1. Clinical Pathology
• Hematology
Leukocytes
Erythrocytes
Bone Marrow
Hemostasis & Platelet abnormalities
Cytology
• Clinical Chemistry
• Integration of hematology, chemistry, cytology

2. Instructor
Mary Anna Thrall
Janet Beeler
jbeeler@rossvet.edu.kn
mthrall@rossvet.edu.kn
Office hours:
by appointment
Dr. Beeler: Pathobiology office #10-08
Dr. Thrall: Pathobiology office no number,
on west end, sea side

3. Course Objectives
• Understand underlying pathophysiology
• Understand how tests are selected
• Interpret and integrate test results
• Understand Technology to generate data
Use laboratory data to MAKE A DIAGNOSIS

4. References
• Hematology
• Hemostasis
• Clinical Chemistry

5. References
• Hematology
• Hemostasis
• Clinical Chemistry

6. References

7. Exams and quizzes
Exam 1 - 95 points
Exam 2 - 95 points
Final and comprehensive - 150 points
Take home and in class quizzes – 20 points
Lab exam- 40 points
Total points possible - 400

8. Learning objectives – Section 1
• Describe components of a CBC and
recognize the information provided by
each.
• Be able to calculate absolute values of
various types of leukocytes if the
differential leukocyte count (percentage),
and the total nucleated cell count is
provided.
• Be able to calculate the absolute
reticulocyte count based on % of RBCS
that are retics, and RBC count.

9. Complete Blood Count
Direct measurements
• PCV
• Hemoglobin
• Red cell count
• Mean cell volume
• White cell count (Total cell count, nucleated cell
count)

10. Complete Blood Count
Direct measurements
• Plasma protein
• Platelet count
• Mean platelet volume
• Reticulocyte (immature erythrocytes) count
• Reticulocyte MCV

11. Complete Blood Count
Microscopic Procedures
Differential White cell count (Differential
nucleated cell count because includes
nucleated erythrocytes)
Red cell morphology
Platelet morphology, assessment of
adequate numbers
Reticulocyte count if anemic

12. Complete Blood Count
Calculations
Erythrocyte indices
Mean corpuscular hemoglobin concentration
RDW
Absolute nucleated blood cell differential values
(% x total nucleated cell count)
Absolute reticulocyte count
(% reticulocytes x RBC count)

14. nucleated cell
count

19. Learning objectives, Section 2
• Remember the types of collection tubes
that are used for CBC, biochemical profile,
coagulation profile, and glucose when
serum can’t be separated from clot.
• Recognize errors that can be introduced
when incorrect tubes, collection methods,
and storage methods are used.

20. Sample collection & processing

21. Color coding of stoppers
Red - no anticoagulant
Blood is expected to clot so that serum can
be harvested for biochemical profile and
many other tests.

22. Color coding of stoppers
Lavender
Contains the anticoagulant EDTA
(ethlenediaminetetraacetic acid with a
potassium salt)
Preserves cell morphology
Used to collect blood for CBC

23. Color coding
Green - heparin - specific tests (ie, lead conc)
Blue - citrate - coagulation tests
Gray - fluoride - inhibits glucose metabolism
Red and black - “Sure-sep” tube
Used to separate serum from RBCs
without transferring serum to different tube

24. Technique for filling tubes
• “Clean” venipuncture with no tissue
contamination from large vein, such as
jugular or cephalic in large dogs.
• CBC and biochemical profile requires
approximately 5 ml blood. Pediatric (small)
tubes are available.

25. Tissue contamination results in activation of platelets with
resulting clot formation, and erroneously low platelet count.

26. Techniques for filling tubes
• Don’t force blood into tube - don’t use
anything smaller than a 20 G needle for
filling.
• Tubes are vacuum tubes - can utilize the
vacuum or can actually take the stopper
off and fill.
• If using syringe to collect blood, then
filling tubes, work quickly, filling tube(s)
that contain anticoagulant first.

27. Ratio of blood to anticoagulant
is designed to be appropriate by
amount of vacuum.
• Inadequate filling of purple top tube
results in excess EDTA, which causes
erythrocytes to shrink, resulting in an
erroneous decrease in PCV and MCV.

28. Sample handling procedures
• Blood for CBC - either analyze within one
hour, or make blood film and refrigerate
tube (Don’t refrigerate blood film -
condensation causes cells to lyse)
• Don’t allow to freeze - freezing lyses cells
• If blood sets at room temperature for 24
hours or more, erythrocytes swell,
resulting in increase in MCV.

