The Peripheral Blood Smear
A peripheral blood smear (peripheral blood film) is a glass microscope slide coated on
one side with a thin layer of venous blood. The slide is stained with a dye, usually
Wright’s stain, and examined under a microscope.
Microscopic examination of the peripheral blood is used to supplement the information
provided by automated hematology analyzers ("blood cell counters"). Hematology
analyzers provide accurate quantitative information about blood cells and can even
identify specimens with abnormal cells. However, the precise classification of abnormal
cells requires a trained microscopist, a well-made peripheral blood smear, and a light
microscope with good optical characteristics. In practice, hematology analyzers of
varying sophistication are used for cell counting in all but the smallest hematology
laboratories. In addition to providing cell counts and graphical displays of the
information recovered, these instruments also provide a warning ("flag") that atypical
cells were found and provide a presumptive identification of the abnormality. The
instrument operator reviews the information from each specimen and decides if smear
preparation and light microscopy are necessary. If not, the information is released to the
clinician.
Peripheral Blood Smear Preparation
The wedge slide ("push slide") technique developed by Maxwell Wintrobe remains the
standard method for the preparation of peripheral blood smears (films). The following
procedure (Fig. 1) is utilized to prepare a peripheral smear.

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





Place a 1" x 3" glass microscope slide with a frosted end on a flat surface
(usually the counter top of a laboratory bench).
Attach a label on the slide or write the patient name, specimen identification
number, and date of preparation on the frosted surface.
Place a 2 - 3 mm drop of blood approximately 1/4" from the frosted slide, using a
wooden applicator stick or glass capillary tube.
Hold the slide by the narrow side between the thumb and forefinger of one hand
at the end farthest from the frosted end.
Grasp a second slide ("spreader slide") between the thumb and forefinger of the
other hand at the frosted end.
Place the edge of the spreader slide on the lower slide in front of the drop of
blood (side farthest from the frosted end).
Pull the spreader slide toward the frosted end until it touches the drop of blood.
Permit the blood to spread by capillary motion until it almost reaches the edges
of the spreader slide.
Push the spreader slide forward at a 30o angle with a rapid, even motion. Let the
weight of the slide do the work.
Table 1
Preparation of Peripheral Blood Smear
Step 1. Placing a small
drop of venous blood
on a glass microscope
slide, using a glass
capillary pipette. A
wooden applicator
stick can also be used
for this purpose.
Step 2. A spreader
slide has been
positioned at an angle
and slowly drawn
toward the drop of
blood.
Step 3. The spreader
slide has been brought
in contact with the drop
of blood and is being
drawn away. Note
layer of blood at the
edge of the spreader
slide.
Step 4. The spreader
slide is further pulled
out, leaving a thin layer
of blood behind.
Step 5. The blood
smear is nearly
complete.
Step 6. End result. A
glass slide with a wellformed blood film. After
drying for about 10
minutes, the slide can
be stained manually or
placed on an
automated slide
stainer.
Fig. 1. Wedge slide technique for preparation of a peripheral blood smear.
A well-made peripheral smear is thick at the frosted end and becomes progressively
thinner toward the opposite end. The "zone of morphology" (area of optimal thickness
for light microscopic examination) should be at least 2 cm in length. The smear should
occupy the central area of the slide and be margin-free at the edges (Fig 2).
Fig. 2. Photograph of the peripheral blood smear prepared above. The arrow points to
the zone of morphology.
Peripheral Blood Smear Examination
Peripheral smear examination requires a systematic approach in order to gather all
possible information. In addition, all specimens must be evaluated in the same manner,
to assure that consistent information is obtained. The following approach is
recommended:

An examination at low power (10X ocular, 10x objective) is first performed to
evaluate the quality of the smear, ascertain the approximate number of white
blood cells and platelets, and to detect rouleaux formation, platelet clumps, and
leukocyte clumps and other abnormalities visible at low magnification. An optimal
area for evaluation at higher magnification is also chosen. This should be an
intact portion of the smear free of preparation artifact where the red blood cells
are separated by 1/3 to 1/2 of a cell diameter. The red blood cells should stain a
pink color, while neutrophils show "crisp" features, with deep blue-purple nuclear
material and lilac to pinkish to violet cytoplasmic granules. Optimal preparation
and staining of the peripheral blood smear is critical for morphologic examination;
an inadequate smear should not be examined.

Following low power examination of a peripheral blood smear, the 50X or 100X
objective of the microscope is selected (500X or 1000X total magnification when
using a 10x ocular) and the area of morphology is examined in a consistent
scanning pattern (Fig 3) to avoid counting the same cell(s) twice. A differential
count of at least 100 white blood cells (200, 500, or 1000 is even better) is
performed, and any abnormal morphology of RBCs, WBCs, and platelets
observed during the differential count is recorded. Each morphologic abnormality
observed should be quantitated ("graded") separately as to severity ("slight to
marked" or "1+ to 4+"). Medical technologists are well trained in the reproducible
quantitation of morphologic abnormalities; details are available in medical
technology textbooks.
