen.77 They may be lost from mucosal surfaces or die in the tissues and be degraded by macrophages. The average life span of the mature neutrophil is thought to be very short, although an individual cell may survive for as long as 2 weeks.78 The neutrophil life span is further shortened if it takes in bacteria or other particles. Chemotactic stimuli, such as C5a and IL-8, draw neutrophils to areas of infection, where they may die in large numbers.
EVALUATION OF ADEQUACY OF NEUTROPHIL PRODUCTION
THE WHITE CELL COUNT AND MARROW CELLULARITY
The white cell and absolute neutrophil counts are the most widely used guides to the status of neutrophil production. They are useful in evaluating the effects of cytotoxic chemotherapy, although they do not provide quantitative information as to the rate of neutrophil production or destruction, the status of marrow reserves, or the presence of abnormalities in cell distribution.
Gauging neutropoiesis by the appearance of marrow films, clot sections, or biopsies also suffers from the limitations of sampling error and relatively poor correlation with kinetics, as measured by other techniques.64 For example, the morphologic findings in the marrow of a “maturation arrest,” with little neutrophil development beyond the promyelocyte or myelocyte stage, does not distinguish between a true defect in cellular maturation and rapid mobilization of cells from the marrow. Similarly, it is often difficult to distinguish by purely morphologic means neutropenic conditions due to ineffective neutropoiesis from those caused by peripheral destruction of neutrophils. However, despite these limitations, when the absolute neutrophil count and marrow cellularity are used together, they provide a useful guide in most clinical settings. If the absolute neutrophil count is less than 1000/µl (1.0 × 109/liter) and multiple marrow aspirations and/or biopsies are hypocellular, the patient almost invariably has impaired production of neutrophils. Very low neutrophil counts predispose to infections by bacteria and certain fungi (e.g., Candida and Aspergillus). Such infections become especially troublesome as the neutrophil count falls below 500/µl (0.5 × 109/liter). Unfortunately, the converse is not true; the finding of a cellular marrow and a neutrophil count above 1000/µl (>1.0 × 109/liter) does not mean that production is normal. Nevertheless, when marrow cellularity and absolute neutrophil count are considered together, they provide the most clinically useful assessment of neutrophil production.
Several agents that stimulate neutrophil production, including glucocorticoids, endotoxin, and etiocholanolone, have been used in the past to evaluate neutrophil reserves in a clinical setting. These have now been supplanted by recombinant human G-CSF, a remarkably nontoxic cytokine that, when given in therapeutic doses (5 to 8 µg/kg), increases the blood neutrophil count by stimulating neutropoiesis and accelerating neutrophil release from the marrow storage compartment (see also Chap. 15). The increase in neutropoiesis results from a threefold increase in the number of cell divisions in the mitotic compartment, together with a shortening of the maturation time from myelocyte to neutrophil from 4 to 5 days to less than 1 day.79,80 Thus, as a byproduct of its therapeutic action, the administration of G-CSF directly tests an individual’s capacity to produce neutrophils. This effect of G-CSF makes most of the older methods for evaluating neutrophil compartments obsolete.
G-CSF, however, does not test the distribution of neutrophils between the marginated and circulating pools. On the rare occasions when such information is desirable, epinephrine stimulation can be used to assess this distribution. For this purpose, 0.1 mg of epinephrine is infused intravenously over 5 min, and blood for white counts is obtained before and 1, 3, and 5 min after completion of the epinephrine infusion. Normally the neutrophils should increase by approximately 50 percent after epinephrine infusion.81
DNA MEASUREMENT: TRITIATED THYMIDINE AND MICROFLUORIMETRY
Tritiated thymidine ([3H]TdR) is selectively incorporated into the DNA of dividing cells and has the advantages of rapid degradation of unincorporated material, low reutilization of label released by cell death, and weak b-particle emission that is ideal for radioautography.82,83 This DNA marker is used to label S-phase cells (i.e., cells engaged in DNA synthesis) in the mitotic compartment. Labeled cells can be followed as they progress to more mature compartments. For example, this technique showed that myelocytes are the most mature cells of the neutrophil series that are capable of division.
