HEMATOLOGY

HEMOGRAM
A hemogram contains all the information for assessment of hematopoiesis and a visual assessment of
plasma appearance and measurement of total solids
The components of a hemogram include the following:
1. Measurement of cell counts
a. RBC
i. PCV (haematocrit/RBC mass)
ii. RBC count
iii. Hb concentration
iv. Reticulocytes – can assess for regeneration by bone marrow in response to
anemia
b. WBC count
c. Platelets – total count
2. Measurement of RBC and platelet indices
a. RBC
i. MCV – mean corpuscular volume – measure of average RBC size
ii. MCH – mean corpuscular hemaglobin – measure of average Hb content in
individual RBC’s
iii. MCHC – mean corpuscular hemaglobin concentration – percentage of RBC that
consists of Hb
iv. RDW – RBC distribution width – measure of the variation in RBC volume
b. Platelets
i. MPV – mean platelet volume – average platelet size
3. Blood smear evaluation - examination of a blood smear prepared from peripheral blood and
stained with a hematologic stain eg Diffquik
a. Differential leukocyte count – relative proportions of leukocytes in the blood
b. RBC morphologic features – variations in RBC shape, Hb content, presence of inclusions
or pattern of arrangement (eg rouleaux or agglutination)
c. WBC morphologic features and number
d. Platelet morphologic features and number
e. Infectious agents
i. In plasma eg microfilaria
ii. In RBC eg mycoplasma, babesia
iii. In WBC eg Anaplasma morulae
iv. In platelets eg Anaplasma morulae
4. Assessment of plasma appearance and total solids
a. Plasma appearance – for evidence of hemolysis, icterus etc
b. Total solids – estimate of total protein in plasma

LABORATORY ERRORS
Results of hematology tests provide information on the function of the bone marrow and gives clues 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.
Many factors other than disease influence the results of hematology tests. These factors may be
preanalytical, analytical and post-analytical.
1. Preanalytical: variables associated with the patient, sample collection and sample handling. These
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. Occur
in the lab itself due to 3 M’s: Man, Machines/material, Method
3. Post-analytical variables: the different ways data from the laboratory is presented, stored and
transferred to the clinician. Ie transcriptional and translational errors
Whenever possible, these variables should be controlled in order to minimize their effect on test outcome.

Pre-analytical errors
Sampling
Determining the reference intervals, 3 types
1. Population based reference intervals – take a representative of entire population/s
 Used by practitioners
 Compare results to healthy animal
 Eg reference interval for PCV at a lab can be for all populations (eg all vets across Harare)
 Errors made when doing reference intervals: 5% of animals in the population fall outside
the reference intervals
i. Can make a 2.5% error by misclassifying an animal by putting it outside the
reference intervals on either side
ii. Eg PCV with reference interval 37-55 - If an animal is on 37 (low end) and near
death, the lab still sees it as being within reference range
2. Individual based reference intervals - restrict population parameters into very fine limits ie
reference interval is from one animal
3. Diseased-based
 Used by specialists
 When a diseased animal comes in, results are compared to the diseased animal rather
than the healthy animal
i. Disadvantage of comparing to healthy animals – some diseased animals will be
missed
ii. Advantage of comparing to healthy animals – animals that are seen to be
diseased are truly diseased (true positive, high confidence)
 Animals that fall between a and c (intersection) – do you treat them or leave them?
i. Depends on hospital standards and protocols

Analytical errors
Method validation
Need to confirm that the method can be performed
1. Determine accuracy by running known samples/controls
 Positive samples
 Negative samples

Eg 20 animals tested, 10 known to be negative and 10 known to be positive

Test done: get 4 negative animals and 8 positive animals  12 animals have been correctly identified
12/20 = 60% accurate
Normally only accept a test as valid if it shows 85% accuracy
Run more than once, using the same samples

2. Determine precision
 When a test is run more than once and same result is obtained
 Can be precise but not accurate – can be ‘precisely wrong’

3. Determine repeatability
 Should be able to redo the same method on different samples

4. Robustness
 Ability of the test to give consistent results in different environments
 Can be run by different people and in different climates and environments etc

5. Dilution tests
 Analytical sensitivity – lowest point at which a disease can be picked up ie lowest dilution
 High analytical sensitivity means can have higher dilutions with the same results
 Tests with high analytical sensitivity are more expensive
 Tests with high analytical sensitivity are used for monitoring
 Different from diagnostic sensitivity
RED BLOOD CELLS

Functions
1. Transport oxygen around the body
 Hb is membrane bound to RBC rather than free because free Hb is highly oxidative and
can damage the body – kidneys are very sensitive to oxidative damage (oxidative
nephrosis)

Production (erythropoiesis) and replacement

RBC’s live for about 120 days – varies with species:
- Dogs – 110 days
- Horses – 90 days
- Cats – 70 days

RBC’s produced in bone marrow from a single stem cell. So if bone marrow stops working, cats will be
more affected than other animals because of shorter lifespan of RBC’s ie halflife is of RBC’s shorter in cats
than other species.
Hematopoeisis replaces 1-2% of RBC each day, with RBC level remaining constant
In younger animals - fetal Hb needs to be switched over to adult Hb – so there amount formed and
replaced everyday is greater
Process maintained by the kidneys, which produce erythropoietin
It is produced at a constant rate to maintain the normal rate of erythropoiesis.
The circulating concentration of erythropoietin increases when the oxygenation of the renal tissue
decreases. It makes bone marrow more porous to immature RBC in crisis.
There are also factors that inhibit hematopoiesis. The maintenance of normal concentrations of peripheral
blood cells depends on a balance between the factors that promote hematopoiesis and those that inhibit
the process.

Rate of secretion of EPO can increase if there is low oxygen tension eg:
- increased destruction of RBC
- respiratory problems because there is interference with oxygen exchange

Rate of production of EPO decreases with

- kidney disease  PCV decreases because more are dying than being replaced (takes time though)
- can tell if renal failure is chronic or acute by PCV
- acute renal failure – PCV normal
- chronic renal failure – PCV decreased

Bone marrow in young animals is busy – red in colour ie the more red, the younger the animal, the more
yellow the older the animal

Erythropoiesis
1. Rubriblasts
 Divide and one differentiates to a prorubricytes
 One remains as a rubriblast to maintain them at a steady level throughout aging
 Requires hematinics
i. vitamin B12 (cobalamin), B6, vitamin B9 for Hb
ii. iron
2. Prorubricytes
 Divide 3-6 times to form rubricytes
 Become smaller in size with each division
 Termed proliferation
Proliferation process takes 2 ½ days
3. Rubricytes
 In RBC crisis, there will be fewer divisions, so more immature, larger RBCs are released
into blood stream – accelerated erythropoiesis
 4. Reticulocytes
 Nucleated RBC matures to punctuated reticulocytes
 Maturation of rubricytes – takes 2 ½ days
 Non-nucleated – stains pink

Under normal situations, it takes about 5 days for a mature blood cell to reach circulation, less in crisis.
Horses - in crisis, RBC can be bigger (NRBC’s) but do not get immature RBC in circulation

The spleen
The spleen is another site of hematopoiesis – extramedullary hematopoiesis
This organ can increase its production of blood cells when needed but cannot replace the bone marrow as
the primary site of hematopoiesis. Several changes in the peripheral blood occur when the spleen has
increased production of erythrocytes, including increased circulating nucleated
erythrocytes.

The spleen checks RBC’s and removes old cells with poor membranes, so that they do not burst in the
bloodstream and release Hb into circulation, which could damage organs such as the kidneys
Hb is broken down by macrophages into
1. Iron
- Recycled
- Cannot give animal Fe parenterally in crisis, rather give iron orally
2. Protein – recycled
3. Pyrrol ring
- Oxidised to biliverdin (water soluble) then reduced bilirubin (water insoluble)
1. If bilirubin is exposed to light, it get oxidised back to biliverdin
- Receptors on hepatocytes internalise bound bilirubin in an energy dependent process,
and it gets conjugated
1. Indirect bilirubin (IBIL) – unconjugated bilirubin coming from the spleen
2. Direct bilirubin (DBIL) – conjugated bilirubin from the liver
3. Total bilirubin (TBIL) = IBIL + DBIL
- DBIL enters gall bladder then GIT to be excreted
1. Oxidised in GIT to stercobilirubin
2. Some converted to urobilirubin and is reabsorbed – enterohepatic circulation,
and excreted in the urine
- Horses and some dogs are sensitive to bilirubin and accumulates when they don’t eat –
turn yellow
1. Partitions into fat
- Bilirubin levels can increase in the blood if there is:
1. Acceslerated erythrolysis
2. Disturbances in uptake by the liver
3. Disturbances of release into the GIT
4. Liver problems

Hemolysis
Intravascular hemolysis - RBC burst in circulation
Extravascular hemolysis – burst/destroyed in macrophages in the spleen
Hemolysis is a normal process but should not be accelerated

Many haemolytic diseases are associated with both intravascular and extravascular mechanisms. Factors
that can influence the type of hemolysis include:
- the type of antibody causing the lysis in immune-mediated disease
- the severity of infection with erythroparasitism
- the severity of oxidative injury with toxicity.
Intravascular hemolysis often causes a more severe disease because of the toxic effects of free
hemoglobin on the kidneys and the widespread inflammation associated with release of hemoglobin into
the blood
EVALUATION OF RBC’S - ERYTHROGRAM
An erythrogram includes all the tests that evaluate RBC, including the following:
1. Assessment of RBC numbers
a. RBC count
b. Haematocrit/PCV
c. Hb concentration
2. RBC indices
a. MCV
b. MCH
c. MCHC
d. RDW
3. Regeneration
a. Reticulocyte counts – done in dogs and cats
b. RBC changes in blood smear evaluation
i. Number of macrocytes in horses and ruminants
ii. Degree of polychromasia in ruminants
4. RBC morphological features

The three erythrocyte parameters in the erythrogram that are directly measured by most in-clinic
hematology instruments are hemoglobin, red cell count, and red cell volume. The remaining red cell
parameters—MCHC, Hct, and MCH—are calculated from the measured values. As a result, if there are
artifactual changes in the measured values, the calculated values will
be erroneous.
Each of these measurements can help identify and characterize the anemic patient.

Assessment of RBC numbers

1. RBC count
- A count per certain volume ie determine the concentration
- RBC x 1012/L
- Methods
i. Impedance counters
- RBC’s diluted in saline
- Use the principle that cells are poor conductors of an electric current,
and saline is a good conductor
- Tube with a small aperture (20fl) – individual cells are counted as they
flow through the tube through an electrical field
- When saline passes through, there is a complete circuit
- When an RBC passes through, it breaks the circuit
- The size of the cells can be measured because the magnitude of the
change in current is proportional to the cell size
- Smaller cells passing through are counted as platelets
- This method is precise and accurate in counting leukocytes, erythrocytes
and platelets and can produce a reliable but limited automated
differential
- Measures: PCV, MCV, RDW, Hb
- Derived parameters: MCH
ii. Flow cytometer
- Blood sucked into a buff and cells stained
- Cells then passed through a laser beam
- A detector measures reflection depending on the stain of the RBC
2. Hct/PCV
- Expressed as a percentage of the blood
- HCT (%) = (MCV x RBC) ÷ 10
- PCV is a directly measured value
3. Hemaglobin
- Measured in g/dL
RBC indices
1. MCV
- Measured in femtoliters (FL)
- Can be calculated manually: MCV (FL) = (HCT/PCV ÷ RBC) x 10
- Factors that influence MCV:
i. Young animals tend to have smaller erythrocytes and a corresponding low MCV.
ii. MCV is most often increased as a result of reticulocytosis.
iii. Microcytosis, or decreased MCV, can be seen in animals with portosystemic
shunts.
iv. Iron deficiency causes decreased MCV.
v. Macrocytosis, or increased MCV, has been described in cats infected with feline
leukemia virus (FeLV).
2. MCH
- Measure in picograms (pg) per cell
- Can be calculated manually: MCH (pg) = (Hgb x 10) ÷ RBC
3. MCHC
- Measures in g/dL
- Can be calculated manually: MCHC (g/dL) = (Hgb ÷ PCV/HCT) x 100
- Hyperchromasia (increased MCHC) is caused by:
i. Absolute increases in MCHC, or actual increased hemoglobin within erythrocytes,
are not usually observed and often are artifactual
ii. Intravascular or in vitro hemolysis is the most common artifact that causes
increased MCHC.
iii. Hyperlipidemia or Heinz bodies may cause falsely increased hemoglobin
concentration due to interference.
iv. Spherocytosis
- Hypochromasia (decreased MCHC) is caused by:
i. Reticulocytosis leads to hypochromia (decreased MCHC)
ii. Iron deficiency can cause hypochromia
4. RDW
- A high RDW indicates that the RBC’s are more variable in volume than normal, due to
larger or smaller RBC or both
- Increased numbers of immature RBC in a regenerative response to anemia will increase
the RDW
- Increased numbers of smaller cells in iron deficiency anemia will also increase the RDW

Regeneration
1. Reticulocyte count
- Presence in the blood in sufficient numbers indicates the bone marrow is responding to
an anemia and indicates the anemia is not die to defective bone marrow production, but
due to loss (eg haemorrhage) or destruction of RBC (eg hemolysis)

RBC morphology
Red blood cells are assessed for morphologic changes and presence of erythroparasites, which are semi
quantified.
1. Shape (poikilocytes): Acanthocytes, echinocytes, eccentrocytes, keratocytes, schistocytes,
spherocytes, ovalocytes, target cells, stomatocytes. Semi-quantified as few, moderate or many.
Some of these changes are more important than others, e.g acanthocytes and schistocytes
indicate fragmentation injury, whereas keratocytes and eccentrocytes indicate oxidant injury
(along with Heinz bodies). Large numbers of spherocytes are usually seen in immune-mediated
hemolytic anemia. In contrast, echinocytes are frequently artifacts.
2. Size: Macrocytes, microcytes. Semi-quantified as few, moderate or many. Anisocytosis (variation in
size) is semi-quantified as mild, moderate or marked.
3. Color: Hypochromasia (too little hemoglobin), polychromasia (immature cells containing RNA or
reticulocytes). Semi-quantified as mild, moderate or marked.
4. Inclusions: Siderocytes (iron), basophilic stippling (RNA), Howell-Jolly bodies (retained nuclei),
Heinz bodies (oxidized hemoglobin). Semi-quantified as few, moderate or many.
 5. Patterns: Agglutination (noted as present or absent), rouleaux formation. Rouleaux formation is
semi-quantified as mild, moderate or marked.
6. Infectious agents: e.g. Babesia, Anaplasma. Semi-quantified as few, moderate or many.

Other tests
1. Total iron binding capacity TIBC
- Transferrin transports iron around body
- TIBC corresponds to iron – ie how much iron can be carried around the body
- Done when when it appears that an animal has iron deficiency or overload when there are
signs of anemia, especially when a CBC is performed and shows red blood cells that are
microcytic and hypochromic and the hemoglobin and hematocrit levels are low.
- In iron deficiency, the iron level is low but the TIBC is increased, thus transferrin saturation
becomes very low.
- In iron overload states, such as hemochromatosis, the iron level will be high and the TIBC
will be low or normal, causing the transferrin saturation to increase.
- It is customary to test for transferrin (instead of TIBC or UIBC) when evaluating a person's
nutritional status or liver function. Because it is made in the liver, transferrin will be low in
those with liver disease. Transferrin levels also drop when there is not enough protein in
the diet, so this test can be used to monitor nutrition.
2. Serum iron test
- Done to evaluate the amount of iron in the body

Erython
circulating erythrocytes, their precursors and all the body elements concerning their production
High erythron mass – polycythemia
Low erythron mass – anemia
ANEMIA
Decreased circulating erythrocytes, or in particular a reduction in any one of – RBC, Hb or PCV
Clinical signs suggestiveof anemia include pale mucous membranes, increased respiratory rate, and, in
some cases, icterus (yellow coloration of tissues).

Mechanisms:
1. Haemorrhage
2. Accelerated hemolysis
a. Intravascular hemolysis - the rupture of erythrocytes within the vascular system.
Mechanisms for intravascular hemolysis include the following:
i. Oxidative injury
ii. Membrane alteration
iii. Physical damage
iv. Immune-mediated lysis
v. Parasitism eg babesiosis
vi. Traumatic rupture
vii. Enzyme deficiency
b. Extravascular hemolysis - the removal and subsequent breakdown of erythrocytes by
macrophages within the spleen and liver. Causes of include:
i. Reduced RBC deformability
ii. Antibody or complement-mediated phagocytosis
iii. Hypersplenism
iv. Altered cytoplasmic ATP content
c. 11. What is intravascular hemolysis
3. Hypoplasia/decreased production ie rate of hemolysis exceeds rate of production
a. Aplasia – complete lack of production – possible in dogs

Identifying anemic animal is not sufficient – need to identify underlying cause

Only sufficient in emergency – give fluids or blood transfusion before trying to identify cause

Classification of anemia
1. According to bone marrow response ie can the bone marrow increase production of erythrocytes
in response to the decrease in PCV
a. Appropriate response – can rule out hypoplasia
i. BM is functioning normally – bone marrow can respond by increased production
of erythrocyte
ii. Normal production of erythropoietin by the kidney interstitium – EPO is the
hormone released by the kidneys in response to the decrease in oxygen-carrying
capacity of the blood, is the stimulus for the bone marrow
i. to increase production of erythrocytes
b. Inappropriate response – rule in hypoplasia
i. No evidence that the BM can respond

Bone marrow response is time dependent.

- There is a window of time before can see if bone marrow is responding
- First 2 days, may not see response – can be used to identify timing ie onset of acute anemia
The lack of response by the bone marrow can be due to a disease that directly affects the bone marrow or
decreased production of erythropoietin by the kidney, usually due to chronic renal failure.

2. According to cell morphology – shape and inclusions

a. MCHC
i. Hypochromic
ii. Normochromic
b. MCV
i. Microcytic
ii. Normocytic
iii. Macrocytic
Inclusions – basophilic stippling, internal parasites, lead poisoning

3. Pathogenesis ie mechanisms

Appropriate BM response Inappropriate BM response

Macrocytic Haemorrhage Leukemia – releasing RBC before
Hemolysis (eg IMHA) properly matured
MCV Clumping of RBC – machine reads as
macrocytic

Normocytic Hypoplasia
Microcytic Iron deficiency - hypochromic

Blood component therapy – transfusions

For dogs, every 1kg of BW, there is 8-10% blood (6-8%) or cats
So amount of blood per 1kg = 10/100 x 1kg
X 1000 to get to ml

 1kg x 100
Per kg, there is 100ml
So if a dog is x kg, it has 100x ml blood

RBC = wt/10 x (total PCV-observed PCV) x 10 = RBC(ml)

Eg 17kg dog, with PCV of 13. What RBC does it require?

17/10 x (21-13) x 10 = 136ml packed red cells required

However, you may not have packed cells, so instead use donor dogs and take whole blood
45% PCV blood in 100ml = 45ml RBC
Ie n/45 x 100

136/45 x 100 = 302ml donor blood needed

BLOOD TYPING & CROSS MATCHING
Dogs
Dog erythrocyte antigens
Given numbers
NULL - rare
DEA-1.1 (40% of dogs)
DEA -1.2
DEA-1.3
DEA-3
DEA-4
DEA-5
DEA-7
DEA-8

**There is no DEA 2 or 6

Can only do transfusions if donor and recipient have not given/received blood before
- No naturally occurring antibodies
- Blood must first be cross matched: check for antibodies against donor RBC’s

Cats
Almost all the same
A or B
Cats with blood type A have naturally occurring anti-B antibodies at a low titer and vice versa
A third, very rare, AB type can occur
A is most common
B is quite common in certain pedigree breeds
A is dominant to b, so:
AA = type A
Ab = type A
bb = type B

Therefore cats should always be blood typed or cross matched before transfusion

Horses
There are over 30 blood groups, but only 8 major blood groups: A, C, D, K, P, Q, U and T
A, C and Q are most likely to stimulate an antibody response when given to a horse that is negative for
them – important in neonatal isoerythrolysis

Difference between cross matching and blood typing

- Cross-matching is looking for sensitization: detection of antibodies against donor blood type by
testing the serum of the recipient)
- Blood typing is looking for blood type: done by measuring the reaction of a small sample of blood
to certain antibodies

The two are different in dogs and horses

In cats, cross-matching is as good as blood typing and vice versa because can only be one or the other
blood type
WHITE BLOOD CELLS
1. Neutrophils
2. Monocytes
3. Basophils
4. Eosinophils
5. Lymphocytes

First 4 are from BM, called myeloid cells

Lymphocytes are from lymphoid tissue

Formation of myeloids
Myeloblasts in bone marrow
Divide and form promyelocytes (round nucleus)
Then divide to form myelocytes (round nucleus)
Then divide to form metamyelocytes (kidney shaped nucleus)
Metamyelocytes mature to form band cells (band nucleus)
Band cells are immature neutrophils
Mature neutrophils (segmented nucleus)

Takes 3 days for cell maturation to get neutrophils in the circulation

Neutrophils have got granules (can’t see them except in cattle and birds)
Granules contain the enzyme lysozyme – kill bacteria
Granules up to myeloblasts are proteinaceous and can be seen up to this stage in other animals – useful in
staging leukemia (protein stains pink)
Metamyelocytes – granules become glycated and no longer see them (don’t stain – are clear)
In cattle and avians and reptiles, glycation does not occur to the same extent so can still see pink granules
Storage of neutrophils in BM depending on species
Small animals in general (dogs and cats), store a lot of neutrophils in the BM – called large storage pool
animals

Neutrophils work outside the blood – move out the blood stream

Pressure increase

Lymphocytes
T and B

High endothelial venules (HEV)

Eosinophils
Eosinophilic chemotactic factor, which is produced by mast cells

Basophils
Eventually become mast cells??

