Download Chemistry challenge cases Leslie Sharkey DVM, PhD, DACVP

Chemistry challenge cases
Leslie Sharkey DVM, PhD, DACVP (Clinical Pathology)
Introduction
The clinical chemistry profile is an extremely valuable diagnostic tool from which extensive
information can be extracted when assessed using a sophisticated approach. This can be
especially critical when other diagnostic tools such as advanced imaging are not immediately
available. The clinical chemistry profile will provide the most information when integrated with
the other elements of the “minimum laboratory data base” (CBC, profile, UA) along with the
clinical history and physical examination findings.
The following outline is designed as a reference/review for the chemistry challenge cases. The
outline is focused on use of liver and renal indicators in the interest of time and space.
Approach to evaluation of hepatic disease and liver indicators
Broadly divided into 4 mechanistic categories based on lab data, history, and physical
Hepatocellular damage (increased ALT, AST)
This may be mild, moderate or severe. Typically the magnitude of the change is not
necessarily prognostic, and evaluations of trends over time should be integrated with
clinical condition to determine patient status.
Mild to moderate elevations can be nonspecific and associated with poor perfusion,
hypoxia, “spillover” pathology from the intestines and pancreas, occur secondary to posthepatic cholestasis, or occasionally reflect ongoing damage from chronic inflammatory or
neoplastic processes with elevations blunted by decreasing numbers of residual
hepatocytes.
Marked elevations should prompt consideration of toxicity, trauma, severe ischemia,
acute infection, torsion, etc.
Cholestasis (increased bilirubin)*remember that bilirubin is first eliminated in the urine and
serum bilirubin only becomes elevated once that mechanism is overwhelmed, so ALWAYS
check the urine! Keep in mind that different types of cholestasis are not mutually exclusive.
Pre-hepatic
This is due to increased production of bilirubin associated with hemolysis. ALWAYS
check a CBC to look for anemia, which will be regenerative with sufficient time. Also be
sure a thorough microscopic evaluation for red cell morphologic indicators associated
with hemolytic disease is performed (agglutination, spherocytes, Heinz bodies,
keratocytes, fragments, ghosts, etc). Pre-hepatic cholestasis may be mild, moderate or
severe depending on the degree of hemolysis. Pre-hepatic cholestasis does not directly
cause increases in the “cholestasis enzymes” (ALP and GGT), however hepatocellular
damage secondary to hypoxia can be observed.
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Hepatic
This is due to failure of bilirubin to be taken up by hepatocytes, and again, does not
directly cause increases in ALP and GGT. Causes include sepsis, decreased liver
function, and anorexia in cats to a minor degree. Elevations in bilirubin due to hepatic
causes are typically mild (not exceeding 4-5 mg/dl). Evaluation of the CBC for evidence
of severe inflammation can be helpful.
Post-hepatic
This occurs when the canalicular flow of bile is obstructed and is usually associated with
increased ALP and GGT, with serum bilirubin elevations observed once renal elimination
is exceeded. Subtypes are 1). intrahepatic bile duct obstruction due to accumulation of
inflammatory or neoplastic cells, fibrosis, lipid, or cellular swelling secondary to
hepatocellular damage, most often resulting in mild to moderate elevations and 2)
extrahepatic bile duct obstruction due to bile sludging, stones, tumors, torsions, and
pancreatic disease, which may be associated with mild to severe increases, and some
causes may require surgical intervention.
Decreased liver function
>2/3 liver function must be lost to detect common abnormalities on routine chemistry.
Associated abnormalities are due to 1) decreased synthetic function: low urea, albumin,
glucose, cholesterol, prolonged PT/PTT, 2) decreased clearance function: increased
bilirubin, serum bile acids (cannot use if there is concurrent post-hepatic cholestasis),
blood ammonia and 3) other: decreased urine specific gravity, note that liver enzyme
activities do not accurately reflect liver function
Liver disease “mimics” must be considered to avoid over diagnosis of liver disease and to
prevent unnecessary expensive or invasive procedures.
Hormone and drug-induced enzyme induction
Poorly regulated diabetes mellitus, hyperthyroidism, hyperadrenocorticism, anticonvulsant treatment. ALP is often the most commonly increased, but other
enzymes may become elevated as well. these should not elevate serum or urine
bilirubin
Appearance of decreased liver function
The two most common conditions include 1)Protein losing enteropathy:
characterized by low albumin and cholesterol, however globulins also tend to be
low and glucose and bilirubin are normal and 2) atypical hypoadrenocorticism:
characterized by low glucose and cholesterol, however albumin, urea, and
bilirubin should be normal
Process for categorization
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1. Determine the presence or absence of each component
2. Try to assess predominant process if multiple are present
3. Additional diagnostics and differential diagnoses based on 1 and 2
Determining more specific differential diagnoses
1. Hepatocellular damage predominant
a. Trauma: history, physical, concurrently elevated CK and/or amylase
b. Hepatotoxin: history
c. Anemia/hypoperfusion: concurrent anemia (usually acute or severe), tachycardia,
tachypnea or cyanosis or other signs of cardiopulmonary compromise
2. Post-hepatic cholestasis predominant
a. Gall bladder or pancreatic disease
3. Decreased liver function predominant
a. Portovascular abnormalities
b. End stage inflammatory liver disease
c. Massive infiltration by neoplastic cells (often lymphoma)
4. Mixed/balanced
a. Most often a mix of hepatocellular damage and post-hepatic cholestasis
b. Often inflammatory or neoplastic disease present this way
i. Hepatocellular swelling leads to canalicular pressure and secondary posthepatic cholestasis
ii. Post-hepatic cholestasis leads to hepatocellualar damage by components of
bile
c. Imaging and biopsy/cytology often required for diagnosis
**Use of cytology for diagnosis: highly sensitive and specific for vacuolar change; neither
sensitive nor specific for inflammatory liver disease, specific but not sensitive for neoplasia.
Infiltrative round cell neoplasia IS SOMETIMES DIAGNOSED ON
ULTRASONOGRAPHICALLY NORMAL livers, so consider aspiration even when ultrasound
studies are unremarkable.
Laboratory characterization of renal disease
Types of classification of renal disease
Classification by structural lesion-often mixed later in disease
Tubular dysfunction
This is often predominant in chronic kidney disease and with acute renal disease
due to toxicity or hypoperfusion. It is characterized by: decreased urinary
concentrating ability, azotemia, electrolyte abnormalities (Na and Cl variable,
with a tendency for hypokalemia when polyuric and hyperkalemia when
oliguric/anuric), uremic acidosis +/- mild proteinuria, azotemia, and the potential
for glucosuria with normoglycemia when tubular damage is acute
Glomerular barrier damage
This is often seen with immune-mediated/antigen-antibody complex disease.
Initially characterized by proteinuria with hypoalbuminemia, concentrating ability
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can be preserved and the patient may not be azotemic until disease progresses, at
which point a pattern of tubular and glomerular barrier damage is observed.
Loss of entire nephrons
Decreased GFR with increased solute load on remaining functional units, leading
to less than optimal concentrating ability +/- mild proteinuria and azotemia,
electrolyte/acid-base abnormalities ensue late in disease or if complicated by other
factors. Damaged nephrons can repair and remaining nephrons can hypertrophy to
a new higher plateau of function, however ongoing damage due to “overwork”
leads to progressive renal disease
Other causes of impaired urinary concentrating ability that may mimic renal disease
Hypercalcemia (ionized only) inhibits ADH action
Hypercalcemia of malignancy (lymphoma, anal sac adenocarcinoma, other) and
primary hyperparathyroidism will lower urine specific gravity (may be
hyposthenuric), sometimes cause azotemia, and phosphorus may be low due to
law of mass action.
Pyometra
Bacterial toxins impair ADH action, leading to impaired urinary concentrating
ability and the potential for azotemia. History, physical exam, intact female with
recent heat, marked inflammatory leukogram in many cases
Medullary washout
GI or other losses of electrolytes impair medullary concentration gradient,
reducing the capacity for optimal urine concentration. Look for low Na, Cl, K
Hyperadrenocorticism
Or treatment with corticosteroids. Decreased USG +/- mild proteinuria, usually
not azotemic unless water intake is restricted, look for clinical signs, liver enzyme
induction, corticosteroid leukogram.
Hypoadrenocorticism
Clinical signs can be vague and waxing and waning. Electrolyte wastage due to
lack of aldosterone decreases urinary concentrating ability but USG may be
slightly higher than isosthenuria. Look for history, lymphocytosis, eosinophilia,
possible hypoglycemia and hypocholesterolemia. Hypercalcemia can be observed,
and rarely may be the only laboratory abnormality, causing confusion with
hypercalcemia of malignancy.
Liver dysfunction
Low urea diminishes medullary concentration gradient, altered central set points
for ADH release, and increased thirst lower urine specific gravity. Look for
Hypoglycemia, hypoalbuminemia, hyperbilirubinemia, hypocholesterolemia,
prolonged PT/PTT.
