Hematologic Laboratory facts

In a typical automated blood cell counter, the blood sample is aspirated and separated into two portions: one is lysed and diluted to permit measurement of hemoglobin concentration and leukocyte enumeration, and the other is diluted without lysis to enable counting and sizing of red cells and platelets

classic method to count and determine the volume of a particle or a cell is electrical impedance in which a specific volume of an electrolyte solution containing a dilute suspension of blood cells is aspirated through a small orifice across which a current is flowing. The electrical impedance produced as a cell passes through the orifice is registered as a particle for counting purposes and the height of the pulse generated by the electrical impedance can be made proportional to the volume of the particle. Automated hematology instruments today rely heavily on analysis of light scattered at different angles from an incident laser beam striking passing cells. Cell count, volume, and internal structure can be determined by multivariate analysis of these data.

In electronic instruments, the hematocrit (HCT) (proportion of blood occupied by erythrocytes) is calculated from the product of direct measurements of the erythrocyte count and the MCV (HCT [u1/100 u1] = RBC [x 10^–6/u1] x MCV [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 (cold agglutinins and some cases of autoimmune hemolytic anemia).³ This causes red cells to clump and by affecting the accuracy of both red blood cell (RBC) count and MCV, also affects the resultant hematocrit.

The "spun" hematocrit includes plasma trapped between red cells in the packed cell volume,⁵ typically about 2 to 3 percent of the packed volume.⁶ Microhematocrits from polycythemic samples (HCT greater than 55) or blood containing abnormal erythrocytes (sickle cell anemia, thalassemia, iron deficiency, spherocytosis, macrocytosis) are increased because of enhanced plasma trapping that generally is caused by increased red cell rigidity.^6,7 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 microcytosis⁸). In general, the automated hematocrit is more accurate and easier to obtain than the spun hematocrit, although the hemoglobin determination is preferred to either, because it is measured directly and is the best indicator of the oxygen-carrying capacity of the blood.

Hemoglobin is intensely colored, and this property has been used 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 blood, red cells are lysed and hemoglobin variants are converted to the stable compound cyanmethemoglobin for quantitation by absorption at 540 nm.

The MCHC, the concentration of hemoglobin per unit with red cell volume, is calculated by the formula MCHC (g/dl of red cells) = hemoglobin (g/dl) / hematocrit (ml/100 dl) x 100. An MCHC greater than 35 g/dl red cells is associated with hereditary spherocytosis,²³ and a low MCHC is typical of iron deficiency,²⁴ but its diagnostic usefulness is limited.

For example, patients with sideroblastic anemia usually have a dimorphic blood picture, with both hypochromic and normochromic cells. The MCH and MCHC may be in the normal range, and the important finding of the mixed-cell population, both normochromic and hypochromic cells, would not be detected unless a blood film is examined. A normal MCV might occur in the setting of iron deficiency coupled with folate deficiency, but examination of the blood film would show cells characteristic of each, such as oval macrocytes and hypochromic microcytes. 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^27,28 and although proposed as an aid in distinguishing iron deficiency from other causes of microcytic anemia,²⁹ such as thalassemia, the RDW is not sufficiently specific to obviate the need for more specific tests.³⁰ The RDW can be used in the laboratory as a flag to select those samples submitted for automated blood count that should have manual review of the blood film for red cell morphology.

The reticulocyte is a newly released anucleate red cell that enters the blood from marrow with residual detectable amounts of RNA. The number of reticulocytes in a volume of blood permits an estimate of marrow erythrocyte production and is thus useful in evaluating the pathogenesis of anemia, distinguishing inadequate production from accelerated destruction (hemolysis). Various proprietary combinations of light scatter and other parameters are used to minimize interferences such as nucleated red cells, nuclear remnants (Howell-Jolly bodies), malaria parasites, or platelet clumps.

The theoretical advantage is that acute changes in red cell function would be detected more rapidly and reliably in the reticulocyte fraction as opposed to the whole red cell population

Most high-end hematology analyzers now report some quantitative measure of reticulocyte RNA content.³³ Increase in the immature reticulocyte fraction (those with highest RNA content) is an early sign of marrow recovery³⁸ and has been used as a marker of ineffective erythropoiesis, distinguishing macrocytosis caused by megaloblastic anemia or myelodysplasia from other causes.³⁹ A limitation at present is that both the methods and reference ranges for these parameters are instrument dependent.

Leukocyte counts are performed by automated blood counters on blood samples appropriately diluted with a solution that lyses the erythrocytes (e.g., an 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 because of technical and statistical factors. Automated leukocyte counts may be falsely elevated as a result of cryoglobulins or cryofibrinogen,⁴¹ clumped platelets or fibrin from an inadequately anticoagulated or mixed sample,⁴² ethylenediaminetetraacetic acid (EDTA)-induced platelet aggregation,⁴³ nucleated red blood cells,⁴² or nonlysed red cells. These interferences cause a population of small-size particles to appear in the leukocyte volume histogram, and trigger a flag for manual review.

Platelets are usually counted electronically by enumerating particles in the unlysed sample within a specified volume window (e.g., 2–20 fl), where volume may be measured by electrical impedance or light scatter. 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 smaller red cells.

Because platelet volumes in health or disease follow a log-normal distribution,⁵⁷ volume histograms inconsistent with such a distribution are flagged for manual review. Automated platelet counting by current instrumentation is accurate and reliable, even in the thrombocytopenic range,⁵⁸ and far more precise than manual methods.⁵⁸ 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 or "satellitism" (falsely decreased count). Platelet clumping, or platelet "satellitism" (adherence of platelets to neutrophils), may occur as a result of platelet-reactive antibodies,⁵⁹ which typically cause no clinical symptoms. 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.⁵⁹ This condition occurs in approximately 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.⁶⁰ Platelet counting under these conditions is difficult, but can be minimized by collecting blood in citrate⁶⁰ or estimating platelet count from a freshly prepared fingerstick blood smear.

The number of platelets with high RNA content ("reticulated platelets"), measured using RNA-binding fluorescent dyes such as thiazole orange, is a marker of marrow megakaryopoiesis and has been proposed as a way of differentiating hypoproductive from destructive causes of thrombocytopenia, in an analogous fashion to the reticulocyte count. The percentage, but not the absolute number, of reticulated platelets is increased in destructive thrombocytopenias, whereas the absolute number, but not percentage, is decreased in hypoproductive states.⁶⁸ Reticulated platelet number or RNA correlates with imminent platelet recovery after chemotherapy.

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 blood cells should be sufficiently thin that only a few erythrocytes in a x100 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).

The blood film is first scanned at low magnification (x200) 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 magnification examination (x400). The optimal portion of the film is then examined at high magnification (x1000, oil immersion) to systematically assess the size, shape, and morphology of the major cell lineages.

Anisocytosis is the term that describes variation in erythrocyte size, and is the morphologic correlate of the RDW. The macrocyte, a red cell larger than normal, may be seen in a number of disease states, for example, folic acid or vitamin B12 deficiency. Cells are considered to be macrocytes if they are well hemoglobinized and their diameters exceed 9 um. Early ("shift" or "stress") reticulocytes (i.e., those with the most residual RNA) appear in stained films as large, bluish cells, referred to as polychromatophilic cells. These cells roughly correspond to those quantitated by automated analyzers as the immature reticulocyte fraction. Microcyte, a red cell smaller than normal, is the term used to describe a cell less than 6 um in diameter.

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. 28. Erythrocytes with evenly spaced spikes (crenated cells) can be an artifact caused by prolonged storage, or may reflect metabolic erythrocyte abnormalities. Spherocytes are more densely stained and appear smaller because of their rounded shape; they show decreased or absent central pallor. 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

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 a result of the presence of an increase in immunoglobulin (Ig), especially IgM, and suggests the diagnosis of macroglobulinemia. Occasionally, very high concentrations of IgA or IgG may produce noticeable pathologic rouleaux, as a manifestation of mieloma.

If the platelet count is normal, approximately 8 to 15 platelets (individually or in small clumps) should be visible in each oil-immersion (x1000) field. There should be 1 platelet present for about every 20 erythrocytes. This is a valuable check when the automated platelet count is in question or an unexpected result is obtained

Neutrophils are round cells ranging from 10 to 14 m in diameter (see Color Plate VII). The nucleus is lobulated, with two to five lobes connected by a thin chromatin thread. The defining feature of the segmented neutrophil is the round lobes with condensed chromatin, because the chromatin thread may overlie the nucleus and not be visible

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 Color Plate X-6). 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 Color Plate VII-3). 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.

Lymphocytes on blood films are usually small, about 10 m in diameter, but larger forms up to 20 m in diameter are seen. The small lymphocyte, the predominant type in normal blood, is round and contains a relatively large, round, densely stained nucleus.

Monocytes are the largest normal cells in the blood, usually measuring from 15 to 22 m in diameter. The nucleus is of various shapes—round, kidney-shaped, oval, or lobulated—and frequently appears to be folded (see Color Plates VII-1, 2). The chromatin is arranged in fine strands with sharply defined margins. The cytoplasm is light blue or gray, contains variable numbers of fine lilac or purple granules, and is frequently vacuolated, especially in films made from blood anticoagulated with EDTA.

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 Chaps. 59 and 66).⁷⁴  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.

Light blue round or oval Döhle bodies, about 1 to 2 m in diameter, may be seen in the cytoplasm of neutrophils of patients with infections, burns, and other inflammatory states. The blue staining is caused by RNA of the rough-surfaced endoplasmic reticulum contained in Döhle bodies. Similar blue inclusions are seen in patients with the May-Hegglin anomaly.

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.