VPTH 123 – 1ST LE NOTE

CLINICAL PATHOLOGY - Keep away from formalin

- COVERS THE DISCIPLINE OF CLINICAL BIOCHEM, HEMATOLOGY,
AND CYTOLOGY
- Lab tests include complete blood counts, blood chemistry,
urinalysis, fluid analysis, and cytology examination
SAMPLE COLLECTION
- Good laboratory results – very dependent on the quality of
samples submitted
- Samples collected from field cases – properly transported to the
laboratory
- Samples – properly collected, placed in appropriate containers
and submitted to the laboratory as soon as possible
Collection of Blood
- Proper blood collection or handling – critical
- Improper techniques – result in inaccurate blood cell counts and
morphologic artifacts
- Blood sample quality – major contributor to analytical errors
- Clean venipuncture is essential to minimize artifactual changes in
the results
- Blood should flow freely with minimal interruptions during
collection
- Anticoagulants:
o Hematology: EDTA is preferred
o Hemostasis: Citrate anticoagulant (blue) is
optimal for coagulation assays and platelets
- Blood smears are prepared as soon as possible after collection
- Red cells show increase susceptibility to lysis after 24hrs in EDTA
- Prolonged exposure to EDTA procedures artifacts in neutrophils
and platelets
- Do not fix until ready to stain, but keep covered; flies will
consume blood on air dried smears
Staining
- Romanowsky stains (Wright, Giemsa, and modified quick
stains)
- Diff quik of Giemsa for cytology
- Acidic stain – Eosin; binds basic structures such as eosinophil
granules; stains red
- Basic stain – Methylene blue; binds aciding structures such as
DNA/RNA or basophil granules; stains blue
- H & E stain – for tissues
- Picture, characteristics (acidic or basic)
o New methylene blue
-used to reticulocyte counts and Heinz bodies
Manual Blood counts
- Hemocytometer method, red and white thoma pipette
- Diluents:
o RBC – Hayem’s solution
o WBC – Turk’sFluid (Glacial Acetic Acid)
Automated Blood Analyzer/Counter
- Mainly detects the diameter of the cells with its different
tubing
- Two main sensors used are light detectors; and electrical
impedance. One way the instrument can tell what type of
blood cell is present is by size.
Why sa Merck vet manual masyadong malayo ang upper at lower
limit ng ranges ng blood counts?
-Kasi every lab has different machines with different calibrations.

Hematological assessment
- Figure of 8 CYTOLOGY AND SAMPLE COLLECTION

Handling the sample General considerations:

- Blood should be processed as soon as possible after collection
(<24hrs)
- Blood films should be made immediately. If a delay is anticipated
before processing further, the blood should be refrigerated.
- Excessive time at room temperature (40C) can cause autolysis
- Platelet counts – affected by delays in processing; they have a
short lifespan and tends to clump even in presence of an
anticoagulant
- Blood samples should be mixed again several times immediately
before a portion is removed for testing; avoid prolonged mixing
to prevent physical trauma to cells.
Blood Smears (steps)
- Acute angle, 30 degrees
- Coverslip method
- Slide method
1. Label all samples with the patient identification, date, and site
sampled
a. Smears: of smears from more than one site are
submitted, e.g. submandibular and popliteal ln,
label each slide as wo which site they represent
b. Fluids: For any fluid other than peripheral blood
2. Submit all prepared smears
3. Unstained, unfixed, and unoiled smears are preferred for
cytologic examination
4. Submit all samples with a completed cytology request form,
including relevant patient information, test requests and
history.
5. Avoid exposure of any cytologic specimens to formalin
6. Do not allow cytologic specimens to come in direct contact
with ice because lysis will occur (don’t refrigerate)
7. Submit samples ASAP

Blood Films Tissue organ collection

- Air dry the smears quickly and store at room temp until A. For histopathological and immunohistochemistry
processed Materials:
- Do not blot or wipe dry; this would introduce scratches ✓ Clean knives
- Do not refrigerate; the condensation that forms on cold slides ✓ Eide mouth bottles with cover
can lyse cells ✓ 10% neutral buffered formalin
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 VPTH 123 – 1ST LE NOTE