In vivo administration of [3H]TdR to humans is no longer permitted. In vitro studies, however, provide useful information about initial pulse labeling and generation times of neutrophil precursors. Cell proliferation in vitro may not be equivalent to that in the intact subject but may be useful in acute leukemia, where such data may be of therapeutic and prognostic significance.
Microfluorimetry measures the DNA content of individual cells stained with a fluorescent DNA-binding dye. It can provide rapid information on the cell cycle distribution and proliferative status of normal and leukemic cells.84,85
The mitotic index for any morphologically homogeneous cell pool (e.g., the promyelocyte pool) is the ratio of cells in mitosis to total cells in the pool. Used alone, the mitotic index provides little information on cell kinetics; when combined with [3H]TdR-labeling studies, however, it can give valuable information on neutrophil precursor proliferation.11,44,86 Determining a mitotic index is laborious, and interpretation is somewhat uncertain because of the variability of the mitotic index in humans (7 to 43 per 1000 nucleated marrow cells) and the limitations in defining the morphologic pool.
TRACER TECHNIQUES FOR STUDYING NEUTROPHIL KINETICS IN HUMANS
The kinetics of neutrophil production and use in humans were worked out many years ago using radioactively labeled tracers. Materials formerly used for these in vivo studies included [3H]thymidine, DF32P, and radioactive chromium (51Cr3+). For reasons related to safety and technical difficulties, these tracers have been replaced for in vivo studies by lipophilic complexes of radioactive indium (111In): 111In-oxime and 111In-tropolone.87 To study neutrophil behavior in vivo using an 111In-labeled tracer, the leukocytes are isolated, incubated with the 111In complex, washed, and reinfused into the subject. Leukocyte life span can be determined by measuring the disappearance of 111In from the blood, while the distribution of the labeled leukocytes can be evaluated by imaging. 111In-labeled leukocytes are sometimes used clinically to locate abscesses and other sites of bacterial infection.87
LYSOZYME AND TRANSCOBALAMIN I
Lysozyme (muramidase) is an enzyme found in the granules of neutrophils but not of eosinophils or basophils. Measurements of serum and marrow lysozyme have been used to assess neutrophil production.88,89 Unfortunately, the correlation with cell kinetics is disappointing, and it is not useful in assessing neutrophil production.90 The clearance of the enzyme from the serum is also variable, depending primarily on the proximal renal tubule cells. Serum lysozyme is almost invariably elevated in monocytic leukemia, and serial measurements may be useful in the disease.88,91
Transcobalamin I is a B12-binding protein found in the specific (secondary) granules of neutrophils. It is present in cells as immature as myelocytes but is more abundant in mature cells.88,92 Serum levels tend to be high in patients with chronic myelogenous leukemia, poly-cythemia vera, or inflammatory leukocytosis.92 At extremely high neutrophil counts there is a reasonable correlation with serum concentration of this protein. Measurement of total B12-binding capacity in combination with serum lysozyme determination can provide useful information, particularly in myeloproliferative disorders.88
ACCUMULATION IN INFLAMMATORY SITES
The Rebuck window technique, utilizing the adherence of leukocytes to sterile cover slips overlying areas of superficially abraded skin, was introduced in 1955.93 Because this method is qualitative and does not assess nonadherent cells, attempts have been made to introduce more quantitative techniques by producing and examining skin blisters94 and skin chambers.95 None of these methods, however, is wholly satisfactory, and all are at best semiquantitative.
IN VITRO MARROW CULTURE
The CFU-GM is the cell that forms a mixed colony containing neutrophils and macrophages in semisolid agar culture. This cell is usually regarded as the committed progenitor of these cell lines. The number of colonies formed in agar from marrow aspirates should therefore reflect the number of CFU-GM in vivo. Unfortunately, in human subjects the technique can give information only about the relative concentration of CFU-GM among all the nucleated marrow cells; it does not give data about the total number of these progenitors. The technique therefore suffers from problems with sampling and determining absolute cell numbers. Total circulating CFU-GM can be quantified in blood, but this determination may reflect distribution more than production.96 The in vitro culture method does permit assessment of abnormalities of granulocytic development in various hematologic disorders and may be of particular use in preleukemia (see Chap. 92) and the acute leukemias (see Chap. 93).97 Inhibition of CFU-GM growth has also been used to identify antineutrophil antibodies.98,99
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn