THE 1-3 MINUTE BLOOD FILM REVIEW:
WHY IS IT NEEDED AND HOW EASY IS IT TO PERFORM?
D. B. DeNicola, DVM, PhD, DACVP
Chief Veterinary Educator
IDEXX Laboratories, Inc
INTRODUCTION: Extremely few veterinary practices that have in-house
hematology instrumentation prepare blood films for routine microscopic
evaluation. Much of this is based upon the lack of confidence in either preparing
a well made blood film or the lack of confidence in being able to accurately
identify significant abnormalities. The advancement in technology for the in-
house hematology laboratory has not encouraged blood film evaluations. In fact,
the veterinarian and technician feel more and more confident as more and more
data are collected by these instruments. There are instruments that provide 16,
18 and more parameter evaluations as well as a three-part or full five-part
leukocyte differential count. It should be noted that none of these instruments,
including the large $50-150,000 instruments used in commercial reference
laboratories were designed to eliminate the peripheral blood film evaluation.
Clearly the amount of time spent on the microscope is dramatically reduced in
the large volume laboratories, but blood film evaluation is still critical to accurate
data generation. There are several basic steps to the process. The first and
most important aspect is the preparation of a quality blood film to evaluate. If this
is not possible, the process of analysis is of questionable value. Basic
instructions for slide preparation are listed below:
Steps to preparation of a blood film
Place a small drop of blood on a clean glass slide approximately 2 cm from
one end of the slide.
Place a clean glass “spreader” slide in front of the drop of blood at an angle of
approximately 30o to the blood film slide and back the “spreader” slide into the
drop of blood.
Let the blood spread along the contact line between the two slides and the
blood; this should take place quickly.
With a steady fluid movement of the hand, move the spreader slide down the
blood film slide without lifting the “spreader” slide off the blood film slide.
Blood from the drop will follow the “spreader” slide placing a thin film of blood
on the blood film slide. If enough blood was placed on the slide originally, the
thin film of blood should be 3-4 cm in length.
Let the blood film air dry. Only apply forced (dry) air drying if in a humid
environment.
TIP – For specimens with low hematocrits (anemia), increase the angle
between the slides to make a thicker blood film.
TIP – For specimens with high hematocrits (dehydration, polycythemia, etc.)
decrease the angle between the slides to make a thinner blood film.
Once a well made blood film is prepared (and stained), the examination process
begins and this process should be organized and followed closely with each
blood film examination. The total amount of time spent examining the blood film
should be relatively short, especially if one does not have to perform a manual
leukocyte differential. Manual leukocyte differentials are relatively imprecise and
if a hematology analyzer provides a complete five-part differential, this differential
will typically be of greater value and more representative of the true leukocyte
distribution in the blood. The basic process is outlined below:
Step 1: 20-30 second scan at low magnification (10x objective) – At this
objective one is looking for obvious abnormalities in each of the different zones of
a blood film
1. Feathered edge – 10 seconds maximum
a. Clumps of platelets
b. Clumps of leukocytes
c. Microfilaria
d. Large atypical cells
2. Body of the blood film – 10 seconds maximum
a. Agglutination – three dimensional clumping of cells
b. Rouleaux – organized “stack of coins”
c. Clumping of platelets or leukocytes
3. Monolayer – 10 seconds maximum
a. Validate RBC count / HCT – erythrocyte density
b. Validate WBC count – leukocyte density
c. Assure even distribution of leukocytes
Step 2: 30-45 second scan at an intermediate magnification – A 20x objective is
preferred but if only a 10x and 40x objective combination is available, the 40x
objective can be used; however, the sample needs to be coverslipped. This can
be easily accomplished with putting a drop of immersion oil on the monolayer
region of the blood film and placing a coverslip on top of the oil to allow proper
refractive index for the 40x objective.
1. Validate leukocyte distribution without performing an actual manual
differential remembering the relative imprecision of the 100 leukocyte
manual differential compared to the thousands of leukocytes evaluated by
selected hematology analyzers.
a. If the analyzer reported a 75% neutrophil distribution, approximately
3 out of every 4 leukocytes should be a neutrophil.
b. If the analyzer reports a 10% lymphocyte distribution,
approximately 1 out of every 10 leukocytes should be a
lymphocyte.
c. If the analyzer reports an eosinophilia, eosinophils should be easily
identified on the blood film.
d. If the analyzer reports a lymphopenia, lymphocytes should be very
difficult to identify on the blood film.
2. Validate reticulocyte count if provided by your hematology analyzer
a. If the analyzer reports a significant reticulocytosis, polychromasia
should be easily identified on the blood film at this magnification.
b. If the analyzer reports a normal or extremely low reticulocyte count,
polychromatophils should be only rarely seen
3. Screen for significant WBC morphologic abnormalities – see below
4. Screen for significant RBC morphologic abnormalities – see below
5. Screen for significant Platelet morphologic abnormalities – see below
Step 3: 20-30 second scan at high magnification (100x oil immersion)
1. Validate platelet count if no platelet clumping is present
a. Normally should see 8-10 platelets per 100x objective field of view
b. Average number of platelets per 100x objective field of view x
20,000 should give a good estimation of the number of platelets per
microliter of blood.
2. Validate RBC, WBC, and Platelet morphologic abnormalities noted at the
intermediate magnification.
In practices where blood film microscopic evaluation does take place, both
veterinarians and veterinary technicians are commonly involved. Therefore, the
information provided below includes both clues into the recognition of certain
morphologic features as well as the significance to the finding of these
abnormalities in the peripheral blood. Even those individuals who routinely
examine blood films should regularly re-visit some of the features included below.
The items chosen for this discussion are limited in scope to some of the more
significant morphologic findings in the peripheral blood film. Clearly this is not an
all encompassing discussion. The top ten lists for the erythron, leukon and
thrombon are presented in hopes of making a limited list of significant items to
look for so that the microscopic evaluation period can be limited in time as much
as possible. Theoretically, the evaluation for significant abnormalities should
take less than 1-3 minutes; the more “normal” the patient, the more rapid the
blood film evaluation. The abnormalities listed should be something that is
identified quickly when looking at the blood film. If a greater amount of time than
3 minutes is spent on a microscope slide the chances of seeing things that are
only rarely present and insignificant or things that are really not present at all
increases dramatically.
A top ten list for each of the major cellular compartments of the peripheral blood
are listed below with associated brief discussion of the significance of the finding
of these abnormalities.
TOP TEN LIST FOR THE ERYTHRON
Polychromasia Recognition of polychromatophils is used as a validation of
reticulocyte counts or in cases where reticulocyte counts
are not determined, is used for the recognition of bone
marrow responsiveness (“regeneration”) in the cases where
there is a demand for erythrocytes in the peripheral blood.
Polychromatophils are immature red blood cells that are
typically slightly larger than adult erythrocytes and have a
slight blue stain due to the presence of RNA in the
immature red blood cells.
Spherocytes Spherocytes appear as smaller than normal red blood cells
with increased density and no central pallor compared to
normal red blood cells. They represent extravascular
hemolysis due to partial phagocytosis by macrophages
recognizing surface attached immunoglobulins. Their
presence in significant numbers indicates the presence of
an immune-mediated extravascular hemolytic process.
Agglutination Three dimensional clumping of erythrocytes when
confirmed with a saline agglutination test are supportive of
an immune-mediated process directed against the red
blood cells. They support the presence of surface-related
antibodies that result in tightly bound cross-linking of
erythrocytes. It must be differentiated from loosely attached
organized linear arrays of erythrocytes (Rouleaux).
Acanthocytes Acanthocytes are a specific type of poikilocytosis
characterized by the presence of 2-10, blunt, finger-like
projections from the surface of the erythrocytes. They
support “lipid-loading” of these cells and investigation into
causes of changes in cholesterol:phospholipid ratios in the
plasma (liver disease, underlying metabolic disturbances)
and possible splenic disease is warranted when identified.
Target Cells Target cells or leptocytes are red blood cells with excess
cell membrane to cytoplasmic content ratio commonly
associated with “lipid-loading” by similar mechanisms as
seen with acanthocytosis.
Hypochromasia Hypochromatophils are red blood cells with decreased
cytoplasmic content of hemoglobin. They present as pale
thin cells typically smaller appearing than normal red blood
cells. In veterinary medicine the primary cause for this
finding is the presence of a chronic blood loss situation with
developing or developed iron deficiency. Investigation into
chronic blood loss is warranted when identified.
Schistocytes Schistocytes are irregular fragments of red blood cells due
to mechanical injury to the cells. Oftentimes, this is
associated with a “microangiopathy” with abnormalities
such as fibrin accumulation in small blood vessels /
capillaries. However, conditions where increased turbulent
flow of blood through large vessels (caval syndrome with
Heartworm disease) or within the heart (endocarditis) can
result in similar findings. The presence of schistocytes can
prove to be a helpful parameter to include in the clinical
investigation of Disseminated Intravascular Coagulopathy.
Metarubricytes Metarubricytes are nucleated red blood cells. When found
in low numbers relative to high numbers of
polychromatophils, they are often accepted as being an
“appropriate” component of a strongly regenerative
response by the bone marrow. However, since there is
typically a physical and physiological restriction for
nucleated red blood cells from being released from the
normal marrow, when found without associated
polychromasia, investigation into bone marrow stromal
damage (infiltrative bone marrow disease, endotoxemia /
septicemia, hypoxia, heavy metal toxicity [acute lead
toxicity] is warranted.
Heinz Bodies Heinz bodies represent small, projections on the surface of
the red blood cells. These are small collections of
denatured / oxidized hemoglobin due to oxidant injury.
Conditions such as acute onion toxicity in the dog and
Acetaminophen toxicity in the cat is warranted but other
oxidant stresses including underlying metabolic
disturbances are possible causes also.
Miscellaneous Whenever an identified structure within or on a red blood
inclusions cell, further characterization is required. Often, this will
involve sending a well made blood film to a reference
laboratory for evaluation and confirmation and hopeful
identification is warranted.
TOP TEN LIST FOR THE LEUKON
Neutrophil Proper identification of normal neutrophils is essential for
identification proper validation of automated leukocyte differentials and
interpretation of leukograms. Neutrophils are 10-12
micrometers in size with dense lobed nuclei and moderate
amounts of pale pink to pale blue cytoplasm.
Lymphocyte Proper identification of normal lymphocytes is essential for
identification proper validation of automated leukocyte differentials and
interpretation of leukograms. Lymphocytes are smaller than
neutrophils with high nuclear/cytoplasmic ratios and round
to slightly indented nuclei with smudged nuclear chromatin
patterns. Cytoplasm is often scant in amount and pale blue.
Monocyte Proper identification of normal monocytes is essential for
identification proper validation of automated leukocyte differentials and
interpretation of leukograms. Monocytes are the largest
leukocyte in circulation with irregularly-shaped nuclei with
finely stippled to open chromatin patterns. Cytoplasm is
moderate in amount and moderately blue often with few
clear small discrete cytoplasmic vacuoles.
Eosinophil Proper identification of normal eosinophils is essential for
identification proper validation of automated leukocyte differentials and
interpretation of leukograms. Eosinophils are slightly larger
than neutrophils with less dense and less lobed nuclei and
moderate amounts of pale blue cytoplasm filled with
variable numbers of species-distinctive orange-red
granules.
Basophil Proper identification of normal basophils is essential for
identification proper validation of automated leukocyte differentials and
leukogram interpretations. Basophils are slightly larger than
neutrophils with less dense and less lobed nuclei and
moderate amounts of pale blue cytoplasm filled variably
with species distinctive granules.
Band The recognition of band neutrophils is something that is not
Neutrophils currently available with in-house hematology analyzers.
Their presence is strong support for the presence of
inflammatory disease and since many inflammatory
leukograms have no increase in neutrophils or the increase
in neutrophils with inflammatory disease overlaps
glucocorticoid (“stress”) and epinephrine (“excitement”)
induced neutrophil increases, they may be the only way to
properly identify the presence of inflammatory disease.
Hyposegmented Neutrophil forms between mature neutrophils and true band
Neutrophils neutrophils are “hyposegmented” neutrophils and their
presence indicates inflammatory disease as the band
neutrophils.
Toxic The presence of toxic neutrophils, like the presence of band
Neutrophils or hyposegmented neutrophils indicates the presence of
inflammatory disease. Common toxic changes,
representing abnormalities in the maturation process of
neutrophil development in the bone marrow, include the
presence of blue staining cytoplasm, the possible presence
of Dohle bodies and the possibility of foamy cytoplasm or
the finding of giant neutrophil forms.
Reactive Reactive lymphocytes indicate the presence of systemic
Lymphocytes antigenic stimulation. Specific cause is never identified.
When present, differentiation from a neoplastic lymphoid
population typically lies in the finding of a heterogeneous
population of lymphocytes including normal appearing small
lymphocytes, mildly reactive lymphocytes, moderately
reactive lymphocytes and occasionally seen markedly
reactive lymphocytes. Common morphologic features of
reactivity include the finding of more deeply blue staining
cytoplasm as well as the finding of increased amounts of
cytoplasm and overall cell size.
Abnormal When an abnormal leukocyte form is identified, further
Leukocytes investigation is required. In most cases, submission of a
similar specimen to a pathology service should be strongly
considered. In many cases, if a large immature appearing
leukocyte form is identified, special staining may be
required to definitively identify the cell lineage of origin for
this cell population.
TOP TWO LIST FOR THE THROMBON
Platelet Because of the difficulty in accurately quantitating platelets
Numbers in samples with platelet clumping and the common finding of
platelet clumping, validation of platelet decreases is
essential with a blood film review. Normally a minimum or 8-
10 platelets per 100x oil immersion field of view are seen
with a low normal number of platelets. Thorough evaluation
of the blood film, with particular attention to the feathered
edge of the blood film, is essential to investigate the
possibility of platelet clumps. When identified, platelet
counts must be questioned as a likely underestimation of the
actual platelet count in the specimen of question.
Enlarged In most species, with the cat being the primary exception,
Platelets the presence of enlarged platelet forms in circulation
supports the finding of increased rate of production of
platelets at the bone marrow level in response to a
peripheral demand for platelets. If found associated with or
without a thrombocytopenia, they support the finding of
increased rate of consumption (coagulation) or destruction
(possible immune-mediated destruction) of platelets in the
peripheral blood.