- Tissue samples must not be larger or thicker than 1cm - Species also demonstrate marked variability in size of their
- Tissue blocks should be collected at the interface of gross erythrocyte
lesions and normal portion - There is less variability in leukocytes and platelets in mammals
- Volume of fixative should be 5-10 times the volume of the tissue but different species do have unique responses to inflammation
blocks immersed - Abnormalities in the blood can provide clues as to the presence
of underlying disease (e.g. inflammation) and can be diagnostic
B. For virology tests: viral isolation, Fluorescent antigen test, PCR in itself, e.g. can reveal a leukemia
Materials:
✓ Sterile scissors, forceps, scalpels HEMOGRAM BASICS
✓ Sterile screw capped specimen bottles
✓ Cooler with ice or dry ice - Hemogram contains all pertinent info req for assessment of
- Generous blocks of lymph nodes (ingunal, submandibular) lung, hematopoeisis and visual assessment of plasma appearance and
liver kidney, tonsil etc. should be cut aseptically and individually measurement of total solids (an estimate of total protein) in
placed in a screw capped specimen bottle. plasma.
- Samples should be refrigerated.
Components of Hemogram:
C. For bacterial sample 1. Measurement of cell counts:
Materials: a. Red blood cells (RBC):
✓ Sterile scissors, forceps, scalpels ▪ Hematocrit (Hct) or Packed cell volume (PCV)
✓ Sterile screw capped specimen bottles - measure red blood cell mass. An increase in red
✓ Cooler with ice or dry ice blood cell mass is equivalent to polycythemia and a
- Swab with transport media decrease indicates an anemia.
▪ RBC count
Identification of Samples - concentration of red blood cells expressed in
- Include a complete history of the animal and other important data millions/ul of whole blood.
- Duration of the condition or outbreak ▪ Hemoglobin concentration
- Mortality rate - since RBCs are approximately 33% hemoglobin, the
- Number of animals affected hemoglobin concentration of whole blood normally
- Treatment given and vaccination records is about one third of the Hct ( MCHC = 33g/dl).
- Type of preservative used on the specimen
b. White blood cell count – total number of leukocytes in a
Preservation of samples volume of blood expressed as thousands/ul.
1. Natural Ice c. Platelet counts
- May preserve specimens for 8-12hrs only
2. Measurement of red blood cell and platelet indices
2. Dry ice a. RBC:
- Preferred only if having frozen specimens will not interfere with ▪ Mean corpuscular volume (MCV)
laboratory procedures. - measure of the average RBC size.
▪ Mean corpuscular hemoglobin (MCH)
3. Formalin - measure of the average hemoglobin content in
- Fixing solution of choice individual RBC.
- Neutral buffered formalin – used for histopath samples ▪ Mean corpuscular hemoglobin concentration (MCHC)
examination - percentage of the RBC that consists of hemoglobin
- Buffered Neutral Formalin ▪ RBC distribution width (RDW)
37-40% formaldehyde 100ml - measure of variation in RBC volume.
Distilled water 900ml b. Platelet:
Sodium phosphate monobasic (crystal) 4g ▪ Mean platelet volume (MPV)
Sodium phosphate dibasic (anhydrous) 6.5g - measure of the average platelet size.
Dissolve all the chemicals in 900 ml distilled water 3. Blood smear examination.
- Examination of a blood smear from peripheral blood and stained
BASIC HEMATOLOGY with a hematologic stain (e.g. Wright’s stain or a rapid stain like
Diff-quik).
Hematology
▪ Differential leukocyte count
- Encompasses hematopoeiesis and lab assessment of
- provide relative proportions of leukocytes in the
hemapoietic cells
blood and converted to absolute counts by
- Veterinary hematology is very interesting because of marked
multiplying results by the total WBC count.
species differences in hematopoetic cells
▪ RBC morphologic features
- Birds, amphibians and reptiles have nucleated erythrocytes and
- Changes in RBCs including variations in RBC shape
platelets (thrombocytes) made blood assessment more
(poikilocytosis), hemoglobin content, presence of
challenging than those of mammals
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inclusions or pattern of arrangement (agglutination
or rouleaux formation).
▪ WBC morphologic features and numbers
- Changes in WBCs, including immature (toxic change) or
abnormal (dysplastic) features and presence of inclusions.
▪ Platelet morphologic features and number
- Changes in platelet appearance, including degree of
granulation, presence or absence of clumps.
▪ Infectious agents
- The smear is reviewed for the presence of infectious
agents in plasma (microfilaria), RBC (eg Mycoplasma) and
WBC (Anaplasma morulae) and platelets.
4. Assessment of plasma appearance and total solids
▪ Plasma appearance.
- The plasma is visually examined for evidence of hemolysis,
lipemia and icterus. This gives clues as to the presence of
underlying disease. Hemolysis and lipemia also interfere
with the assays used to measure several hemogram results
(e.g. hemoglobin concentration) and these interferences
must be considered when interpreting results.
▪ Total solids
- estimate of total protein in plasma.
Difference of plasma and serum:
Artificial: serum
Plasma: part of the blood
HEMATOLOGY TEST INTERPRETATION
- Results of hematology tests provide information on the function of
the bone marrow and yields clues or even diagnosis as to the
presence of underlying disease.
- Hematology tests should always be interpreted with respect to what
is known about the patient (signalment, history, clinical signs,
results of other diagnostic testing) and should not be interpreted in
isolation.
Factors other than disease that influence test results:
1. Pre-analytical
- Variables associated with the patient, sample collection and
sample handling. Generally, affect the composition of the body
fluid before analysis and can have a major impact on result
interpretation.
2. Analytical
- Factors which influence the analytical procedure, such as
precision and accuracy.
3. Post analytical
- Involves the different ways data from the laboratory is
presented, stored and transferred to the clinician.
Two ways to interpret hematology test results:
1. On an individual basis:
VPTH 123 – 1ST LE NOTE
- Results for individual hematology tests are generally interpreted
with respect to reference intervals.
- The degree of change above or below the upper or lower
reference limit that is actionable (of diagnostic relevance) differs
between analytes and highly subjective (and a matter of
opinion).
2. Grouping results (pattern recognition)
- Erythrogram: All RBC results, including assessment of the
regenerative response (reticulocyte counts) and morphologic
features.
- Leukogram: All WBC results, including total and differential
counts and assessment of WBC morphologic features.
- Thrombogram: platelet number and size.
SAMPLE COLLECTION
- Blood for hematologic testing must be collected into an
anticoagulant, preferably EDTA (purple top tube).
- Venipuncture should be minimally traumatic to minimize platelet
activation. Use a 23G needle (21-22G is ideal for small animals
and 18G is ideal for large animals) and 3-5 mL syringe (depending
on the desired volume).
- Blood must be adequately mixed with the anticoagulant during
and after collection to prevent clotting. Done by gentle inversion
of the tube (tubes should never be shaken).
Blood Sample Handling Guidelines
✓ All samples should be kept cool during storage and shipping to
minimize changes in cells that can occur with storage.
✓ The sample should be wrapped in paper towels to prevent direct
contact of the tube with ice to prevent freezing and lysis of red and
white blood cells.
✓ Blood smears should be made from freshly collected blood and
submitted along with the tubes to help facilitate blood smear
examination and ensure provision of the most accurate results.
ANTICOAGULANTS used in hematologic studies
1. EDTA
- Cell preservation is optimal in this anticoagulant, which chelates
calcium, preventing clotting
- High concentrations of EDTA are hypertonic in comparison to
red blood cells, so if only a small amount of blood is collected
(e.g. 0.5 mL) and placed into a standard 5 mL EDTA tube, the red
blood cells will shrink.
2. Citrate
3. Heparin
ARTIFACTS
Artifacts in blood samples for hematologic testing stem mainly from
either
✓ Sample collection
✓ Aging of sample
✓ Poor maintenance of the staining solutions (stain precipitate
and water artifact).
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A. COLLECTION ARTIFACT:
1. Difficult venipuncture
- Slow or traumatic venipuncture (poking around a lot for the
vein, exiting the vein during sample withdrawal) can precipitate
platelet clumping and induce small microclots within the
sample or even clotting of the sample.
2. Low sample volume
- Collection of a small blood volume (e.g. 0.5-1 mL) with
placement into a standard 5 mL EDTA tube will cause shrinkage
of red blood cells, because EDTA is hypertonic.
3. Inappropriate mixing with anticoagulant.
- The blood should be thoroughly mixed with anticoagulant
during or immediately after sample collection (by several gentle
inversions).
4. Rough handling:
- Shaking of blood tubes, forcing blood through needles, vigorous
expulsion into tubes, can cause shearing of red blood cells
(hemolysis) and platelet clumping.
B. STORAGE (SAMPLE AGE-RELATED) ARTIFACT:
✓ Storage of blood can result in many false changes in hematology
results.
✓ Changes are minimized but not eliminated by cold storage
(refrigerated, shipping on ice packs).
✓ Artifacts of specimen handling and preparation can significantly
impair examination of blood cells.
✓ If a delay in analysis is anticipated (e.g., lab closed or when
sending a sample to a reference lab), smears should be made
and sent along with the EDTA tube.
✓ Smears made from the same blood sample immediately after
collection (right pic) and after overnight storage at refrigerated
temperature (left pic).
✓ Target cells and neutrophils with condensed chromatin are seen
in the freshly made smear.
✓ In contrast, numerous echinocytes (burr or crenated cells) are
seen in the smear from stored blood.
✓ The neutrophil nuclei have also swollen, with lighter (less
condensed chromatin) than normal and there is polarization of
neutrophil granules to one side of the cell. Such swelling of
neutrophil nuclei can result in the formation of “false” bands,
preventing an accurate differential leukocyte count (may be mis-
interpreted as a true left shift or inflammatory leukogram).
C. AGE-RELATED CHANGES
1. RBC
✓ Crenation (echinocyte formation),
VPTH 123 – 1ST LE NOTE
✓ Lysis (will decrease the RBC count and HCT, leading to a falsely
high MCH and MCHC),
✓ Hemoglobin crystallization.
✓ Red blood cells also swell with storage (take up water). Causes a
falsely increased mean cell volume (MCV) and decreased mean
cell hemoglobin concentration (MCHC).
2. WBC
✓ Swellling and smoothing of the nuclear chromatin (mimicking
band neutrophil formation),
✓ Pyknosis and karyhorrhexis of nuclei,
✓ Cell smudging,
✓ Prominence of Döhle bodies (mimicking toxic change).
✓ Pyknotic leukocytes resemble (and can be misinterpreted as)
nucleated red blood cells. These changes can decrease a white
cell count (lysed cells are not counted) and can affect the
accuracy of a differential cell count.
3. Platelets
✓ Clumping,
✓ Degranulation (makes platelets difficult to see and enumerate).
✓ Platelet clumping decreases a platelet count and increases the
mean platelet volume (MPV), since a small clump of platelets is
seen as a single large platelet. Large platelet clumps are
excluded from the count altogether.
D. STAINING ARTIFACT
✓ Diff-Quik®, Hemacolor®, and other commonly used quick stains
for hematology and cytology can provide good staining quality if
properly used and maintained:
- Keep tightly capped when not in use. This prevents
evaporation, minimizes contamination of solutions and
prevents water from the air getting into the fixative.
- Do not “top-off” the solutions. When fluid level drops or
staining quality declines, empty, clean and dry the jars,
then refill with fresh solutions.
E. WATER ARTIFACT
The red blood cells in this image have irregular punched out areas.
Other water artifacts can be more refractile and mimic an infectious
agent, such as Mycoplasma species.
F. STAIN PRECIPITATE
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 VPTH 123 – 1ST LE NOTE

Stain precipitate mimics bacteria, including cocci and Mycoplasma or hyper-osmolality (excess EDTA – common,
species. Generally, the stain precipitate is purple, whereas bacteria (hypernatremia, hyponatremia).
are blue (with a Wright’s stain). Bacteria are also more uniform and hyperglycemia etc), o Relative change to blood
would be in the same plane as focus of the cells, whereas stain agglutination. water IV fluid dilution, splenic
precipitate is slightly outside the focal area of the smear ✓ Relative change to blood relaxation (anesthetic agents,
water Dehydration tranquilizers).
BLOOD TESTS (common), splenic o Absolute ↓ in RBC mass
contraction (horses Anemia – Hemorrhage,
A. RBC particularly). hemolysis, ↓ production
▪ Red blood cell number: ✓ Absolute ↑ in RBC mass
- Hematocrit (Hct) Primary polycythemia
- Packed cell volume (PCV) (polycythemia vera or
- Red blood cell count chronic erythroid leukemia,
- Hemoglobin (Hgb) concentration rare), secondary
▪ Red blood cell indices: polycythemia due to
- Mean cell volume (MCV) appropriate secretion of
- Mean cell hemoglobin concentration (MCHC) erythropoietin (hypoxia) or
- Red blood cell distribution width (RDW). inappropriate secretion of
▪ Nucleated RBC: erythropoietin (renal
- How nucleated RBC are identified and how they affect cysts/neoplasia , other
the WBC count. neoplasia
▪ Reticulocyte counts:
- Reticulocyte percentage
- Absolute reticulocyte count that are used to assess RED BLOOD CELL COUNTS
regeneration in dogs and cats.

B. WBC
▪ Assessment of leukocyte numbers:
- Total WBC count, relative (%) and absolute (cells/uL),
differential leukocyte count (WBC separated by type). INCREASED DECREASED
▪ WBC morphologic features: ✓ Artifact Thrombocytosis with o Artifact Hemolysis (in vitro),
- These can give clues as to underlying disease large platelets (marked, rare) clotting of sample,
pathogenesis or can identify the cause of the anemia, ✓ Relative change to blood agglutination.
including parasites. water IV fluid dilution, splenic o Relative change to blood
relaxation (anesthetic agents, water IV fluid dilution, splenic
C. Platelet tests tranquilizers). relaxation (anesthetic agents,
▪ Platelet counts: this can be done via various methods and is ✓ Absolute ↑ in RBC mass tranquilizers).
part of most routine mammalian hemograms. Primary polycythemia o Absolute ↓ in RBC mass
- Manual counts: using a hemocytometer (polycythemia vera or Anemia – Hemorrhage,
- Automated counts by hematologic analyzers chronic erythroid leukemia, hemolysis, ↓ production.
- Blood smear estimation rare), secondary
▪ Mean platelet volume: obtained through blood analyzers polycythemia due to
▪ Other platelet tests: includes plateletcrit (like the hematocrit), appropriate secretion of
like the hematocrit), platelet component (cytoplasmic erythropoietin (hypoxia) or
granularity or complexity), reticulated platelets and platelet- inappropriate secretion of
associated IgG. erythropoietin (renal
cysts/neoplasia , other
QUICK TEST INTERPRETATIONS neoplasia).

HEMOGLOBIN
HEMATOCRIT

INCREASED DECREASED
INCREASED DECREASED
✓ Artifact: False increases in o Artifact Hemolysis (in vitro),
MCV: uptake of water with false decreases in MCV
storage (delayed processing)
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 VPTH 123 – 1ST LE NOTE

✓ Artifact Lipemia (common), hemolysis (in vitro or in vivo), hyperosmolality (eg.

Heinz bodies, nRBC.
Iatrogenic Oxyglobin therapy
✓ Relative change to blood
water IV fluid dilution, splenic
relaxation (anesthetic agents, o Absolute ↓ in RBC mass
tranquilizers).
✓ Absolute ↓ in RBC mass
Anemia – Hemorrhage,
hemolysis, ↓ production.
MEAN CELL VOLUME
o Relative change to blood
water IV fluid dilution, splenic
relaxation (anesthetic agents,
tranquilizers).
Anemia – Hemorrhage,
hemolysis, ↓ production.
oxyhemoglobin, nRBC
(uncommon), agglutination,
excess EDTA (common).
hypernatremia).
o Anemia Regenerative anemia
(normal or high MCV), iron
deficiency anemia (low MCV).
o Functional iron deficiency
Portosystemic shunts, (low
MCV).
o ↓Hgb production Lead
poisoning, B6 deficiency,
copper deficiency
o RBC swelling Heriditary
stomatocytosis.

BASIC HAMATOLOGY PART 2

RED BLOOD CELSS

INCREASED
✓ Artifact RBC clumping or
agglutination, storage-changes
(swelling – common),
hyperosmolality (e.g.
hypernatremia).
✓ Physiologic Breed
(Greyhounds, Miniature and
Toy Poodles), fetal RBC.
✓ Anemia Regenerative anemia
(reticulocytosis)
✓ Osmotic/membrane
abnormalities Hereditary
stomatocytosis (Alaskan
Malamutes, Miniature
Schnauzers,
✓ Pomeranians, Drentsche
Patrijshond)
✓ Defects in nuclear
maturation/DNA synthesis
Primary myelodysplasia, folate
and Vitamin B12 deficiency (all
uncommon), hydroxyurea
(interference with DNA
metabolism)
✓ Inherited abnormalities in
erythropoiesis Congenital
dyserythropoietic anemia
(Polled Hereford cattle,
Poodles) uncommon
✓ Idiopathic Hyperthyroidism
(some cats)
MEAN CELL HEMOGLOBIN CONCENTRATION
INCREASED
✓ Artifact Lipemia (common),
Heinz bodies (uncommon),
DECREASED
o Artifact Excess EDTA
(common), hyponatremia.
o Physiologic Age (puppies,
kittens, foals), breed (Shar Pei,
Siberian Husky).
o Iatrogenic Chloramphenicol,
lead (secondary iron
deficiency).
o Iron deficiency Iron
absorption, nutritional
deficiency (copper,
pyridoxine), excess zinc
(interferes with iron
absorption).
o Hepatic dysfunction
Portosystemic shunts.
o Idiopathic Inherited
dyserythropoietic disorder in
English Springer Spaniels
(rare).
DECREASED
o Artifact RBC swelling with
storage (common),
- The `erythron' is the name given to the organ of the body,
technically classified as connective tissue. Comprises all the red
cells plus all the red cell producing tissue.
- The single function of the erythronis oxygen/carbon dioxide
transport between the tissues and the lungs, with
haemoglobinas the O2/CO2 carrier.
- Any thoughts why hemoglobin is contained within RBCs rather
than being free in the plasma?
ERYTHROPOIESIS
- Takes place in the red bone marrow.
- Total maturation time varies between species from about 4±5
days in cattle to about 1 week in the dog.
- Normally about 10±15% of developing red cells die before
reaching maturity.
- When there is an increased demand for red cells (e.g.
hemorrhage, oxygen starvation) production is increased. First -
allowing younger forms (reticulocytes, normoblasts) to enter the
circulation, and secondly by allowing the maturation stages to
merge and skip so that erythropoiesis speeds up.
CONTROL OF ERTHROPOIESIS
✓ Production and destruction of red cells are kept in balance.
✓ Hormone responsible for the regulation of the rate of
erythropoiesis is a glycoprotein with a molecular weight of about
60000±70000 daltons_______?______
✓ The principal site of EP production is _______?________.
✓ Erythropoietin affects red cell production in four ways:
- More stem cells differentiate to red cell precursors
Page 6 of 17

- Stages of red cell development are speeded up.
- Transit time out of bone marrow is reduced.
- Immature red cells are released (depending on species)
NORMAL ERYTHROCYTES
Canine
- Relatively large, uniform, biconcave disc; lifespan id 110-120 days
- Small numbers of polychromatophilic erythrocytes are observed
in blood smears from healthy dogs (<1.5%reticulocytes).
- Occasional nucleated red blood cells and Howell-Jolly bodies may
also be seen in blood smears from healthy, non-anemic dogs.
- The canine erythrocyte life span varies from 110-120 days
Feline
- Smaller and more variable in size and shape than those of dogs
- They have little to no central pallor as normally seen in routine
blood films
- Polychromatophiliccells are very few in health (<1%).
- Lifespan is 65-76 days; forms rouleaux
Equine
- Smaller size as feline and lacks central pallor
- Occasional Howell-Jolly bodies are observed in blood smears from
healthy horses
- Polychromatophils are not observed in blood from healthy horses
and are rarely observed in blood from anemic horses
- Therefore, the degree of erythroidregeneration in response to an
anemia cannot be assessed by quantifying reticulocytes in horses.
- 140-15 days lifespan; rouleaux formation
Bovine
- Marked poikilocytosis (variation in red cell shape), thrombocytosis
(often >1millioncells/uL) and microcytosis are features of healthy
calf blood (usually under 3 months of age)
- Calves can remain microcytic for up to 1 year of age, which is
attributed to a physiologic iron deficiency.
- The approximate erythrocyte lifespan is 160 days.
VPTH 123 – 1ST LE NOTE
Goat
- Goat erythrocytes are the smallest of the domestic animal
species, with mean corpuscular volume ranging from16-25fL.
- Marked poikilocytosis can be a normal feature in the blood of
some goats (especially Angora) and is especially prominent in kids
(<3 months of age).
- Goats do not display a prominent reticulocytosis in response to
an anemia and lack polychromasia in peripheral blood in health.
- The caprine erythrocyte lifespan is approximately 125 days.
NUCLEATED RBC
- Nucleated RBC are usually not seen in the blood of healthy
mammals (low numbers maybe seen in dogs, particularly
Dachshunds and Schnauzers, and camelids).
- Erythroblastosis – presence of nucleated cells of the erythroid
line in peripheral blood.
- Often seen in dogs and cats in the context of strongly
regenerative anemia.
- Nucleated RBC must also bed is distinguished from apoptotic
white blood cells (WBC).
CHANGES IN SHAPE
- “Poikilocyte” -a generic term to describe erythrocytes with
abnormal shape.
- Diagnostic relevance of poikilocytesis number-, shape-and
context-dependent.
- Presence of moderate or many misshapen cells is of more clinical
relevance and yield clues as to the cause of anemia in affected
animals.
▪ Acanthocytes
- Acanthocytes (or spurcells) are spherical cells with blunt-
tipped or club-shaped spicules of different lengths
projecting from their surface at irregular intervals.
- These morphologic changes are most frequently seen in
dogs and cats, where they are of diagnostic relevance.
- Seen in: Hemangiosarcoma in dogs, liver disease,
disseminated intravascular coagulation, Iron deficiency
anemia, other diseases, normal finding in young ruminants
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 VPTH 123 – 1ST LE NOTE

▪ Drepanocytes
- Drepanocytes are sickle-shaped erythrocytes that occurs in
certain deer breeds (excluding reindeer and Montjacdeer),
antelope, some breeds of sheep (e.g.Barbary), goats,
antelope, mongoose and genet.
- The changes are seen in blood exposed for more than a few
seconds to atmospheric oxygen, e.g. during mixing of a
blood sample or blood smear preparation.
- Alkalosis can also induce red blood cell sickling, and the
invitro sickling can be prevented by acidification of the
blood.
CHANGES IN PATTERN
- Rouleaux formation is a term describing groups of red blood cells
that form stacks, such as stacks of coins.
- This is a normal finding in the blood of healthy horses and to a
lesser extent cats, but is not normally seen in dogs or cattle (in
health or disease).
- Excessive rouleaux formation in any species indicates
hyperglobulinemia. Note that a low albumin will promote
rouleaux formation (but not cause it).

INCLUSION BODIES

ANAPLASMA and EHRLICHIA

▪ Eccentrocytes and Pyknocytes Old name New name Species Infected
- Eccentrocytes form under conditions of excess oxidant stress to affected blood cell
the erythrocytes, which induces cross-linking of membrane Anaplasma - Bovine RBC
proteins. marginale (extravascular
- They are seen in association with Heinz bodies, which provide hemolytic
evidence of an oxidant effect on hemoglobin. anemia)
- Seen in: oxidant-induced hemolytic anemia, endogenous Anaplasma - Sheep, RBC (non-
oxidants, inherited enzyme defects. ovis goats pathogenic)
Ehrlichia Anaplasma Horse, dogs, Neutrophilia,
equi phagocytophilium cats. eosinophilia
Camelids. (pethogenic)
Humans
Ehrlichia Anaplasma platys Dogs Platelets
platys (cyclic
thrombocyte)
CHANGES IN SIZE
ANAPLASMA
- Changes in red blood cell (RBC) size on a blood smear correspond - Anaplasm aspecies are intracellular bacteria that are within the
to changes in diameter of the cell. family of Anaplasmataceae of the order Rickettsiales.
- Variation in red blood cell size on a blood smear is called - Anaplasma marginale is an intracellular parasite of cattle that
anisocytosis. produces severe hemolytic anemia and is of major economic
importance.
▪ Macrocytes - The clinical syndrome is one of acute onset, severe anemia with
- These are larger red blood cells than normal. icterus, fever, anorexia, dehydration and depression.
- Macrocytes have a normal content of hemoglobin and very - The organism must be differentiated from Howell-Jolly bodies.
little RNA. Infact, there is insufficient RNA (blue) to offset the - Serology is also used for diagnosis (IFA, complement fixation, ELISA)
red hemoglobin, so macrocytes are red in color. as is bacterial DNA detection by polymerase chain reaction (PCR).
- Mechanisms: Regeneration, Abnormal DNA synthesis, Red - Effective vaccines have mitigated the economic impact of the
blood cell swelling. disease.

EHRLICHIA
- Vector: Rhipicephalus sanguineus, Dermacenter variabilis.

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- Cells: Monocytes, macrophages, lymphocytes. Morulae rarely seen in
infected dogs and usually only in acute infection.
- Clinical signs: Fever, lethargy, anorexia, excess hemorrhage
(attributed to platelet dysfunction as well as thrombocytopenia –
thrombocytopenia is usually not severe enough to induce
hemorrhage alone –platelet counts are usually moderately
decreased, i.e. 50-150,000/uL). Some dogs have neurologic disease
and may have increased numbers of granular lymphocytes in their
cerebrospinal fluid (this finding is not specific for this organism).
- Diagnosis: serologic testing, PCR
POLYCYTHEMIA AND ANEMIA
POLYCYTHEMIA
- Polycythemia: increase in red blood cell (RBC) numbers.
- Synonym: Erythrocytosis
- On hemogram:
✓ Increased hematocrit
✓ Increased PCV
✓ Increased hemoglobin concentration
✓ Increased RBC counts
- Can be distinguished through:
✓ Clinical signs and signalment (dehydration, young horse),
✓ Response to fluid therapy (relative polycythemia should
correct with appropriate fluid treatment),
✓ Total protein concentration (usually only increased with
relative polycythemia due to dehydration),
✓ Detection of hypoxia (arterial blood gas analysis),
✓ Cardiovascular and pulmonary evaluation, and searching for
underlying causes of polycythemia (liver and renal disease,
including neoplasia).
- Types:
✓ Relative polycythemia: proportional changes of RBC numbers
in relation to plasma water.
✓ Absolute polycythemia: a true increase in RBC numbers due
to erythropoiesis
RELATIVE POLYCYTHEMIA
- Due to changes in RBC numbers with respect to plasma.
- Causes of relative polycythemia:
✓ Loss of intravascular water
o Dehydration and increased vascular permeability
(secondary to endotoxemia).
o Increased vascular permeability (secondary to
endotoxemia).
o Elevated total protein in relative polycythemia due to
water losses (externally or into the extravascular
space).
✓ Proportional increase in RBC numbers:
o Splenic contraction. Up to 30% of RBCs can be stored
in the spleen and these RBC can be released upon
splenic contraction.
ABSOLUTE POLYCYTHEMIA
- Due to increased erythropoiesis.
- Can be
a. Primary (a bone marrow disorder)
- Can be a familial disorder or neoplastic process.
VPTH 123 – 1ST LE NOTE
- Familial erythrocytosis: reported in Jersey calves and was
suspected in a 2 year-old Arabian gelding. The cause is
unknown.
- Polycythemia vera: chronic myeloproliferative disorder
(chronic erythroid leukemia) and is a neoplastic
condition in which RBC production is autonomous and
independent of erythropoietin concentrations.
b. Secondary, due to appropriate or inappropriate production
of the erythropoietic cytokine, erythropoietin.
- Due to increased erythropoietin production.
- Physiologic stimulus for erythropoietin production is
hypoxia.
- Appropriate secondary polycythemia: Hypoxia is the
principal stimulus for erythropoietin production.
- Inappropriate secondary polycythemia: due to enhanced
erythropoiesis independent of a hypoxic stimulus, and
mediated via erythropoietin.
ANEMIA
- Defined as a decreased hematocrit (HCT) or hemoglobin.
- Anesthetic agents and tranquilizers can cause splenic relaxation
which can result in apparent anemia.
- Young animals frequently have physiologic anemia (due to rapid
growth rate with hemodilution from plasma volume expansion,
dilutional from ingested colostrum, destruction of fetal RBC,
decreased production due to low erythropoietin concentrations)
in the first few months of life (generally < 4 months old).
CHARACTERIZATION OF ANEMIA
1. Severity
- Degree in the HCT and will vary between species. Depending on
the lower limit of a reference interval.
Grade Canine Feline
None (reference interval) 41-58 31-48
Mild 30-40 25-30
Moderate 20-30 15-25
Severe <20 <15
2. Red Cell Indices
- Allows categorization of an anemia on the basis of cell volume
and hemoglobin concentration.
- RBC indices include mean corpuscular volume (MCV) and mean
corpuscular hemoglobin concentration (MCHC).
a. MCV (size)
- Macrocytic: MCV above the reference interval.
- Normocytic: MCV within the reference interval.
- Microcytic: MCV below the reference interval.
b. MCHC (color)
- Hyperchromic: MCHC above the reference interval. Usually a
false increase (not a true in vivo finding), e.g. lipemiacan
falsely increase the hemoglobin concentration relative to the
HCT, thus falsely increasing the MCHC.
- Normochromic: MCHC within the reference interval.
- Hypochromic: MCHC below the reference interval
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 VPTH 123 – 1ST LE NOTE

3. Regenerative response IS the ANEMIA REGENERATIVE??

- Assessment of regeneration is the very first step in evaluating
an anemia.
- If an anemia is regenerative, it is due to hemorrhage or
hemolysis.
- If the anemia is non-regenerative, then decreased bone
marrow production is causing the anemia, although
hemorrhage or hemolysis could also be occurring.

MECHANISMS OF ANEMIA

1. Hemorrhage
- These anemias are usually regenerative (given sufficient time for
a bone marrow response). Can be:
▪ Internal
- Into a tissue or body cavity, such as the peritoneum.
▪ External
- In this case, blood (RBC, plasma) is lost from the body,
e.g. hemorrhage from the skin or into gastrointestinal,
urinary, respiratory tracts.

2. Hemolysis
- These anemias are usually regenerative (given sufficient time for
a bone marrow response). Due to:
▪ Intravascular
- This is when RBC lyse (“pop”) within blood vessels,
releasing free hemoglobin, which is rapidly cleared
through the urine.
- Diagnosed by the presence of hemoglobinemia and
hemoglobinuria.
- Most causes of intravascular hemolysis have concurrent
extravascular hemolysis.
▪ Extravascular:
- RBC are phagocytized by macrophages (usually in the
spleen, but also liver and bone marrow).
- This almost always accompanies intravascular
hemolysis, but more frequently, is the sole mechanism of
hemolytic anemia.

3. Decreased Production
- These anemias are usually non-regenerative. Decreased
production can be due to: Including evidence of hemolysis (no branch of internal or external
▪ Intramedullary disease hemorrhage)
- A problem within the bone marrow preventing a bone
marrow response, e.g. acute leukemia. CAUSES OF ANEMIA
▪ Extramedullary disease
- A disease outside the bone marrow that secondarily
suppresses the ability of the bone marrow to respond to
an anemia or produce erythrocytes.

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 VPTH 123 – 1ST LE NOTE

ERYTHROCYTOSIS

PANCYTOPENIA

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 VPTH 123 – 1ST LE NOTE

HEMOSTASIS a. Life span: 5-10 days, shorter in cats

b. 1/3 of platelet mass is located in the spleen.
Interaction of blood vessel, platelets, and coagulation factors to
achieve hemostasis without obstructing blood flow. ▪ Morphology and Function
- Small granular discs (2-4 μ) with a cytoskeleton, α and dense
I. Fibrin Formation granules, canalicular and tubular systems, and membrane
glycoproteins (integrins, leucine-rich glycoproteins,
immunoglobulins, and selectins) that act as receptors for
ligands, such as von Willebrand factor and fibrinogen
- High metabolic activity
- Function: PRIMARY HEMOSTASIS
- Formation of primary hemostatic plug: 3-5 minutes
- Platelets adhere to subendothelium (exposed vascular
collagen), undergo activation (including shape change),
secrete their granules, and aggregate to form a platelet
II. Fibrinolysis plug.
a. Adhesion factors include von Willebrand
factor9vWf) and collagen.
b. Shape change (from smooth discs to spheres
with numerous processes) increases the surface
area of the thrombin to the platelet surface.
c. Platelets secrete α granule products (fibrinogen,
factors V and VIII, growth factors) and dense
granule products (ADP, thromboxane A2, Ca++)
that recruit more platelets, mediate platelet
aggregation, facilitate coagulation, and mediate
repair of the vessel.
d. Platelets irreversibly aggregate as a result of
fibrinogen binding to activated platelets and
bridging adjacent platelets. Calcium (CA++) is
required.
e. Platelets provide a surface for formation and
depostion of the definitive fibrin plug; then
platelets “contract”, via actomyosin filaments,
to cause clot retraction (facilitates wound
closure and vessel patency).
- Role of platelets in coagulation pathway: localize
coagulation and facilitate generation of thrombin (the
PLATELETS enzyme that converts fibrinogen to fibrin, the definitive
hemostatic plug)
Thrombopoiesis - Role of platelets in inflammation
▪ Bone marrow a. Chemotaxis, enhancement of neutrophil
- Stem cell differentiates into megakaryoblast. function
- Megakaryoblast differentiates in to mature megakaryocyte by b. Antimicrobial activity
process of endomitosis. c. Source of inflammatory mediators, vasoactive
- Megakaryocytes are located at the sinus wall where they substances, and mitogenic agents
probably shed cytoplasm as long proplatelet processes
directly into sinus lumen. LABORATORY EVALUATION OF PLATELETS AND PLATELET
- Maturation time from megakaryoblast to platelet release: PRODUCTION
- 4-5 days.
▪ Regulation QUANTITATIVE
- Thrombopoietin (TPO) ✓ Use EDTA tubes; venipuncture technique in important.
a. Stimulates megakaryocyte differentiation and platelet ✓ Estimate platelet number from peripheral blood smear
production - Normal: Minimum of 7-10/1000x field (Horse:4-10/1000x
b. TPO is constitutively produced and binds to receptors field)
on platelets. - Scan feathered edge for platelet clumps (common in cats,
c. TPO is highly conserved among people, dogs, and mice. cows).
- Other growth factors that affect thrombopoiesis include IL-6 - Note platelet size and morphology.
and GM-CSF. a. More size variation in cats
- Platelets in circulation b. Pale-staining in horses
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c. Large platelets megakaryocytes)
✓ Platelet concentration: electronic cell counter or hemacytometer
counts
- Reference intervals:
a. Dogs 200,000-500,000/μL
b. (Healthy Greyhounds have lower platelet
concentrations)
c. Cats 200,000-600,000/μL
d. Horses 100,000-350,000/μL
e. Cattle 150,000-800,000/μL
- Thrombocytopenia: platelet concentration below lower end
of reference interval for species
a. mild: 80,000-150,000/μL
b. moderate:30,000-80,000/μL
c. severe:<20,000-30,000/μL
- Spontaneous hemorrhage: <20,000-30,000/μL
- Thrombocytosis: Platelet count above upper and pf reference
interval.
- MPV (mean platelet volume in fL): indicates the average size
of platelets; MPV is often increased if thrombopoiesis is
accelerated.
✓ Function Tests
- Buccal mucosal bleeding time (BMBT)
- Standardized in vivo test of the platelet plug formation;
coagulation factor defects do not result in prolonged
bleeding time.
- Measures the time from inflicting standardized wound to
cessation of bleeding.
- Normal time: dogs 1-5 minutes, cats 1-3.5 minutes,
horses/cattle 8-10 min when test done in lip.
- Prolonged BMBT:
 Platelet function defect
 Vascular defect
 Von Willebrand’s disease
 Thrombocytopenia (,80,000/μL) naturally can
result in a prolonged bleeding time, and the BMBT
provides no additional information; therefore, only
perform the test in animals with normal or
increased platelet concentrations.
✓ Bone Marrow Examination: number and morphology of
megakaryocytes
✓ Platelet surface-associated immunoglobulin (PSAIg) and other tests
for immune-mediated thrombocytopenia.
- Anti-platelet antibody tests are available, but can be difficult
to interpret.
- Immunofluorescent anti megakaryocyte antibody test:
detects megakaryocyte-associated immunoglobulin.
DISORDERS OF PLATELETS
-- THROMBOCYTOPENIA
✓ Decreased concentration of circulating platelets
✓ Clinical sign, not diagnosis
✓ Clinical features:
- Signs often associated with mucosal surface bleeding:
- Petechiation/ecchymosis
- Melena, hematuria, epistaxis, gingival bleeding,
hyphema
- Spontaneous hemorrhage it platelet count is <20,000-
30,000/Μl
VPTH 123 – 1ST LE NOTE
- Other clinical signs depend on the cause of thrombocytopenia
and the presence of an underlying disease.
✓ Mechanisms of thrombocytopenia
▪ Decreased production of platelets by bone marrow
 Associated with decreased numbers of megakaryocytes in the
bone marrow
 Bone marrow hypoplasia, aplasia
- Drugs, hormones (e.g. anticancer drugs, estrogens)
- Toxins (bracken fern poisoning in cattle)
- Infectious agents (Ehrlichia spp., FeLV,
parvovirus/panleukopenia viruses)
- Immune-mediated disease (anti-megakaryocyteic
antibodies)
- Idiopathic
 Space occupying lesions ion the bone marrow, e.g. neoplasm
 Myelonecrosis or myelofibrosis
 Pathophysiology:
- Disruption of cell cycle
- Production of abnormal growth factors, inhibitory factors
- Antibody-mediated destruction
- Stem cell defects
- Competition with neoplastic cells for nutrients
 Clinical and laboratory features:
- Clinical signs are associated with thrombocytopenia and
the primary disease process.
- Peripheral blood:
- Thrombocytopenia may be mild, moderate, or
severe; depending on degree of bone marrow
disease.
- MPV (mean platelet volume) is usually within
reference interval (not increased as would be
expected if bone marrow was responsive).
- Bone marrow disease can be a) selective and affect
only megakaryocytes or b) more generalized and
cause decreased production of other cell lines (I.e.,
erythroid, granulocytic).
- If there is a neoplasm in bone marrow, abnormal
cells may be found in circulation
- Bone marrow
- Megakaryocytes
- Decreased or absent
- May have morphologic abnormalities
- Other cell lines may be decreased or absent.
- Abnormal cells or inclusions
- In myelofibrosis, marrow is replaced by fibroblastic cells
and collagen.
▪ Decreased platelet survival (destruction) resulting from:
 Immune-mediated destruction
- Primary (idiopathic)
- Secondary
- Neonatal alloimmune thrombocytopenia
 Direct injury to platelets
- Drugs
- Infectious agents (Ehrlichia platys)
- Physical damage from vascular disease
*IMMUNE MEDIATED THROMBOCYTOPENIA
✓ Classifications:
A. Primary or idiopathic
- Anti-platelet autoantibody attaches to platelet membrane.
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- Platelet is cleared from circulation, primarily by splenic
macrophages.
- May occur as part of a systemic-immune-mediated disorder;
e.g., systemic lupus erythematosus (SLU), or concurrently with
immune-mediated hemolytic anemia (IMHA)
B. Secondary
- Platelets are altered or coated by:
- Drugs or drug-antibody complexes (e.g., trimethoprim-
sulfa)
- Viruses, modified-live virus vaccines
- Other infectious agents (e.g., Ehrlichia canis)
- Circulating immune complexes associated with neoplasia
or sepsis
- Increase in platelet-associated immunoglobulin may cause
clearance of platelets from circulation
C. Alloimmune thrombocytopenia from maternal alloimmunization
✓ Clinical and laboratory features:
 Signalment: Primary IMT primarily occurs in young to middle-
aged female dogs. IMT is also seen in cats, horses, and rarely
cattle.
 Clinical signs:
- Associated with thrombocytopenia anemia
- In primary IMT, no other physical abnormalities are
detected.
- In secondary IMT, signs of the primary disease are
present.
 Laboratory features of primary (idiopathic) IMT
- Platelets
- Decreased or absent on blood smear
- Count is often < 20,000 μL
- MPV initially may be decreased, normal or
increased; it increases as bone marrow responds.
- Anemia (regenerative)
- Leukocytosis (neutrophilia)
- Coagulation tests (PT, aPTT, FDP) are normal.
- Bone marrow: usually increased megakaryocytes
- Anti-platelet antibody tests should be positive.
- Tests to detect other autoantibodies may be appropriate
in some cases: antinuclear antibody (ANA), Coombs’ test,
and Rheumatoid factor.
- Serum biochemical analytes are usually normal.
✓ Treatment of primary IMT
 Control bleeding
- Blood transfusion
- Depends on severity of anemia
- If platelets required, fresh whole blood or platelet-
rich plasma must be used.
- Avoid drugs that inhibit platelet function.
- Provide atraumatic environment.
 Re-establish normal platelet count.
- Immunosuppressive therapy
- Depends on severity of anemia
- If platelets required, fresh whole blood or platelet-
rich plasma must be used.
- Intravenous human IgG: mechanism of action
unclear
- Splenectomy if medical therapy is not effective
VPTH 123 – 1ST LE NOTE
▪ Decreased platelet survival (consumption of platelets)
✓ Definition: Consumption refers to utilization of platelets
during platelet aggregation/coagulation.
✓ Causes:
- Disseminated intravascular coagulation (DIC)
- Vasculitis (some rickettsial agents, FIP)
- Viral infection
- Modified-live virus vaccination
✓ Pathophysiology: Consumption of platelets results from their
utilization in the hemostatic process. If the process is severe
and ongoing, production may not match demand.
✓ Features:
- Platelet count is usually in the range of 50,000-
150,000/μL.
- Hemorrhage may occur if 1) thrombocytopenia is
severe (<20,000-30,000/μL) or 2) coagulation defects
are also present
- Other signs are associated with the primary disease and
its sequelae.
▪ Abnormal distribution of platelets
✓ Definition: Abnormal distribution usually refers to reversible
sequestration of platelets in a large vascular bed, 3.g., spleen,
liver, dilated vessels, lung
✓ Causes:
- Splenomegaly, splenic torsion, neoplasia, severe
hypothermia
- Hepatomegaly, hepatic disease, portal hypertension,
hypothermia
- Vasodilation in endotoxic shock (lung, liver)
▪ Pseudothrombocytopenia (false thrombocytopenia): not all
platelets detected because of clumping or exclusion by analyzer
THROMBOCYTOSIS
✓ Definition: Increased concentration of circulating platelets caused
by increased production of platelets or redistribution of platelets
into circulation
✓ Classifications:
 Primary thrombocythemia
- Marked elevation of platelet count from uncontrolled
production of platelets
- Rare in animals; maybe a component of
myeloproliferative disorders in cats
 Secondary or reactive thrombocytosis
- Associated with certain disease processes
- Inflammatory diseases, especially chronic
- Iron deficiency anemia
- Hemorrhage, especially chronic (from cause
other than thrombocytopenia)
- Immune-mediated hemolytic anemia
- Some neoplasms
- Present in the following situations:
- Rebound from thrombocytopenia
- Post-splenectomy
- Response to certain drugs, e.g. vincristine,
epinephrine
- Excitement and exercise
- Animals are usually asymptomatic
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- Thrombosis and, paradoxically, bleeding are
possible if platelet counts reach exceedingly high
levels.
- Clinical signs are associated with the primary
disease.
- Pseudohyperkalemia (false increase in serum
potassium) may occur as an in vitro artifact when
platelet concentrations are increased in blood
because platelets release K+ during clotting in the
tube.
QUALITATIVE DISORDERS OF PLATELETS
I. Acquired
 Uremia: Toxins in uremic plasma inhibit platelet function
 Drugs: AVOID THESE AGENTS IN ANIMAL WITH BLEEDING
DISORDER
- Aspirin
- Inhibits production of thromboxane
- Irreversible: lasts lifetime of platelet
- Phenylbutazone, acetominophen, NSAIDS
- Some anesthetics, xanthine derivatives (theophylline,
aminophylline), and calcium channel blockers
 Fibrin degradation of products (DIC): Inhibit platelet function
 Paraproteins, e.g., in plasma cell myeloma, coat platelet
surface and inhibit function.
II. Inherited
 Intrinsic platelet defects include absence of glycoprotein
receptors; absence or reduction in platelet granules and
signal transduction defects.
 VON WILLEBRAND DISEASE (vWD)
- Deficiency (types 1 and 3) or qualitative abnormality
(type 2) of von Willebrand factor (vWf, also called
Factor VIII-Related Antigen) resulting in reduced
platelet adhesion
- vWF is a carrier for Factor VIII (the coagulation factor,
also called Factor VIII:C); in type 3 disease (severe
quantitative deficiency of vWf), therefore, Factor VIII
activity may be reduced.
- Species affected: dogs (may breeds), rabbits, pigs, cats,
cattle, horses
- Several modes of inheritance
- Clinical and laboratory features:
- Mild to moderately severe bleeding
- Exacerbated by surgery or trauma
- Signs decrease with age and successive
pregnancies
- Platelet/coagulation studies
- Normal platelet count
- Prolonged bleeding time (tests capacity of
platelets to form a primary platelet plug)
- vWf: Quantitative or structural assays
- aPTT and ACT are usually normal, but may be
prolonged if reduction in Factor VIII activity is
pronounced.
- PT is normal.
CLINICAL CASE
•History: A 7 year old castrated male dog presented due to weight
loss, lethargy, inappetance, intermittent epistaxis and ocular
VPTH 123 – 1ST LE NOTE
inflammation which began about two months prior. The patient
initially presented due to clear discharge from his right eye and
change of color of the eye (red, brown and gray discoloration).
Treatment was initiated with oral and topical antibiotics but the eye
lesions progressed and the patient became lethargic, inappetant and
began exhibiting epistaxis.
•On his most recent presentation, his right eye was hyperemic with
diffuse corneal cloudiness. A complete ophthalmic exam revealed
chronic anterior uveitis and an indolent ulcer in the right eye and
retinal detachments in the left eye.
•Laboratory tests done:
•CBC and blood chemistry panel
•Abdominal ultrasound was performed, which revealed a mildly
enlarged liver and spleen.
•Bone marrow biopsy
Image 1 is a blood smear (Wright’s stain). Image 2 is bone marrow
smear (Wright’s stain).
1. What is the likely cause of the rouleaux formation on the blood
smear? (Clue: Refer to the chemistry panel.)
- Increased rouleaux formation is often due to an increase in plasma
macromolecules, specifically globulins.
2. What is the predominant cell type in the bone marrow smear?
- Increased plasma cell numbers in the bone marrow smear. The
plasma cells have eccentric nuclei, deep blue cytoplasm and a
perinuclear clear zone; they represent approximately 50% of the
marrow cellularity with focal areas having close to 80% plasma cells.
3. What other test could be done to facilitate a definitive diagnosis in
this case?
- Serum protein electrophoresis to confirm if the hyperglobulinemia
is due to an increase in one type of immunoglobulin (monoclonal
gammopathy) or several different globulin types (polyclonal
gammopathy).
- The presumptive diagnosis is Multiple myeloma. The finding of over
20% plasma cells in the bone marrow is suggestive of multiple
myeloma. The neoplastic plasma cells usually produce an
overabundance of one type (or clone) of immunoglobulin resulting in
a hyperglobulinemia and a monoclonal gammopathy.
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LEUKOCYTES
LEUKOGRAM CHANGES:
- Changes in the leukogram include morphologic features of
leukocytes and changes in numbers
1. Individual leukocytes
2. Leukocyte patterns
Left shift: presence of immature neutrophils in neutrophils; indicates
severe inflammation
 Degenerative left shift: there are more immature than mature
neutrophils; indicates severe inflammation
 Regenerative left shift: there is neutrophilia with increased
numbers of band neutrophils (compared to reference intervals)
Toxic change: indicates inflammation and only evident in the
neutrophil lineage.
 Toxic neutrophil – sign of inflammation and NOT a sign of
toxemia
NEUTROPHILS
Released from marrow in an age-dependent manner, i.e., the most
mature cells are released before less mature cells
▪ INCREASED – NEUTROPHILIA
-An increased number of mature neutrophils (absolute number not
percentages)
MECHANISMS:
✓ Shift from marginating → circulating pool: corticosteroids and
epinephrine cause movement of neutrophils from the
✓ Increased release from bone marrow:
o Inflammation: cytokines stimulate release of the storage
pool of neutrophils (lower in ruminants)
o Corticosteroid: can also cause the release of some mature
(not usually band) neutrophils from the post-mitotic
storage pool in marrow
✓ Increased production by bone marrow (3-5 days): a. Reponse to
inflammatory cytokines (inflammation), which stimulate
granulopoiesis
✓ Delayed apoptosis: a. Prolonged survivals of neutrophils can be
seen under some situations
CAUSED BY:
1. Include inflammation (either infectious or non-infectious) →
trauma, surgery, burns
VPTH 123 – 1ST LE NOTE
2. Exogenous or endogenous corticosteroids & epinephrine.
Immune-mediated diseases such as immune-mediated hemolytic
anemia are associated with neutrophilia due to actions of
inflammatory cytokines
3. Neutrophilia is a feature of several different leukogram patterns:
- Including an inflammatory leukogram
- Stress leukogram
- Physiologic leukocytosis
DECREASED – NEUTROPENIA
- Decreased numbers of neutrophils (absolute numbers not
percentages)
- Most common cause is inflammation (increased movement into
tissue), particularly when there is evidence of neutrophil
immaturity.
MECHANISM:
✓ Shift from circulating → marginating pool: occurs in acute
endotoxemia. This shift contributes to short-term (1-3 hrs)
neutropenia seen after initial exposure to endotoxins.
✓ Decrease release from bone marrow: congenital or inherited
defects in bone marrow release in humans have called
myelokathexis
✓ Increased migration into tissue: In response to inflammatory
cytokines and chemokines o Due to tissue inflammation (bacterial
sepsis, tumor necrosis, abscess, endotoxemia). Most common cause
of neutropenia particularly in cattle with severe acute inflammation
(metritis, mastitis), which have decreased marrow stores of
neutrophils that they can release in response to inflammation
LYMPHOCYTES
- Small, mature lymphocytes are the most common lymphocyte in
peripheral blood
- Feature: dense, round to slightly indented nucleus with smooth
(clumped or blocky chromation), a small amount of clear to pale
blue cytoplasm with a high nuclear: cytoplasmic ratio
✓ Reactive lymphocytes:
- have increased amounts and generally deeper blue cytoplasm.
- Some may be larger than a normal lymphocyte but they still
have clumped chromatin (responsible for antigenic stimulus
that is not specific)
INCREASED – LYMPHOCYTOSIS
- Increase in absolute numbers not percentage of lymphocytes
CAUSED BY:
1. Physiologic lymphocytosis
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 VPTH 123 – 1ST LE NOTE

•An epinephrine response, cats and horses, and in young animals

that results in a transient lymphocytosis of mature cells
2. Physiologic due to youth
• Young animals (<6 months of age) can have lymphocyte counts
than adults
3. Hypoadenocorticism - the lymphocytosis is usually mild, but can
be marked (up to 30,000/μL) in some animals
DECREASED- LYMPHOPENIA
- Decrease in absolute numbers
CAUSED BY:
1. Stress Leukogram – due to endogenous or exogenous
corticosteroids
2. Acute infection – could be mediated by corticosteroids or a
consequence of the infection (decreased production, increased
margination and emigration into tissues, reduced egress from
lymphoid tissue into blood)
INCREASED – EOSINOPHILIA
CAUSED BY:
1. Allergies or hypersensitivity reaction
2. Parasites - ectoparasites or those with a migrating tissue phase.
Heartworm (Dirofilaria immitis) is also a common cause of
eosinophilia in dogs
3. Paraneoplastic response: certain neoplasms can also promote
production and release of eosinophils
4. Hypoadrenocortism: Addison’s disease
Lack of corticosteroids can result in an eosinophilia that is usually
mild
BASOPHILS

3. Loss of lymphocytes: loss into lymphocyte-rich effusions (e.g.

chylothorax) or loss of lymph (usually from the gastrointestinal
tract e.g. lymphangiectasia)
INCREASED – BASOPHILIA
MONOCYTES
✓ Increased absolute numbers (not percentage) of basophils
(basophilia) is a relatively rare finding
✓ Basophils without eosinophils can be seen in chronic
myeloproliferative disease, such as chronic myeloid leukemia and
essential thrombocythemia (chronic platelet leukemia)
- Monocytes share a common committed stem cell with neutrophils
- They are produced in marrow, circulate briefly in blood, and migrate
into tissues where they differentiate further to become
macrophages
- There is no storage pool of monocytes in marrow; their numbers in
marrow at a given time are very small

INCREASED – MONOCYTOSIS

CAUSED BY:
1. Stress response: this can cause a monocytosis, particularly in dogs
2. Inflammation (either infectious or non0infectious, acute or non-
acute)
3. Recovery from acute marrow injury: e.g. secondary to
chemotherapeutic agents. The monocytosis can be quite marked
and mimic a leukemia
4. Paraneoplastic response: various types of tumors, e.g. lymphoma,
can induce a monocytosis through cytokine secretion (presumable,
granulocyte monocyte colony stimulating factor)

EOSINOPHILS
Eosinophils are produced from the common myeloid progenitor in
bone marrow, which differentiates to eosinophils under the influence
of eosinophilopoietic cytokines, such as interleukin-5

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