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CBC Challenge cases
Leslie Sharkey DVM, PhD, DACVP (Clinical Pathology)
Introduction
The complete blood count is a critical part of the assessment of sick patients in
your practice. It is used to evaluate plasma protein, red blood cells, white blood cells,
and platelets. While the measurement of numbers of each of these components is
important, description of their morphology on the blood smear also can provide you with
important information regarding the patient that is not apparent based on only the
quantitative data. The results of the complete blood count should be interpreted in the
context of the history and physical examination and any medications the patient may be
taking. Be careful to always note the breed of your patient; several types of purebred
dogs have hematologic values that fall outside most reference ranges as a normal
finding for that breed. Only sometimes are the results of the CBC diagnostic for a
particular disease. More often, the results reflect general categories of pathology, such
as anemia, inflammation, etc. Frequently other diagnostic work is required to obtain a
specific diagnosis for treatment.
The following outline is designed as a reference/review for CBC challenge cases, which
will be reviewed during the session. The majority of the challenge will be to integrate
morphology with quantitative data to arrive at appropriate diagnostic conclusions. See
Pearls section below for a few key take home points for pattern analysis.
Collection & Handling
Your ability to prepare and accurately interpret a blood film will depend on your
collection technique.
1.
2.
3.
4.
Minimally traumatic venipuncture procedures will prevent in vitro
hemolysis and platelet clumping that can interfere with analysis of CBC
results.
When transferring blood from the syringe to the tube containing
anticoagulant, remove the stopper of the tube and take the needle
off of the hub of the syringe to minimize trauma to erythrocytes and
platelets.
Label all samples carefully. In an emergency situation, this small but
critical step can be missed.
Always try to fill EDTA tubes at least half full with blood. Excess
EDTA will cause dilution and cell shrinkage and can decrease red and
white cell counts, PCV and MCV but may increase mean corpuscular
hemoglobin content because of erythrocyte shrinkage.
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5.
6.
7.
8.
Examine the blood in the tube for evidence of hemolysis, lipemia,
methemoglobinemia, icterus, and agglutination. If there is a clot in the
tube, the sample should be discarded and a new one collected.
Once the sample is collected and you are ready to make your smear, be
sure to GENTLY rotate the tube for even mixing of blood constituents.
This will help ensure that your white blood cell estimates are accurate.
ALWAYS ALWAYS ALWAYS prepare a blood film just after
collection of the sample, especially if the tube of blood will be sent to
a reference laboratory for analysis. In vitro artifacts can develop, and a
freshly prepared blood film will still be interpretable even if the sample in
the tube is unusable. (Same is true for fluid analysis!)
Be sure to adequately maintain your stains. Stain may precipitate out
of solution or become contaminated; often filtration or replacement of
stains at regular intervals is required.
Importance of blood film review!
The availability of in-house automated analyzers has, in some cases, promoted a false
sense of security regarding CBC evaluation. Even the best automated analyzers fail to
provide data about diagnostically significant morphologic abnormalities of blood cells or
the presence of parasites which may be present despite quantitative results that fall
within reference intervals. Just as importantly, examination of the blood film allows the
clinician to double check the results of automated analyzers, which may have their
results “flagged” as potentially erroneous in up to 30% of clinical patients (Mitzner). My
clinical impression is that this rate may be higher for sick animals. Look at normal
films often! This helps get you familiar with normal variation and artifacts. Review of
sufficient numbers of normal blood films at regular intervals is a pre-requisite for skilled
examination of abnormal films.
Mitzner BT. Why automated differentials fall short. J Am Anim Hosp Assoc.
2001;37:117-118.
Approach to Examination of the Blood Film
A consistent, methodical approach will maximize your ability to identify all potential
abnormalities. This is similar to reading a radiograph in that the order of evaluation is
not as important as having a routine that ensures that nothing is forgotten. I
recommend that each clinician or technician develop a method that works for him or her
, but a suggested pattern is the following:
1.
Scan the entire smear at 10X, with emphasis on the feathered edge.
At this power, try to identify large parasites (such as microfilaria),
platelet clumps, and large or unusual cells such as blasts or mast cells.
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2.
Still using the 10X objective, find the counting area. The counting
area is between the feathered edge and the body of the smear where
erythrocytes appear in a monolayer, neither touching nor having
excessive space between them. This area may be small in severely
anemic or polycythemic patients.
3.
In the counting area using the 10X objective, estimate the leukocyte
count. There are various methods for leukocytes estimation. One
quantitative method is to count the leukocytes in at least 3 10X fields
and determine the average. Then multiply the average you obtained by
2.5 to give you the approximate number of leukocytes X 10 3/ul. I am
generally less quantitative, and estimate the leukocyte numbers as
very low, low to normal, normal, normal to high, and high. Some
authors report that a 10X field from a “normal” dog or cat should
contain 18-51 white blood cells (Weiss). BEWARE!: The accuracy of
white blood cell estimates will depend on proper mixing of the blood
sample prior to smear preparation, even distribution of leukocytes
throughout the smear (verified by scanning the smear at 10X), and
how thick or thin the smear is, which may be influenced by the total
protein and PCV as well as interpersonal variation in smear technique.
4.
In the counting area (monolayer) using a 50X or 100X oil immersion
lens, perform a differential cell count on 100-200 leukocytes. Note
any morphologic abnormalities by examining cells at 100X.
a. The two most common reasons for difficulty performing a differential
cell count include attempting to identify broken or distorted cells and
performing cell identification outside of the counting area, where cell
morphology is optimal.
b. If you find a cell you cannot identify, continue on. If there are very small
numbers of those cells (you only find one or two on the smear), they
may not be significant. If there are more, send the smear to a
reference lab for examination.
c. When first beginning to evaluate smears, it may be helpful to describe
the unidentified cell to yourself (size in relation to a neutrophil, nuclear
size and shape, quantity and characteristics of the cytoplasm).
Although you may not have recognized the cell immediately by sight,
the words you use to describe it will allow you to compare it to
reference materials that include descriptions of cells.
d. Be sure that cell morphology is characteristic for the patient’s species
(labeling errors can be detected by seeing feline eosinophils in a
supposedly canine blood film). Cat neutrophils occasionally contain
Dohle bodies in the absence of significant inflammation, in the dog
Dohle bodies are more often interpreted as evidence of toxic change.
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5.
6.
a.
b.
c.
d.
7.
8.
In the counting area, using a 100X oil immersion lens, examine
erythrocytes for morphologic abnormalities. Keep in mind normal
species characteristics in terms of size and shape. The degree of
expected anisocytosis and polychromasia is also species specific. Like
with leukocytes, very small numbers of a particular type of abnormality
may not be significant (fragments, Heinz bodies, spherocytes, HowellJolly bodies), but occurrence at higher frequencies may be
diagnostically significant. For example, small numbers of Howell-Jolly
bodies or Heinz bodies are often present as non-specific findings in the
cat.
Do a platelet estimate in the counting area using the 100X oil
immersion lens.
Be sure to consider that any platelet clumping in the tube or at the
feathered edge will artifactually decrease the numbers of platelets in
the counting area.
In dogs and cats, each 100X oil immersion field should contain 10-25
platelets. Qualitative assessments based on these numbers can allow
estimates of very low, low, normal, or increased numbers of platelets
unless platelet clumps are present.
If platelet clumps are present and adequate numbers of platelet are
present, platelet numbers should be interpreted as adequate. If platelet
clumps are present and numbers of platelets in the counting area
appear to be decreased, estimation should not be attempted and a
new sample should be collected in an attempt to avoid platelet
clumping.
In some fractious patients or in patients that may have increased
platelet activation as part of their disease process, it can be very
difficult to completely avoid platelet clumping, especially in cats and
horses. (Just so you know we appreciate that some things the lab
recommends are easier said than done!)
Write a morphological description that includes erythrocytes,
leukocytes, and platelets.
Generate a list of differential diagnoses and any additional tests that
may be required to confirm or rule them out.
Weiss D and Tvedten H. The complete blood count and bone marrow examination:
General comments and selected techniques. In: Small Animal Clinical Diagnosis by
Laboratory Methods, 4th ed. MD Willard and H Tvedten, eds.Saunders, St. Louis, MO.
2004. pp 14-37.
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Body of smear
Counting Area
Feathered Edge
Leukocytes and leukocyte morphology
Neutrophils
1. Normal morphology
a. Larger than mature lymphocyte, smaller than monocyte
b. Highly lobated nucleus with condensed chromatin, Barr body may be
seen in females as a nuclear appendage
c. Granules are often non-staining, however some individuals may have
granules that stain a faint pink
2. Aging changes
a. May be in vivo (extended time in circulation associated with
glucocorticoids) or in vitro
b. Nuclear hypersegmentation or pyknosis
c. Excessive in vitro aging change may complicate differential cell count
and bias result
d. All leukocytes are susceptible to aging changes
3. Toxic changes associated with systemic inflammation or bone marrow
disorders
a. Are DEVELOPMENTAL changes that occur in the bone marrow (vs
degenerative neutrophils that acquire morphologic abnormalities in the
peripheral tissues).