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A fairly accurate estimate of the white blood cell count (cells/mL) can be obtained
by counting the total number of leukocytes in ten 500X microscopic fields,
dividing the total by 10, and multiplying by 3000. These estimates should
approximate that obtained by the cell analyzer. If the estimate does not match
the automated cell count, obtain the original blood specimen, confirm patient
identity, repeat the automated analysis, and prepare a new smear.
Fig. 3. Scanning technique for peripheral blood differential count and morphologic
evaluation. (a) Ten microscopic fields are examined in a vertical direction from bottom to
top (or top to bottom). (b) The slide is horizontally moved to the next field (c) Ten
microscopic fields are counted vertically. (d) The procedure is repeated until 100
leukocytes have been counted (for a 100-cell count).
A peripheral smear must be interpreted in the context of the clinical situation. That is,
only limited information can be obtained unless the following information is available
with the peripheral smear.

The age and sex of the patient must be known, since absolute cell numbers and
the significance of some findings vary with age. For example, relative
lymphocytosis with NRBCs and atypical lymphocytes would be unusual and
pathologic in an adult, but appear in any infant under stress.

The red blood cell count (RBC), hemoglobin, hematocrit, mean corpuscular
volume (MCV), and red cell distribution width (RDW) cannot be accurately
determined by manual smear examination and should be available. The white
blood cell count (WBC) and platelet count can be approximated manually, but an
automated ("machine") count is helpful.

Graphical information provided by the hematology analyzer is helpful but not
essential for peripheral blood smear interpretation. Hematology analyzers utilize
light scatter, electrical impedance, and other physical parameters to count cells,
determine cell size and differentiate different types of blood cells. For example,
many modern hematology analyzers measure electrical impedance, light scatter,
cell viability, and other parameters during the evaluation process. Light scatter at
0 o roughly corresponds to cell size, 10 o light scatter to cellular internal
"complexity," 90 o light scatter to nuclear lobularity, and 90 o depolarized light
scatter to cytoplasmic granularity. An example of a light scatter histogram
produced by a modern hematology analyzer is shown in Fig 4.
Disadvantages of the Peripheral Blood Smear
Peripheral blood smear examination provides information that cannot be obtained from
automated cell counting. However, peripheral smear evaluation has some limitations
and special considerations. These include:
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Experience is required to make technically adequate smears.
There is a non-uniform distribution of white blood cells over the smear, with
larger leukocytes concentrated near the edges and lymphocytes scattered
throughout.
There is a non-uniform distribution of red blood cells over the smear, with small
crowded red blood cells at the thick edge and large flat red blood cells without
central pallor at the feathered edge.
Automated Hematological Evaluation
The total red cell count (RBC), RBC size (mean corpuscular volume, MCV), and red cell
distribution width (RDW) are determined from analysis of electrical impedance and/or
light scattering data by the hematology analyzer. These measurements are used to
calculate the hematocrit, MCH, and MCHC.
In unusual circumstances the automated hematology analyzer produces cell counts
which are falsely increased or decreased. Fortunately, in almost all cases the
instruments "flag" the specimen as abnormal so that the operator can verify the results
manually or perform necessary corrections. Causes of spurious red blood cell counts
include:
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
Very small red blood cells (microcytosis) may be counted as large platelets and
result in a falsely decreased RBC.
Autoagglutination or cryoglobulins lead to RBC clumping, which may falsely
increase the RBC.
Spurious elevations in the RBC may also occur in patients with very high WBCs
(> 100 x109).
Fig. 4. A modern hematology analyzer (Cell Dyn 4000, Abbott Laboratories, Chicago,
IL). Sample analysis is performed in the
RBC Evaluation
Importance of the MCV and RDW
The MCV is the median value of the histogram distribution obtained when red blood cell
size is plotted against the number of cells ("red cell histogram")(Fig. 5). The MCV,
measured in femtoliters (fL, or 10-15 L), is the most important of the red cell indices. It is
used to classify anemias as normocytic (normal MCV), microcytic (decreased MCV), or
macrocytic (increased MCV). However, the MCV may be falsely elevated in patients
with red blood cell agglutination, since the hematology analyzer may identify some of
the cell clumps as single cells. In patients with severe hyperglycemia (glucose > 600
mg/dL), osmotic swelling of the red blood cells may also spuriously elevate the MCV.
A related parameter, the red cell distribution width (RDW) is the coefficient of variation
of the red blood cell distribution histogram. As a quantitative measure of variation in red
blood cell size (anisocytosis), the RDW is elevated in iron deficiency anemia,
myelodysplastic syndromes, macrocytic anemia secondary to vitamin B12 or folate
deficiency, and some malignancies. In contrast, the RDW is usually normal or only
mildly elevated in the microcytic anemia of thalassemia.
Fig. 5. Red cell distribution
histograms. In these histograms,
RBC volume (x-axis) is plotted vs.
the cell count (number of events
counted (y-axis). The mean
corpuscular volume (MCV) is the
median value of the histogram
distribution. The red cell distribution
width (RDW) is the coefficient of
variation of the curve. Microcytic red
cells (a) fall to the left portion of the
curve, while macrocytic red cells fall
to the right (c). The histogram in the
center is from a normocytic,
normochromic specimen with an
MCV of 88 fL.
Measurement of Hematocrit
The hematocrit (Hct, "crit") is the ratio of the volume of red blood cells to the volume of
whole blood. In the past, the hematocrit was determined by centrifugation of whole
blood in a narrow glass tube (capillary blood tube) sealed at one end ("spun
hematocrit"). The spun hematocrit is spuriously elevated if plasma becomes trapped in
the red cell layer. This phenomenon occurs in patients with polycythemia, macrocytosis,
spherocytosis, hypochromic anemias, and RBC fragment syndromes. Improper mixing
of the specimen or the addition of excessive anticoagulant can also lead to false Hct
values. Since "crit" tubes are also fragile and dangerous to use, spun hematocrits are
rarely used today.
The automated hematology analyzer calculates the Hct from the RBC and MCV by the
following formula:
Hct (L/L, %) = RBC (cells/L) x MCV (L/cell)
Since the Hct is a calculated value, it is less accurate than either the RBC or Hb, and is
affected by errors in either or both of these measurements.
The MCV, mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin
concentration (MCHC) are the red blood cell indices. The MCH is the hemoglobin
concentration per cell (hemoglobin mass/red blood cell), expressed in picograms per
cell (pg, 10-12 g). The MCH is calculated from the hemoglobin and RBC by the
following formula:
The MCH is decreased in patients with anemia caused by impaired hemoglobin
synthesis. The MCH may be falsely elevated in blood specimens with turbid plasma
(usually caused by hyperlipidemia) or severe leukocytosis.
The MCHC is the average hemoglobin concentration per total red blood cell volume
(ratio of hemoglobin mass to RBC volume), as determined from the following equation:
The MCHC is decreased in microcytic anemias where the decrease in hemoglobin
mass exceeds the decrease in the size of the red blood cell. It is increased in hereditary
spherocytosis and in patients with hemoglobin variants, such as sickle cell disease and
hemoglobin C disease).
Measurement of Hemoglobin
Nearly all automated hematology analyzers utilize the cyanomethemoglobin method to
measure the hemoglobin content of the red blood cell. In this method, hemoglobin is
converted to cyanated methemoglobin (cyanmethemoglobin) by the addition of a
solution (Drabkin solution) containing potassium ferricyanide and potassium cyanide.
Cyanated methemoglobin maximally absorbs light at 540 nm, and the total amount of
hemoglobin is determined by spectrophotometry. The hemoglobin concentration is
measured in grams per deciliter (g/dL) of whole blood.
Hyperlipidemia, fat droplets (from hyperalimentation), hypergammaglobinemia,
cryoglobulinemia, and leukocytosis (> 50 x 109/mL) can result in spurious elevations of
hemoglobin concentration. Spurious hemoconcentration or hemodilution, occurring with
improper specimen collection, may falsely elevate or decrease hemoglobin
concentrations. In addition, the age, sex, and race of the patient must also be
considered in the interpretation of hemoglobin levels. Hemoglobin levels fall during the
first month of life and remain relatively low until after puberty. The mean male
hemoglobin level is 1 - 2 g/dL higher than the mean female level. Blacks of both sexes
and all ages have hemoglobin levels which are 0.5 - 1.0 g/dL lower than whites of the
same age and sex.
White Blood Cell Evaluation
The total white blood cell count (WBC, leukocyte count) includes all circulating
nucleated hematopoietic cells with the exception of nucleated red blood cells (NRBCs).
The WBC is of great importance in the diagnosis and management of patients with
hematologic and infectious diseases. It is also used to monitor patients receiving
cytotoxic drugs, radiation therapy, and some antimicrobial drugs.
The WBC is determined on EDTA-anticoagulated blood. RBCs are removed by lysis,
and the total WBC is measured by electrical impedance or light scatter techniques.
Unlysed red blood cells, nucleated red blood cells, platelet clumps, large platelets, and
cryoglobulins may result in spurious WBC results. If these conditions are detected by
the hematology analyzer, the specimen is "flagged" for a manual peripheral smear
evaluation. Occasionally, a manual WBC, using a hemacytometer, may be necessary to
verify the accuracy of an automated WBC. If NRBCs are present, a relative estimate of
their number is obtained by light microscopy and expressed as # NRBCs/100 WBC.
Under these circumstances, the total WBC must be corrected by use of the following
formula:
Differential Leukocyte Count
The differential leukocyte count (leukocyte differential, white blood cell differential) is
probably the least understood and overutilized of all hematologic assays. Until the
1980’s, the relative number (%) of each type of white blood cell was determined by
manual examination of the peripheral blood smear and multiplied by the white cell count
to obtain the absolute leukocyte count (cells/mL). Unfortunately, the manual differential
is labor intensive, subjective, statistically unreliable (only 100-200 cells are counted),
and inaccurate because of nonrandom distribution of cells on the smear (monocytes at
the edge, lymphocytes in the middle).
Hematology analyzers are more accurate than the manual count for leukocyte
elaboration under normal circumstances. For example, the hematology analyzers used
in the Hematology Laboratories at WVU Hospitals generates a five-part differential
based on electronic impedance, conductivity, and light scatter measurements (Fig. 6).
Cell counting with these instruments is rapid, objective, statistically significant (8000 or
more cells are counted), and not subject to the distributional bias of the manual count.