29. Blood for biochemical profile
Blood allowed to clot for 15 to 30 minutes
Centrifuge
Separate serum from clot using pipette
Refrigerate harvested serum until analyzed
Freeze if can’t analyze within two days
Some serum enzymes are not stable, but
most are.

31. Basic Hematologic Techniques
• Blood mixing
• Packed cell volume by centrifugation
• Plasma protein estimation by refractometry
• Leukocyte concentration by Unopette
• Preparation of blood film
• Differential leukocyte count
• Blood film examination

32. Hematologic techniques
Blood mixing
• Important before any analyses -
performed because cells settle out.
• Manually or with a tilting rack or wheel.

35. Packed Cell Volume
• Percentage of whole blood composed of
erythrocytes
• Also called “hematocrit”
• Measured after centrifugation that
maximally packs RBCs

36. Fill tube 70 - 90% of its length

41. Buffy coat
• Leukocytes
• Nucleated erythrocytes
• Platelets

42. Plasma
• yellow pigmentation - suggestive of
icterus (increased bilirubin concentration
in the blood) In large animals, may be due
to carotene pigments associated with diet

43. Plasma
• White, opaque - lipemia (chylomicrons) -
may be due to postprandial collection or
may be due to diseases associated with
abnormalities in lipid metabolism

44. Plasma
• Red coloration - due to presence of
hemoglobin in plasma
• Result of hemolysis
• May be in-vitro due to technique or
presence of lipemia
• May be in-vivo due to hemolytic anemia
(intravascular hemolysis)
• IF PCV not decreased, likely in-vitro.

46. Plasma protein by refractometry
Refractometers estimate the concentration
of solute in fluid, since solute bends slight
passing through the fluid proportionate to
the solute concentration.
Measures the refractive index relative to
distilled water.
Used in clinical diagnostics to estimate
plasma protein estimation & urine specific
gravity

47. Plasma protein by refractometry
• Protein concentration is an estimate,
assuming that other solutes in the serum
are present in normal concentrations.
• Lipemia will artificially increase the
estimate.
• Urea, glucose, cholesterol may also
influence (artificially increase) the
estimate.

52. Plasma protein abnormalities
Follow up with biochemical measurement of albumin
and globulin in serum
Decreases may be due to decreased albumin,
decreased globulin, or both
Increases may be due to increased albumin,
increased globulin, or both.
Increased albumin always due to dehydration.
If total protein and PCV both increased, suggests
dehydration
If total protein and PCV both decreased, suggests
blood loss

54. Total Leukocyte concentration
• Actually Total nucleated cell count
• Technique detects all nuclei in solutions
from which RBCs have been removed by
lysis (or in some systems, centrifugation)

55. Unopette system
• Also need hemocytometer counting
chamber and microscope.
• Unopette capillary is used to transfer 20µl
blood into 1.98 ml acetic acid diluent,
resulting in a 1:100 dilution.
• Acid lysis cytoplasmic membranes,
eliminating RBCs, leaving behind only
nuclei.
• Incubate, mix, place onto hemocytometer.

59. Unopette system
• After filling hemocytometer system, allow
to settle, then examine, using the 10x
objective of the microscope. The volume
of fluid present is 0.9 µl. Nuclei are
counted in all 9 grids. After counting
nuclei, add 10% to yield the number of
cells in 1.0 µl. Multiply the result by 100
(the dilution factor). This results in the
total number of nuclei/µl.

63. Total leukocyte
(nucleated cell) count
By itself is NOT very useful for
interpretative purposes. It is used
primarily to CALCULATE the
concentration of specific leukocyte types,
which is determined after performing the
DIFFERENTIAL CELL COUNT

64. BLOOD FILM PREPARATION
Stained blood film is essential tool for
1) determining concentration of individual
leukocyte types (differential cell count)
2) evaluating morphologic abnormalities of
leukocytes, erythrocytes, and platelets

65. Blood film preparation
• “Push” technique best
• Two slides needed.
• Increase angle if animal is anemic
• Push QUICKLY

69. Staining the slide
• Wright Stain
• Wright-Giemsa Stain
• Variety of quick stains that are
modifications of Wright stain are
available, such as “Diff-Quick”
• Best to use “dipping” procedures.

71. Examination of blood film
Success depends on:
• Quality of blood film preparation
• Examination of correct part of blood film
• Recognition of morphologic abnormalities
• Recognition of artifacts
• Ability to interpret diagnostic significance
of morphologic abnormalities. For
example, spherocytes = IMHA.

73. Nucleated RBC
Neutrophil

75. F
E
A
T
H
E
R
E
D
E
D
G
E

81. Examine with 50x or 100 x objective
• Systematic examination of all three major
cell types (RBCs, leukocytes, platelets)
• Differential count of leukocytes
• Evaluation of RBC morphology
• Estimation of platelet concentration & size

83. Differential nucleated cell count
• Count a minimum of 100 nucleated cells within
the counting area
• Classify nucleated cells as
Segmented neutrophils
Band neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Nucleated RBCs
Other

85. Conversion of % to absolute
Multiply the total nucleated cell
concentration by the percentage of each
leukocyte type to yield the absolute
concentration of each type of nucleated
cell within the blood sample.
Eg, total nucleated cell count = 10,000 µl
80% of cells are segmented neutrophils
80% x 10,000 µl = 8,000 µl segs

86. Erythrocyte morphology
Color
Shape
Inclusions
Arrangement on slide

87. Platelet evaluation
• Estimate concentration
• 6 - 10 platelets per oil immersion field,
depending on microscope’s field of view
• Remember to examine feathered edge for
clumps if platelets appear decreased.
• When platelets approach the size of RBCs
they are termed macroplatelets or giant
platelets.

88. Hematologic techniques performed
with automated instrumentation
Cell particle counting and sizing
• RBC count (x106/µl
• MCV (fl)
• Nucleated cell count (µl)
• Differential cell count (µl)
• Platelet count (µl)
• Reticulocyte count and size (µl, fl)
Spectrophotometry
• Hemoglobin concentration (g/dl) (usually approx
1/3 of the PCV)

89. Hematologic techniques performed
with automated instrumentation
Calculations
Hematocrit (PCV)
MCV x RBC = HCT (PCV)
10
MCV 70 FL x 7.0 RBC = HCT 49%
10
Also, MCV can be calculated if PCV and RBC are
known. PCV/RBC x 10 = MCV

90. Mean Cell Hemoglobin Concentration
not particularly useful diagnostically
Hgb (g/dl) x 100 = MCHC (g/dl)
PCV (%)
10 g/dl x 100 = 33.3 g/dl
30%
Normal is approx 32 to 36 g/dl in all species
except camel family members, which have
approx 41 -45 g/dl

91. Mean Cell Hemoglobin Concentration
Increases are always artifactual
If increased, is due to erroneously high hemoglobin
reading due to hemolysis, lipemia, or presence of
Heinz bodies (pieces of denatured hemoglobin as
a result of oxidation).
Decreases may be due to iron deficiency, but not
unless very severe.
Usually decreases due to presence of many
reticulocytes that are still making hemoglobin,
usually associated with a regenerative anemia.

92. Cell sizing and counting
Light scatter measurement (Flow)
Cells are passed through a flow cell that is
intersected by a focused laser beam.
Physical properties of the cells scatter light to
different degrees and different angles relative
to the light source. Scatter events are counted
to derive the cell concentration. The degree of
scatter in the direction of the light beam, known
as forward angle scatter, is proportional to the
size of the cell. Light scattered in other angles
is correlated with other cellular properties,
leading to the ability to differentiate cell types.

93. Cell sizing and counting
Electronic cell counting (impedence)
Cells are suspended in an electrolyte medium
that conducts electricity.
Cells are relatively poor conductors of
electricity.
Deflections in current are proportional to
the size of the cell. Cells are thus
identified and measured.

94. Size distribution curves
• Established for each population of cells
(eg, erythrocytes, platelets).
• Must be set for each species, since
erythrocyte sizes are so variable for
different species of domestic animals.
• Primary reason that instruments set up to
measure human blood are inappropriate for
domestic animals.

95. Erythrocyte Size
Normal values vary with species
Dog: 60 - 72 fl
Cat, horse, cow: 39 - 52 fl
Sheep: 25-35 fl
Llama: 21 - 29
Goat: 15-25
Human: 80 - 100 fl

96. ERYTHROCYTE SIZE
MCV determined electronically
Rel # of
RBCs
Cell volume (fl)
MCV

97. Red cell distribution width (RDW)
• Describes the relative width describes
the relative width of the size distribution
curve.
• It is the standard deviation of most of
the erythrocytes divided by the MCV.
The tails of the erythrocyte distribution
are usually excluded from this calculation.