b. Most commonly seen toxic changes are CYTOPLASMIC
i. Cytoplasmic basophilia-retained ribosomes
ii. Cytoplasmic vacuolization-lysosomal rupture/leakage
iii. Dohle bodies-aggregates of rough endoplasmic reticulum (small
numbers can be normal in cats)
iv. Giant neutrophils or bands-may be the same size a monocytes
and can be difficult to differentiate from monocytes. Remember,
monocytes will have vacuoles and bluer cytoplasm, but DO
NOT contain Dohle bodies.
v. Toxic granulation-retained mucopolysaccharides in primary
granules, this is a less commonly seen change than i-iv.
c. Nuclear toxic changes are less common
i. Hypersegmentation
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ii. Hyposegmentation-can mimic Pelger Huet anomaly (generally
benign nuclear segmentation defect occasionally seen in dogs
and cats with mature cytoplasm and poorly segmented nuclei).
iii. Nuclear ring forms-nuclei look like donuts.
4. Left shift-presence of immature neutrophils, most often band neutrophils
a. Be conservative in calling bands
b. There is considerable inter-observer variation here. If a patient has a
left shift, it is best for a single technician or veterinarian to review all
blood films to be sure that changes in the magnitude of the left shift
reflect changes in patient status rather than subjectivity in classification
of cells.
c. Definition of a band: no nuclear segmentation, parallel sides, the
thinnest part of the nucleus is no less than 50% of the width of the
thickest segment.
d. Left shifts are often, but not always, associated with some degree of
toxic change
e. Metamyelocytes and more immature forms of neutrophils are
occasionally seen. When present, the blood film should be reviewed by
a pathologist.
5. Infectious agents are rarely present in circulating neutrophils
a. Histoplasma capsulatum
b. Hepatozoon canis
c. Ehrlichia-nuclear appendages may be confused with Ehrlichia
d. Distemper inclusions
6. Miscellaneous inclusions-RARE
a. Birman cat neutrophil granulation anomaly- fine azurophilic granules in
neutrophils that are not associated with functional problems or clinical
disease.
b. Chediak Higashi syndrome-associated with abnormal pigmentation in
many cases, characteristic giant granules form from aberrant organelle
fusion within cells.
c. Lysosomal storage diseases
Lymphocytes
1. Normal morphology
a. Small lymphocytes predominate in small animal species and are
typically smaller than a normal neutrophil
b. Small lymphocytes are classically characterized by a scant rim of pale
blue cytoplasm and a single round nucleus with stippled chromatin and
high nuclear to cytoplasmic ratios. Nucleoli should not be present.
c. “Normal” variations
i. Some lymphocytes may contain a few small reddish cytoplasmic
granules: these are granular lymphocytes and may be slightly
larger with lower nuclear to cytoplasmic ratios. Granular
lymphocytes may be confused with mast cells.
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ii. Some lymphocytes may have a few small clear cytoplasmic
vacuoles
iii. Some lymphocytes may have indented or cleaved nuclei; some
T-cells may have lobated nuclei similar to monocytes
iv. Other domestic species may have medium or large lymphocytes
present in circulation
2. Reactive lymphocytes
a. These lymphocytes have been stimulated by cytokines or antigens
b. These cells are often slightly larger than normal lymphocytes
c. Cytoplasm may be greater in volume, darker blue, contain small clear
vacuoles, reddish granules or a prominent perinuclear clear zone
d. Reactive lymphocytes may have slightly more pleomorphic nuclei than
quiescent cells
e. These cells should not be excessively large or contain prominent
nucleoli
3. Lymphoblasts
a. These cells share characteristics with reactive lymphocytes, however
they are larger (often larger than neutrophils) and contain nucleoli.
b. Occasionally, cells are too immature to be sure that they are lymphoid,
and are categorized nonspecifically as “blasts” or “unidentified cells”.
Sometimes special stains or immunocytochemistry are required to
identify the lineage of these cells.
c. Lymphoblasts are often associated with the presence of neoplasia,
however severe antigenic stimulation can result in the presence of very
small numbers of these cells in the peripheral blood.
d. “Atypical cells” are cells that are considered intermediate between
reactive cells and lymphoblasts. These cells are interpreted to be a
non-specific finding often associated with severe inflammation, sepsis,
necrosis or neoplasia.
4. Inclusions in lymphocytes
a. Lysosomal storage diseases
b. Distemper
Segmented
Neutrophil
Small
Lymphocyt
e
Reactive
Lymphoblast
Lymphocyte
Atypical
Lymphocyte
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Monocytes
1. Normal morphology
a. Normally the largest appearing cell in the peripheral blood-cell spreads
along the glass
b. The cytoplasm is abundant and blue-grey with a slightly granular
texture. It may contain small clear vacuoles.
c. The nucleus is indented to band shaped to lobated, thus the
morphologic characteristics of the monocyte may overlap with enlarged
toxic segmented or band neutrophils
2. Abnormal monocyte morphology
a. Monocytes may become reactive, with similar cytoplasmic changes as
those described in lymphocytes
b. Monocytes may phagocytose other cells or contain iron or infectious
agents
Eosinophils
1. Normal morphology
a. These cells are often slightly larger than neutrophils but also contain a
lobated nucleus with condensed chromatin
b. When the cytoplasm can be visualized around the granules, it is more
blue-grey than neutrophil cytoplasm
c. Band eosinophils do occur, but are rarely counted separately because
of uncertain diagnostic significance
d. Eosinophil granule morphology is influenced by species and can be
used as a reference to confirm the species of the individual patient.
e. Eosinophil granules may coalesce or granule contents may be lost
during preparation. Eosinophil granule drop out leads to a highly
vacuolated appearance that could be confused with a monocyte and is
more common in greyhounds but can be seen in individuals of any
genetic background.
c. Extremely rarely, infectious organisms may be identified in peripheral
blood eosinophils
Basophils
Normal morphology
a. Basophil cytoplasm is pale blue and the nucleus may be slightly less
segmented than neutrophil nuclei of the same patient.
b. Like eosinophils, granule morphology varies with species. Dog
basophil granules are generally dark purple, while cat basophil
granules are oval and stain pale lavender.
Mast cells
1. Mast cells are mononuclear cells characterized by the presence of variable
numbers of very dark purple granules that may obscure the nucleus
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2. Mast cells are not normally found in the circulation; when they are seen they
are a NON-SPECIFIC finding and may be associated with inflammatory
disease, hypersensitivity conditions, tissue necrosis, or neoplasia.
Erythrocytes
1. Estimation of numbers:Erythrocyte numbers/PCV cannot be accurately
estimated from blood film examination, although anemia patients will tend to
have thinner films than normal patients, and polycythemic or
hyperproteinemic patients tend to have thicker films.
2. Red blood cell associations
a. Rouleaux is red cell association resulting in a “stacked coin”
appearance that is normal in some species such as the horse. In dogs
and cats, rouleaux is associated with increased fibrinogen or gamma
globulins in the blood as a result of inflammation or neoplasia. These
associations are loose, and rouleaux will disperse if the blood is diluted
with saline.
b. Agglutination is immune-mediated association of erythrocytes into
grape-like clusters. It may be grossly visible and does not disperse
when the blood is diluted with an equal volume of physiologic saline.
3. Rouleaux
Normal erythrocyte morphology
Agglutination
a. Erythrocytes in dogs and cats are biconcave discs, with a prominent
central pallor evident in canine erythrocytes.
b. Low numbers of polychromatophilic cells are present in dogs because
up to 1.5% of erythrocytes may be slightly immature under normal
circumstances. Very slight to no polychromasia is typical in most
healthy cats.
c. Mild anisocytosis may be present in normal dogs and cats.
4. Morphologic abnormalities of erythrocytes
a. Increased polychromasia
i. This is the presence of more blue and less red erythrocytes in
the blood and is associated with retention of organelles and
incomplete hemoglobinization.
ii. Increased polychromasia is expected as part of the regenerative
response to anemia.
1. In dogs the following is suggested
a. 1% polychromatophilic cells: normal
b. 2-4%: slightly increased
c. 5-20% moderately increased
d. 0-50%: markedly increased
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2. In cats the following is suggested
a. 0.5% normal
b. 0.5-2% slightly increased
c. 3-4% moderately increased
d. >5% markedly increased
iii. Quantitative evaluation of the response to anemia is best
accomplished by performing reticulocyte counts
b. Increased anisocytosis (variation in red cell size)
i. This is often associated with regeneration as immature
erythrocytes are larger than their more mature counterparts
ii. Microcytes can be present with iron deficiency anemia or
portovascular anomalies.
iii. This may accompany dyserythropoiesis
iv. Spherocytes appear smaller on blood films because of the loss
of the biconcave disc morphology, however the cell volume
does not change significantly if measured by automated
instruments.
c. Nucleated erythrocytes
i. Most normal healthy animals do not have nucleated
erythrocytes in the circulation, however occasional animals may
have rare nucleated erythrocytes on the blood film, especially in
young patients.
ii. Nucleated erythrocytes have variable amount of blue to red
cytoplasm (depending on the degree of hemogloblinization of
the cell). Nuclear chromatin is very dark and condensed. It can
be easy to confuse these cells with small lymphocytes. Small
lymphocytes will have smoother chromatin and scant pale blue
cytoplasm with no red or pink tinge.
iii. Increased numbers of nucleated erythrocytes are present in a
variety of conditions that may cause endothelial damage
(including sepsis, shock, transient hypoglycemia or hypoxia
associated with seizures or acute onset anemia), or lesions in
hematopoietic tissues.
d. Spherocytes
i. Spherocytes are erythrocytes that have lost their biconcave disc
morphology because of swelling or loss of cell membrane. They
can be difficult to identify in cats, who already have smaller
erythrocytes and lack prominent central pallor
ii. Spherocytes appear to have a smaller diameter on the blood
film (see anisocytosis).
iii. Spherocytes are most often associated with immune-mediated
damage, but other potential causes include snake
envenomation (coral and rattlesnake), bee stings, zinc toxicity,
parasites, and genetic biochemical defects of erythrocytes.
iv. Some of the causes of spherocytosis other than IMHA may
cause positive Coombs tests
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e. Schistocytes (fragments)
i. Fragments of erythrocytes look like irregularly shaped pieces of
cytoplasm that may have ragged borders or sharp projections
ii. The causes of mechanical disruption or biochemical
abnormalities that cause fragmentation are numerous, but
important and common diseases associated with red cell
fragmentation include vascular abnormalities such as valvular
stenosis, heart worm, hemangiosarcoma, and portosystemic
shunts with or without therapeutic intravascular device
placement. Vasculitis, disseminated intravascular coagulation
and renal disease are also implicated.
iii. Fragments may form with oxidative injury or severe iron
deficiency
f. Heinz bodies
i. Heinz bodies are variably sized knob-like projections of oxidized
precipitated hemoglobin that stain red to pale pink and stain with
reticulocyte stains, which are help identify small Heinz bodies
that may be difficult to appreciate on Wright Giemsa stains.
ii. Heinz body formation is most often associated with exposure to
some type of oxidant, although small numbers are normal in
cats, and metabolic diseases like diabetes mellitus in cats may
increase numbers significantly.
iii. Cats are more likely to have a single large Heinz body per
erythrocyte, while dogs may have multiple smaller Heinz bodies
iv. Be sure to check the patient for evidence of
methemoglobinemia if Heinz bodies are noted. The blood will
have a brownish discoloration and the patient may appear to be
more hypoxic than the PCV would suggest.
g. Howell-Jolly bodies
i. Howell-Jolly bodies are small, round, dark purple retained
nuclear remnants that could be confused with infectious agents
or other inclusions
ii. Howell-Jolly bodies may be present in small numbers under
normal circumstances, and may increase when there is
accelerated erythropoiesis or when the pitting function of the
spleen is suppressed by drugs (glucocorticoids) or disease
h. Basophilic stippling
i. Aggregated ribosomes form irregular blue staining inclusions
throughout the cytoplasm in Wright Giemsa stained films. These
cells resemble reticulocytes.
ii. Basophilic stippling may be seen during regenerative anemias,
but is also associated with lead intoxication.
i. Hypochromasia
i. Erythrocytes with decreased hemoglobin content
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ii. When mild, this may be reflected in decreased MCHC, however
when more severe, erythrocytes will be pale or have increased
central pallor. The cells may be fragile and become fragmented
(see fragments or schistocytes)
iii. Hypochromia must be differentiated from “punched out cells” in
which the normal hemoglobin content is artifactually
concentrated at the periphery of the erythrocyte, also causing
an increased zone of central pallor
iv. Hypochromasia is a feature of iron deficiency
j. Echinocytes
These erythrocytes are spiculated cells that have uniform pointed
or blunt projections from the cell membrane. They are usually
artifactual (drugs, fatty acids, erythrocyte dehydration, altered pH),
but have been described in association with snake envenomation
and renal disease.
k. Acanthocytes
These cells contain variably-size, irregularly shaped cytoplasmic
projections that are generally larger and less consistently shaped
than those in echinocytes. They are the result of membrane
abnormalities associated with altered lipid metabolism, and thus
may be seen in patients with liver disease, however the finding is
nonspecific and patients with a variety of disease may have
acanthocytes.
l. Target cells (codocytes)
Target cells resemble targets because of excess membrane,
resulting in a prominent zones of hemoglobin at the center and the
periphery of the cell separated by a clear ring. They are frequently
seen in sick animals, but have little diagnostic significance.
m. Red blood cell ghosts
Occasionally red cell membranes devoid of hemoglobin are noted
on blood films. These reflect in vitro or in vivo hemolysis
n. Infectious agents
i. Babesia in dogs and Mycoplasma felis are the most common
and must be distinguished from artifacts such as stain
precipitate, platelets superimposed on erythrocytes, Howell-Jolly
bodies, or drying and fixation artifacts.
16
Dog RBC
Cat
RBC
Polychromatophili
c cell, also slightly
larger
Spherocy
te
Nucleated RBC vs
Lymphocyte
Hypochromic cell vs punched
out cell
Echinocyte vs.
Acanthocyte
Howell-Jolly
body
(nuclear
Target
cell
Fragments
(Schistocytes
)
Heinz body
(Oxidized
hemoglobin)
Basophilic
stippling
Mycoplasma felis and Babesia
Platelets: Besides examining the film for platelet clumps and estimating platelet counts,
note any unusually large platelets, especially if they approach the size of erythrocytes.
These are macroplatelets, which can be associated with a number of conditions, but the
release of macroplatelets can be evidence of platelet regeneration.
Reference materials
17
Atlas of Veterinary Hematology,
Blood and Bone Marrow of Domestic
Animals
JW Harvey, ed
W.B.Saunders, Philadelphia, PA 2001
Diagnostic Cytology and
Hematology of the Dog and Cat
2nd ed. RL Cowell, RD Tyler, JH
Meinkoth eds
Mosby, St. Louis, MO 1999
Fundamentals of Veterinary Clinical
Pathology
SL Stockham and MA Scott eds
Iowa State Press, Ames, IA 2002
Hematology, Quick Look Series in
Veterinary Medicine
Susan M. Cotter
Teton New Media
Jackson Hole, WY 2001
Schalm’s Veterinary Hematology
5th ed, BF Feldman, JG Zinkl, NC Jain
eds
Lippincott, Williams and Wilkins,
Philadelphia, PA 2000
Small Animal Clinical Diagnosis by
Laboratory Methods
4th ed. MD Willard and H Tvedten, eds.
Saunders, St. Louis, MO. 2004.
Veterinary Hematology: Atlas of
Common Domestic Species
WJ Reagan, TG Sanders, DB DeNicola
eds
Iowa State University Press, Ames, IA
1998
Veterinary Hematology and Clinical
Chemistry
MA Thrall, ed
Lippincott, Williams, and Wilkins
Philadelphia, PA 2004
Veterinary Laboratory Medicine
Clinical Pathology
4th ed KS Lattimer, EA Mahaffey and
KW Prasse eds
Iowa State Press, Ames, IA 2003
Pearls:
Paper back
Many photos, quality not as high as
some of the other atlas books
Hard back
Many color photos
Good practice reference
Hard cover text reference
Few photos, not an atlas
Paper back, no photos. Good, very
concise text reference, but not an atlas
Hard back, expensive
Some color photos
Excellent reference, but not practical
for most clinical situations
Paper back, some color plates
Good practice reference
Hard cover. Excellent reference with
numerous excellent quality photoshighly recommended
Hard cover. Good reference, much text
but some good photos. Includes exotic
species
Hard cover. Good reference text,
few color plates, not an adequate atlas
18
1. Automated counts can be in error and should be verified by routine rapid
assessment of blood films.
2. Significant inflammation can be present with NORMAL quantitative data, so
morphology is a critical component of review
3. Neoplastic cells can be present in small numbers, be sure to scan the feathered
edge and whole smear carefully to avoid overlooking them. Total white cell
counts may be normal.
4. Hematoparasites can be present prior to SNAP tests becoming positive, be sure
to look carefully for them in symptomatic animals, again, concentrating on the
feathered edge and making sure to scan the whole smear.
5. Species matters! Cats often have significant numbers of Heinz bodies due to
endogenous metabolic stress (look for diabetes, hepatic lipidosis, and lymphoma,
but can be seen with conditions) with a very low risk of hemolysis, however the
same finding in dogs should prompt an extensive search for toxin exposure,
consider radiographs to look for zinc. In contrast, the presence of mast cells can
be nonspecifically associated with hypersensitivity or mucosal pathology in dogs,
but is fairly specific for mast cell neoplasia in cats.
19