In addition, the precision of the automated differential makes the absolute leukocyte
count reliable and reproducible.
Hematology analyzers cannot yet correctly identify all abnormal white blood cells, and
manual examination of the peripheral smear is still needed under some circumstances
(blasts and immature cells, atypical lymphocytes, leukopenia, etc.). Specimens that
meet one or more of abnormal criteria are flagged for manual examination. Depending
on the patient population, a manual smear examination is required in only approximately
25% of peripheral smears.
Fig. 6. Normal leukocyte
differential histogram from
a modern hematology
analyzer (Cell-Dyn 4000,
Abbott Diagnostics).
Scatterplot obtained from
laser light scatter analysis
of white blood cells. y-axis
represents data obtained
by light scatter at 0o
(measure of cell size),
while x-axis represents
laser light scatter at 7o (cell
internal complexity). Each
"dot" represents data from
a single cell. Clusters of
cells represent neutrophils
(66.6%), monocytes
(8.63%), lymphocytes
(22.2%), eosinophils
(2.23%), and basophils
(0.35%) present in the
specimen. The total white
blood count was 6.89 x
109/L.
Absolute vs. Relative Leukocyte Counts
The absolute leukocyte count provides clinical information of much greater value than
the relative differential count. In fact, the relative count can be misleading, and the sole
use of this parameter can conceal the diagnosis of certain cytopenias or obscure
clinically significant trends that are occurring. The absolute neutrophil count, (ANC) and
not the relative count, is helpful in monitoring chemotherapy patients, and the absolute
neutrophil count is a superior indicator of infection and inflammation.
The report of an abnormal blood count is often the first clue to an abnormality of the
white cell series; less commonly, the patient presents with an infection or other clinical
problem. However, the peripheral blood leukocyte count is only one measure of white
cell activity, and several factors must be considered in data interpretation. For example,
a patient with a total white blood cell count of 2000/mL and 100 neutrophils/mL has a
different problem from a patient with a white blood count of 2000/mL and 100
lymphocytes/mL. Absolute leukocyte numbers must be always be reviewed. In addition,
the peripheral blood is only a conduit for leukocytes, and only a small percentage of the
total white blood cells in the body are present in the peripheral blood at any one time.
Therefore, the total white blood count and absolute leukocyte count must be interpreted
in light of the physical findings and other laboratory data. Common causes are
summarized in Table I.
Table I
Common Causes of Altered Leukocyte Counts
Decreased
Increased
Neutrophil Congenital
Hereditary neutropenia
Acquired
Infections
Acquired
Tissue destruction
Bone marrow disease
Corticosteroids, lithium
Immune reactions
Neoplastic growth
Drugs
Leukemoid reaction
Gram-negative septicemia
Myeloproliferative disorders
Lymphocyte Congenital
Acquired
Congenital immunodeficiency
Viral infection (EBV, hepatitis,
disease
etc.)
Some fungal, parasitic infections
Acquired
Rare bacterial infection
Severe infection
(Pertussis)
Drugs (Corticosteroids, alkylating) Allergic reactions/drug
GI disease
sensitivities
Immunodeficiency
Immunologic disease
Monocyte
Acquired
Mycobacterial infection
Acquired
Tuberculosis, syphilis
Hairy cell leukemia
Subacute bacterial endocarditis
Corticosteroids
Inflammatory responses
Recovery phase of neutropenia
Myeloproliferative disorders
Eosinophil Acquired
Acquired
Bacterial infection
ACTH administration
Basophil
Acquired
Corticosteroids
Parasitic infections
Drug therapy
Hypersensitivity reactions
Pulmonary disease
Myeloproliferative diseases
Acquired
Myeloproliferative syndromes
Lymphoproliferative disease
Hypersensitivity reactions
Hodgkin’s disease
Some viral infections
Myxedema
Influence of Age on Leukocyte Count
The lymphocyte is the predominant white blood cell in young children, while the
neutrophil is predominant in normal adults. Each laboratory is required to establish
"normal" leukocyte ranges for different ages, and this information must be displayed on
the report from the laboratory. Age-related changes in the cellular composition of the
blood must be considered during the interpretation of leukocyte counts.
Platelet Evaluation
The platelet count is one of several laboratory assays of importance in the functional
evaluation of the hemostatic system. A decreased platelet count (thrombocytopenia)
can result from a marrow production problem or a peripheral platelet destructive
process. Bleeding complications or even death can result in the presence of a severely
decreased platelet count. Elevated platelet counts (thrombocytosis) can result from a
reactive or neoplastic process. EDTA-anticoagulated blood is preferred for platelet
counts, but the specimen must be thoroughly mixed to prevent platelet clumping and
falsely decreased platelet counts (spurious thrombocytopenia). However, rare
individuals have EDTA-activated, nonpathogenic anti-platelet antibodies and require
citrate-anticoagulated blood specimens for platelet counting. Other than platelet
clumping, red and white blood cell fragments are most often responsible for interference
with accurate platelet counts. Modern hematology analyzers calculate the mean platelet
volume (MPV), based on a platelet distribution histogram. The MPV is increased in
patients with peripheral platelet destructive processes, in whom young, large platelets
are rapidly released from the bone marrow. The MPV is decreased in cases of
thrombocytopenia due to marrow suppression.