98. ERYTHROCYTE SIZE
MCV determined electronically
Rel # of
RBCs
MCV
RDW

101. Reticulocyte concentration
• Can now be determined by Flow technology
• Can be determined manually
• Immature erythrocytes (reticulocytes) still have
organelles for protein synthesis and aerobic
metabolism (ribosomes and mitochondria).
Certain stains cause these residual organelles to
aggregate, resulting in clumped material that can
be seen.
• Reticulocytes are polychromatic RBCs when
stained with Wrights stain.

103. Reticulocyte count
• New Methylene Blue
• Brilliant cresyl blue
• 1000 RBCs are counted. The percentage
of RBCs that are reticulocytes
• Percentage is multiplied by the RBC count
to obtain an absolute reticulocyte
concentration.

105. Interpretation of reticulocyte count
• Dogs: 0 - 60,000/µl
• Cats: 0 - 40,000/µl
• Cows: 0, but can respond
• Horses: Do not release reticulocytes

106. Reticulocyte interpretation
Non regenerative anemia: 0 - 10,000µl
Poorly regenerative anemia: 10,000 - 60,000 µl
Mild to moderate regeneration: 60,000-200,000 µl
Maximal regeneration: > 200,000 µl

107. Reticulocyte maturation
• Dogs - 24 -48 hours from release to
maturation
• Cats - Aggregate reticulocytes become
punctate reticulocytes. Punctate forms
are not polychromatophilic with Wrights
stain, and are not counted in the
reticulocyte count. Aggregates become
punctates in approx 12 hours. Punctates
persist for 12 days.

110. Options for laboratory service
• In-house (performed on the premises)
• Commercial veterinary laboratory
Examples: Idexx, Antech Diagnostics
• Human laboratory or community hospital

111. Advantages of In-house testing
• Rapid turnaround time
• Control over when tests are performed

112. Disadvantages of In-House tests
• Cost of equipment, technician
• Expertise required for quality assurance,
trouble-shooting
• Will still need to send out some samples
for specialized tests
• Will need to consult with clinical
pathologist on difficult blood films, bone
marrow aspirates, and cytology samples

113. In-House Testing considerations
• $25,000 to set up hematology and chemistry
systems. Consider instrument amortization,
supplies, personnel, training, cost of quality
assurance program, time for supervision of tech)
• Practice volume adequate to make it profitable?
• Biochemical profiles usually more expensive ($3
per test vs $25 for complete profile)
• Hiring and retaining technician that is able,
willing, interested, & will seek consultation
• Commitment to quality assurance
– Monitoring of equipment accuracy and precision

114. Advantages of commercial
veterinary laboratories
• May be less expensive
• Complete menu of testing services
• Professional support of technical
performance, quality control, species
differences
• Clinical pathologist usually on-site to
review abnormalities, and for consultations

115. Disadvantages of commercial
veterinary laboratories
Fixed turnaround times
Sample transportation logistics
Cost of sample transportation
Variable quality in pathology support and
consultation.

116. Advantages of human
laboratories or hospitals
• May be only option in remote areas
• Sample transportation may be simple

117. Disadvantages of human
laboratories or hospitals
• Instrumentation not modified for animals
(eg, difference in RBC size will result in
significant abnormalities in hemogram)
• Technicians and pathologist not familiar
with normal and abnormal blood cells
• Turn around time may be increased due to
low priority of animal samples.

118. Out-House testing
considerations
• Community hospital not usually viable option.
Species specific testing is critical.
• Sample pick up serviceconvenient?
• Appropriate turn-around time? - most labs are
located in large cities on either coast, and
Chicago. Results usually faxed or e -mailed.
• Telephone consultation available?

119. Leukocytes & Leukograms
Numeric data + morphologic abnormalities =
leukogram
Abnormal leukograms can describe pathologic
processes (eg, the presence of inflammation)
Leukogram + other lab data and clinical
findings may lead to diagnosis

120. Leukocyte Morphology
neutrophil
monocyte

121. Common blood leukocytes
• Neutrophils - participate in inflammatory
responses by migrating into tissue sites
and phagocytizing and killing organisms.
• Immature neutrophils released from bone
marrow when inflammation present
(Band neutrophils, metamyelocytes)

124. NEUTROPHILS
&
BANDS
• CONSTRICTION, LENGTH OF NUCLEUS?

126. Bands vs metas
length, width of nucleus

128. Lymphocytes
• Numerous lymphocyte subpopulations, such
as B- cells (responsible for humoral
immunity), T- cells (responsible for cell-
mediated immunity), large granular
lymphocytes (null cells or T cells), etc.

