CHAPTER 2 EXAMINATION OF THE BLOOD
CHAPTER 2 EXAMINATION OF THE BLOOD
DANIEL H. RYAN
Quantitative Measures of Hematopoietic Elements in the Blood
Morphologic Examination of the Blood
Red Cell Morphology
The Need for Examination of the Blood Film
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.
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
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
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 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.
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.”
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.
Leukocytes may contain abnormal inclusions as a result of genetic or acquired disorders.
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
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.
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.
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
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn