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 anemia27,28 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.