Morphologic Evaluation of Red Blood Cells
Normal red blood cells ("normocytes," "discocytes") are round to very slightly ovoid cells
with a mean diameter of approximately 7 mm and a central pale area ("area of central
pallor") approximately 1/3 the diameter of the cell that gradually fades towards the more
deeply stained periphery. The RBC is approximately the same size as the nucleus of a
mature lymphocyte. Any deviation in size, volume, or shape represents an abnormal red
blood cell.
Clinical Importance of RBC Morphology
Since different types of abnormal red blood cells arise by different etiologic processes,
disease diagnosis can often be made by interpretation of red blood cell pathology in
conjunction with CBC data and other clinical and laboratory information. The following
diagram (Fig. 5) shows the etiology of the more common abnormal red blood cells, and
they are individually discussed below.
Classification of RBC Morphologic Abnormalities
A summary table of red blood cell morphologic abnormalities is included on another
page (go there).
Acanthocytes
Acanthocytes ("spur cells, spicule cells") are spheroid RBCs with a few large spiny
(thorny) projections. There are usually 5-10 spicules per cell, which show irregular
spacing and thickness. Acanthocytes must be differentiated from echinocytes, which
have shorter and more regular spicules (see below). Occasional acanthocytes can be
seen after splenectomy, in patients with alcoholic cirrhosis, and in hemolytic anemias
caused by pyruvate kinase (PK) deficiency. microangiopathic hemolytic anemia,
autoimmune hemolytic anemia, sideroblastic anemia, thalassemia, severe burns, renal
disease, McLeod phenotype, or infantile pyknocytosis. The majority of erythrocytes form
acanthocytosis in the rare disease abetalipoproteinemia. Therefor, serum lipid
evaluation is recommended if large numbers of acanthocytes are seen in the absence
of an obvious clinical cause.
Agglutination
True agglutination is irregular clumping and agglutination of red blood cells into grapelike clusters. True agglutination must be differentiated from the rouleaux formation
(pseudoagglutination) seen in patients with paraproteins or marked
hypergammaglobulinemia or fibrinogenemia, which produces more regularly spaced
clusters of red blood cells adhering side-to-side ("coin stacks," see below). True red cell
agglutination usually indicates the presence of a cold reactive anti-red blood cell
antibody ("cold agglutinin") found in cold agglutinin syndrome or paroxysmal cold
hemoglobinuria, although some warm-reactive autoantibodies with wide temperature
specificity may produce similar agglutination. True agglutination and
pseudoagglutination cannot always be differentiated by light microscopy, but the
Coomb’s test, cold agglutinin titer, and serum/urine protein analysis can provide
additional information.
Basophilic stippling
Basophilic stippling is the occurrence of fine, medium, or coarse blue granules uniformly
distributed throughout some red blood cells. Fine stippling may be associated with
polychromatophilia, while coarse stippling usually indicates impaired erythropoiesis.
Heavy metal poisoning (e.g. lead and arsenic), hemoglobinopathies, thalassemias,
sideroblastic anemias, pyrimidine-5’-nucleotidase deficiency, and other diseases should
be excluded when coarse basophilic stippling is found.
Bite cells
Bite cells (degmacytes) are RBCs with peripheral single or multiple arcuate defects
("bites"). They are usually accompanied by at least a few blister cells (RBCs with
vacuoles or markedly thin areas at periphery of membrane), acanthocytes, and
schistocytes. Bite cells are associated with oxidant stress to the red blood cell. They can
be found in normal individuals receiving large quantities of aromatic drugs (or their
metabolites) containing amino, nitro, or hydroxy groups, or in patients with red-cell
enzymopathies involving the pentose phosphate shunt (most notably G-6-PD
deficiency, pyruvate kinase deficiency) during acute hemolytic episodes following
exposure to oxidant stress. If indicated, a Heinz body test, G-6-PD level, and other
studies of red blood cell metabolism may be indicated.
Blister cells
Blister cells are red blood cells with vacuoles or markedly thin areas at periphery of
membrane. These cells are characteristic of glucose-6-phosphate dehydrogenase (G-6PD) deficiency and other conditions imposing oxidant stress on the erythrocyte.
Codocytes
Codocytes ("target cells") are thin, hypochromatic cells with a round area of central
pigmentation. Codocytes are characteristically seen after splenectomy, and in patients
with thalassemia, and certain hemoglobinopathies (hemoglobin SS, SC, CC, EE, AE,
sickle cell-thalassemia). They are also found in association with iron deficiency anemia,
liver disease, and familial lecithin-cholesterol acyltransferase (LCAT) deficiency. If
indicated, hemoglobin electrophoresis, liver function evaluation, serum iron studies,
serum lipid profile and/or other studies may be indicated.
Dacrocytes
Dacrocytes ("tear drop cells") are red blood cells in the shape of a teardrop.
Microcytosis and hypochromia usually accompany them. Dacrocytes are
characteristically seen in relatively large numbers in patients with myelophthisic anemia
(particularly myelofibrosis with myeloid metaplasia), but can be found in moderate
numbers in megaloblastic anemia, beta-thalassemia, renal failure, tuberculosis, Heinz
body disease, acquired hemolytic anemia, hypersplenism, and other hematologic
diseases. Teardrop cells are pathologic and usually indicate significant bone marrow
dysfunction. Clinical correlation and patient follow up is essential.
Drepanocytes
Drepanocytes ("sickle cells") are curved, irregular red blood cells with pointed ends,
which are characteristic of the "sickle" hemoglobinopathies. Diseases with Hb S (sickle
cell anemia, hemoglobin SC disease, hemoglobin S-beta-thalassemia, hemoglobin SD
disease, hemoglobin Memphis/S disease) are the usual cause, but drepanocytes can
also be seen in other hemoglobinopathies (especially Hb I, Hb C-Harlem, Hb C
Capetown). A sickle cell screen and/or hemoglobin electrophoresis may be indicated.
Echinocytes/Burr Cells
Echinocytes ("sea urchin cells") are red blood cells with multiple tiny spicules (10-30)
evenly distributed over the cell surface. These cells result from exposure of the red cell
to fatty acids, lysolecithin, amniotic compounds, elevated pH, and other substances.
They occur post-splenectomy, after the administration of heparin, in the hemolyticuremic syndrome, and in hepatitis of the newborn, pyruvate kinase deficiency,
phosphoglycerate kinase deficiency, uremia, and malabsorption states.
Burr cells ("crenated cells") are similar in appearance, but show an uneven distribution
of spicules. Burr cells are characteristically seen in uremia, where they represent
damaged or fragmented red blood cells.
Fig. 7. Representative examples of red blood cells with abnormal morphology.
Elliptocytes
Elliptocytes are cells with an elliptical shape, while ovalocytes have an oval shape.
Severe elliptocytosis (> 10% elliptocytes) is characteristic of hereditary elliptocytosis,
but can be prominent in thalassemia, sickle cell trait, and Hb C trait. Elliptocytic
hemolytic anemia (< 10% has been reported in association with cirrhosis, decreased
erythrocyte glutathione, and with glucose-6-phosphate deficiency. Other diseases
where elliptocytosis occurs include iron deficiency anemia, megaloblastic anemia,
myelophthisic anemia, and mechanical trauma. Rare elliptocytes (< 1%) occur in normal
peripheral blood smears. If clinically indicated, osmotic fragility evaluation, hemoglobin
electrophoresis, and studies of red blood cell metabolic activity may be indicated for
further evaluation of patients with elliptocytosis.
Howell-Jolly bodies
Howell-Jolly bodies are small (1 mm) dense, perfectly round basophilic red cell
inclusions which represent nuclear material derived from nuclear fragmentation
("karyorrhexis) or incomplete nuclear expulsion during normoblastic maturation. HowellJolly bodies produced in non-diseased individuals are effectively removed by the spleen
and are not visible in the circulation. However, Howell-Jolly bodies are readily identified
in splenectomized patients and may also be seen in smaller numbers in patients with
megaloblastic anemia, severe hemolytic processes, hyposplenism, and myelophthisitic
anemia.
Hypochromia
Hypochromia is a decreased amount (MCH) and concentration (MCHC) of hemoglobin
in red blood cells. In the peripheral blood smear, hypochromic cells have an expanded
central zone of pallor. Small hypochromic red blood cells (microcytes) are usually
present, and the mean corpuscular volume (MCV) is decreased. Microcytosis and
hypochromia are characteristic of iron deficiency anemia and other microcytic,
hypochromic anemias [anemia of chronic disease, hereditary hemoglobinopathies with
diminished globin synthesis (thalassemias, hemoglobin E, hemoglobin H), red blood cell
enzyme deficiencies (sideroblastic anemias, lead poisoning, pyridoxine deficiency)].
Serum iron studies, erythrocyte sedimentation rate (ESR), hemoglobin electrophoresis,
bone marrow examination, and serum and urine lead quantitation are other laboratory
studies may be indicated.
Fig. 8. Comparison of normal peripheral blood smear and smear from a patient with
severe microcytic, hypochromic anemia.
Hyperchromia
Hyperchromia is an increase in the red blood cell hemoglobin concentration (MCHC >
36 g/dL). Since it is usually associated with spherocytosis, peripheral smear
examination reveals many spherocytes and microspherocytes. Consideration of
hereditary spherocytosis is recommended, but spherocytes are also seen in patients
with isoimmune and autoimmune hemolytic anemias, Heinz body hemolytic anemia,
hereditary pyropoikilocytosis, and severe burns. If indicated, an osmotic fragility assay,
Coombs’ test, serum bilirubin, LDH, and haptoglobin, and other laboratory assays may
be indicated.
Keratocytes/ schistocytes
Keratocytes ("horn cells, helmet cells") and schistocytes ("fragmented cells") are
damaged red blood cells. Such damage characteristic occurs from fibrin deposits (DIC,
microangiopathic hemolytic anemia, thrombotic thrombocytopenic purpura (TTP),
prosthetic heart valves, severe valvular stenosis, malignant hypertension, or march
hemoglobinuria. However, keratocytes and schistocytes also occur in normal newborns
and in patients with bleeding peptic ulcer, aplastic anemia, pyruvate kinase deficiency,
vasculitis, glomerulonephritis, renal graft rejection, severe burns, iron deficiency,
thalassemia, myelofibrosis with myeloid metaplasia, hypersplenism and postsplenectomy, and other diseases. Clinical correlation is recommended, with the
appropriate diagnostic studies. These cells are pathologic and should never be ignored.
Knizocytes
Knizocytes ("pinch bottle cells") are characteristically seen in patients with hemolytic
anemia, including hereditary spherocytosis. An osmotic fragility assay, Coombs’ test,
serum bilirubin, LDH, and haptoglobin, and other laboratory assays may be indicated.
Macrocytes
Oval macrocytes ("macroovalocytes, megalocytes") are large oval red blood cells (> 8.5
mm) with an elevated MCV (> 100 fL, frequently > 120 fL) and normal MCH. The
presence of these cells suggests impaired bone marrow DNA synthesis, and may
indicate a vitamin B12 or folate deficiency. Serum vitamin B12 or folate levels are
usually indicated and a bone marrow examination may be needed.
Round macrocytes are slightly to moderately larger than normal (macrocytosis, MCV
>95 fL but usually < 120 fL) and are round in shape. This finding suggests impaired
bone marrow impaired DNA synthesis, stress erythropoiesis, or excessive surface
membrane. Possible clinical causes include liver disease (obstructive jaundice,
alcoholism), impaired DNA synthesis from chemotherapy or inherited diseases,
myeloproliferative disorders, myelodysplastic syndromes, or splenectomy. Bone marrow
examination, liver function studies, and other laboratory assays if clinically indicated.
Fig. 9. Comparison of normal peripheral blood smear and smear from a patient with
severe microcytic, hypochromic anemia.
Microcytes
Microcytes are small red blood cells (MCV < 80 fL) with decreased amounts of
hemoglobin formed as a result of iron deficiency and defective hemoglobin synthesis,
imbalance of globin chains, or defective porphyrin synthesis. Possible clinical causes of
microcytosis include iron deficiency anemia, thalassemia, the anemia of chronic
disease, lead poisoning, and sideroblastic anemias.
Nucleated red blood cells
Nucleated red blood cells (NRBCs, normoblasts) are immature red blood cells. In an
adult, the presence of NRBCs indicates markedly accelerated erythropoiesis and/or
severe bone marrow stress. Clinical conditions associated with peripheral
normoblastosis include acute bleeding, severe hemolysis, myelofibrosis, leukemia,
myelophthisis, and asplenia. The presence of NRBCs in the peripheral blood of an adult
always indicates a significant disease process, the etiology of which must be delineated.
NRBCs in the peripheral blood of an infant indicates significant stress but does not have
the ominous significance of features of those in an adult.
Poikilocytosis
Poikilocytosis is variation in red blood cell shape, seen in many disorders.
Polychromasia
Polychromasia ("polychromatophilia") is the occurrence of slightly immature red blood
cells, which are larger than normal (increased MCV) and have a blue-gray coloration.
Polychromasia is due to the presence of ribosomal protein in immature red blood cells,
which pick up the basophilic component of the Wright-Giemsa stain. Small numbers of
these cells (0.5 - 2%) are normally present in the peripheral blood and signify the
presence of erythropoietic activity in the bone marrow. They are increased in states of
increased erythropoietic activity in response to anemia or the iatrogenic administration
of erythropoietin or another marrow stimulatory agent. The MCV may increase slightly in
response to significant polychromasia. Decreased polychromasia is seen with
hypoproliferative marrow states.
Rouleaux formation
Rouleaux formation ("pseudoagglutination") is a linear arrangement of RBCs
("coinstack") caused by an increased blood concentration of fibrinogen, globulin, or
paraproteins. Associated clinical disorders include acute and chronic inflammatory
disorders, Waldenstrom’s macroglobulinemia, and multiple myeloma. Serum and urine
protein analysis should be performed in the absence of an acute or chronic
inflammatory disease to determine if a paraprotein is present.
Spherocytes
Spherocytes are small (< 6.5 mm), dense spheroidal RBCs with normal or decreased
MCV and absent central pallor. Hereditary spherocytosis is likely if large numbers of
spherocytes are present and other forms of abnormal RBCs are absent. Small numbers
of spherocytes, in combination with other abnormal RBCs, are seen in patients with
isoimmune and autoimmune hemolytic anemias, Heinz body hemolytic anemia,
hereditary pyropoikilocytosis, microangiopathic hemolytic anemia, hypersplenism and
post-splenectomy, myelofibrosis with myeloid metaplasia, hemoglobinopathies, malaria,
liver disease, recent transfusions, and severe burns. An osmotic fragility assay,
Coombs’ test, serum bilirubin, LDH, and haptoglobin, and other laboratory assays may
be indicated.
Stomatocytes
Stomatocytes are uniconcave red blood cells with a slit-like area of central pallor. A
predominance of stomatocytes is characteristic of hereditary stomatocytosis (a type of
hemolytic anemia). Small numbers of stomatocytes (usually in association with other
abnormal RBCs) occur in patients with acute alcoholism, cirrhosis, obstructive liver
disease, advanced malignancy, severe infections, Rhnull disease, treated acute
leukemia, and other diseases.