130. LYMPHOCYTES
Normal with granules Reactive

133. Monocytes
• Monocytes migrate into tissues to become
macrophages. Functions are many,
including phagocytosis, presentation of
antigens to T-lymphocytes, iron recycling,
cytokine production, etc.
• May be confused with band neutrophils on
blood films.

137. monocytes & bands

138. Monocytes vs metamyelocytes
Color of cytoplasm, chromatin pattern

139. Eosinophils
• Functions are related to modulation of
immune-complex reactions, modulation of
allergic inflammation, defense against
parasites, etc.
• Prominent pink staining granules in
cytoplasm.

140. Basophils
• Basophils contain histamine, heparin, and
numerous other proteins.
• Increased concentration often related to
parasitic infestations.

142. Lymphocyte
Nucleated RBCs
Neutrophil
Howell Jolly body
spherocytes

143. Nucleated RBC
Neutrophil

144. Data Interpretation (Chapter 3)
Reference ranges (intervals) (normal values)
To recognize abnormal results, normal results
must be established, using a population of
apparently healthy animals.
Species, age, breed, gender, pregnancy status,
geographical location, etc.
The more animals that are sampled, the more
accurate the reference interval.

145. Reference intervals
• In some situations, extreme values on
either end should be discarded to
increase the sensitivity of the test.
These are called “outliers”.
• Simple rule: If the difference between
the highest value and the second highest
value in this population exceeds one-third
of the total range, eliminate it.

146. F
R
E
Q
U
E
N
C
Y
BLOOD GLUCOSE CONCENTRATION (MG/DL)

147. Reference intervals
• If the data are normally distributed (Bell-
shaped curve), mean and standard
deviation are calculated, and the central
95% of values (mean + 2 SD) is considered
the reference interval.
• If less than 40 animals are used, then
reference interval is the observed range
of values that remains after outliers.

149. Reference Intervals
• A few (5%) healthy animals will have results
SLIGHTLY outside of the reference interval.
• If result is FAR outside the reference interval,
it probably indicates an abnormality.
• Slight abnormalities in some test results, such
as electrolytes, are more significant than slight
abnormalities in others, such as serum enzyme
activities.

150. Data interpretation
• Use signalment, history, physical exam, and
laboratory data.
• Results may exclude some differential diagnoses,
may suggest additional differential diagnoses, or
may be diagnostic for a specific disease.
• Results that are most outside of the reference
interval are likely the most significant (possibly
w/ exception of enzymes)

151. Data interpretation
• Pattern Recognition of abnormal results is
critical for making diagnoses.
• For example, an increased BUN and a urine
specific gravity indicating that animal is
not concentrating urine is very suggestive
of renal disease. An increased BUN with a
concentrated urine, indicates that animal
is dehydrated or had blood loss into GI
tract or other very high protein meal.

152. Sensitivity of a test
• Measure of the frequency with which the
test result will be abnormal in animals with
the disease for which the test is being used.
If the sensitivity of PCR for lymphoma is
91%, then 91% of animals with lymphoma will
have a positive result. (and 9% will have a
false negative result)

153. Specificity of a test
• Measure of the frequency with which a
test result will be normal in animals
without the disease one wishes to detect.
• If the specificity of a test is 95%, then
95% of unaffected animals will have
normal results. (and 5% will have false
positive results).

154. Sensitivity and Specificity
• Established by applying the test to animals
with known disease status. IE, known to
have or not have the disease in question.

155. Predictive value of a test
• Reliability of a test to detect whether or
not an animal has a certain disease.
• Determined by using both the sensitivity
and specificity of a test. (See pages 49 -
51).

156. Quality control
• Accuracy - gauge of how close the result is to
the true value for that test.
• Precision - gauge of how repeatable the result is
when assaying the same sample.
• Quality control programs assure both accuracy
and precision. This is done by assaying control
samples on which the correct result is known -
control samples are commercially available.
• Problems may be due to instrument, reagents or
operator error.
• CRITICAL that your laboratory has a quality
control program.