Morphologic Evaluation of White Blood Cells
Clinical Importance of WBC Morphology
Light microscopy is of greatest value in confirming the automated white blood cell count
and performing a manual differential count. However, specific morphologic
abnormalities of leukocytes occur, and can provide evidence of disease processes.
Classification of WBC Morphologic Abnormalities
Alder-Reilly granules
Alder-Reilly granules are large, coarse, dark purple, azurophilic granules that occur in
the cytoplasm of most granulocytes. These are characteristically found in the AlderReilly anomaly and in patients with mucopolysaccharidoses.
Auer rods
Auer rods are needle- or rod-shaped, eosinophilic structures which occur in the
cytoplasm of immature leukocytes (blasts) and more mature cells in some patients with
acute myelogenous leukemia (AML). These structures are formed from coalescing
primary (azurophilic) granules. Although Auer rods occur singly in AML FAB subtypes
M1, M2, and M4, cells with numerous Auer rods are common in the hypergranular form
of AML FAB M3 to form "faggot" cells.
Chédiak-Higashi granules
Chédiak-Higashi granules are very large red or blue granules that appear in the
cytoplasm of granulocytes, lymphocytes, or monocytes in patients with the ChédiakSteinbrinck-Higashi syndrome.
Döhle bodies
Döhle bodies are variably sized (0.1 to 2.0 um) and shaped, blue or grayish-blue
cytoplasmic inclusions usually found near the periphery of the cell. Dohle bodies are
lamellar aggregates of rough endoplasmic reticulum, which appear in the neutrophils,
bands, and metamyelocytes of patients with infection, burns, uncomplicated pregnancy,
toxic states, or during treatment with hematologic growth factors such as G-CSF.
May-Hegglin anomaly
Neutrophils contain small basophilic cytoplasmic granules which represent aggregated
ribosomes. Leukopenia and large platelets are also found. An autosomal dominant trait,
the May-Hegglin anomaly is associated with a mild bleeding tendency, but not by an
increased susceptibility to infection.
Neutrophilic hypergranulation (toxic granulation)
Small dark blue to purple granules resembling primary granules appear in the cytoplasm
of methmyelocytes, bands, and segmented neutrophils during inflammatory states,
burns, and trauma, and upon exposure to hematopoietic growth factors such as
granulocyte-colony stimulating factor (G-CSF). Toxic granulation is usually
accompanied by a "shift to the left" in the neutrophilic population, and by the presence
of vacuolations in the cytoplasm (toxic vacuolations) and Dohle bodies.
Neutrophilic hypogranulation
XXX
Neutrophilic hypersegmentation
Increased lobulation of granulocyte nuclei (neutrophilic hypersegmentation) is a
characteristic finding in megaloblastic anemia, but can also be seen as an inherited
autosomal dominant trait (hereditary hypersegmentation of neutrophils).
Neutrophilic hyposegmentation
Single or bi-lobed neutrophils (Pelger-Huët cells) can be inherited (Pelger-Huët
anomaly), or acquired (pseudo-Pelger-Huët cells) in patients with malignant
myeloproliferative disorders (including preleukemia and myelodysplastic syndromes)
and infections or tumors which have metastasized to the bone marrow.
Large, purple or dark-blue azurophilic granules in the cytoplasm of neutrophils, bands,
and metamyelocytes are characteristically seen in patients with severe infection,
septicemia, toxic states, and chemical poisoning. Cytoplasmic vacuolation is seen as
well.
Morphologic Evaluation of Platelets
Clinical Importance of WBC Morphology
Light microscopy is of greatest value in confirming the automated platelet count and
performing a manual differential count in patients with very high or very low platelet
counts where automated counting is inaccurate. However, care morphologic evaluation
may reveal abnormalities which can support other observations.
Classification of WBC Morphologic Abnormalities
Platelet hypogranularity
Many small, reddish-purple granules are normally present in the cytoplasm of the
platelet. These granules, which vary in size and shape, represent dense bodies, alphabodies, and lysosomes. Hypogranular platelets, as the name implies, have little, if any,
of the purple-red granules found in normal platelets. These granules may be decreased
in number or absent in patients with myeloproliferative diseases and myelodysplastic
syndromes. In these disorders, platelet hypogranulation is usually accompanied by
abnormalities in platelet size and shape, anemia, leukocytosis or leukopenia, and
abnormalities in leukocyte morphology.
Large and giant platelets
Platelets are normally 1.5 to 3 microns in diameter. However, platelet size can increase
in patients with increased platelet turnover from bleeding or stress, and in the
myeloproliferative and myelodysplastic disorders. Large platelets are 3 to 7 microns
(roughly the diameter of a normal RBC), while giant platelets are larger than red cells.
Morphology may appear normal or abnormal.
Platelet satellitism
In this unusual phenomenon, normal platelets adhere to the surface of neutrophils, or,
rarely monocytes, to form "platelet rosettes." Platelet satellitism is associated with blood
specimens anticoagulated with EDTA, and disappears when heparin-anticoagulated
blood is collected from the same patient. Although an innocuous finding, significant
platelet satellitism may cause spurious thrombocytopenia, since the cell-bound platelets
are not counted with the platelet fraction of the blood specimen.
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