Number
Differentials
Morphology
WHITE BLOOD CELLS

Neutrophils
- Innate immune cells produced in the bone marrow
- Released in to blood after maturation in the BM, in an age-dependent manner
- Circulate for less than a day (10-15hrs) and migrate out of the vessels into tissues or body cavities
- Circulating pool – neutrophils in large vessels
- Marginating pool – neutrophils in small vessesl
- Total body neutrophil pool = CNP, MNP and the pool of most-mitotic neutrophils in marrow
- Humoral factors at sites of inflammation stimulate increased production and release from marrow
- Chemotactic factors such as IL-8, at sites of inflammation direct migration of neutrophils from
blood vessels into tissues at those sites
- Endotocin can cause sequestration of neutrophils in the spleen, lever and lung lowering neutrophil
count in a blood sample

Neutrophilia
- An increased number of mature neutrophils (absolute numbers not percentages)
- Can occur due to
a. Shift from marginating to circulating pool due to corticosteroids and epinephrine ie
associated with excitement or stress (physiologic or stress leukogram respectively)
b. Increased release from BM
i. Inflammation – inflammatory cytokines stimulate release of storage pool. If
inflammation is moderate to severe, band cells may be released as well
ii. Corticosteroids – release of mature neutrophils
c. Increased production by BM (takes 3-5days) – response to inflammatory cytokines
d. Decreased migration into tissue eg lack of adhesion molecules (uncommon)
e. Delayed apoptosis – prolonged survival of neutrophils eg with inflammation,
corticosteroids

Neutropenia
- A decreased number of neutrophils
- Occurs due to opposite mechanisms to those that cause neutrophilia
a. Shift from circulating to marginating pool in acute endotoxemia – causes short term
neutropenia (1-3hrs) after initial exposure to endotoxins so may not be noted clinically
b. Decreased release from BM – uncommon
c. Decreased production in BM due to
i. Miscellaneous primary BM disease eg idiopathic BM aplasia
ii. Deficiency of cytokines
iii. Congenital or inherited defect eg canine cyclic hematopoiesis in grey collies
iv. Viral disease eg feline leukemia virus and feline immunodeficiency virus
d. Increased migration into tissue in response to inflammatory cytokines and chemokines eg
bacterial sepsis, endotoxemia (longer term), tissue necrosis. Most common cause of
neutropenia especially in cattle with severe acute inflammation eg metritis, mastitis
e. Increased destruction eg in immune medated neutropenia, drugs (chemotherapy), toxins
and viruses (parvo, feline panleukopenia, BVD)
f. Unknown mechanisms
i. Vitamin B12 deficiency in border collies and giant schnauzers
ii. Idiopathic neutropenia in cats – usually negative and asymptomatic for common
viruses associated with neutropenia
iii. Breed associated – some breeds of dogs have lower neutrophil counts eg
Australian Shepherds and related Labrador retrievers

Immature neutrophils
- A left shift indicates the presence in blood of neutrophils less mature than segmented neutrophils
ie bands
Lymphocytes
- Produced in lymphoid tissue rather than in bone marrow
- Most lymphocytes in blood are long lived cells that recirculate between the blood and tissue
- Changes in lymphocyte number usually reflect changes in distribution rather than changes in
production or loss
- Dense, round to slightly indented nucleus with a small amount of clear to pale blue cytoplasm
- Granular lymphocytes involved in cell mediated cytotoxicity – cytotoxic T cells or NK cells

Lymphocytosis
- Increase in absolute numbers of lymphocytes can be due to:
a. Physiologic leucocytosis – epinephrine response, particularly in cats and horses and young
animals. Results in a transient rise in mature lymphocytes
b. Physiologic due to youth – animals < 6 months of age can have higher lymphocyte counts
c. Antigenic stimulation
i. Infectious agents eg Ehrlichia ccanis, bovine leukemia virus, Theileria
ii. Chronic inflammation (uncommon)
d. Hypoadrenocorticism – usually mild, but can be marked lymphocytosis
e. Non-lymphoid neoplasia eg thymomas
f. Chronic lymphocytic leukemia

Lymphopenia
- Decrease in absolute numbers of lymphocytes due to
a. Stress leukogram (most common) – endogenous or exogenous corticosteroids causing a
shift of lymphocytes from the circulating to other pools
b. Acute infection: This could be mediated by corticosteroids or a consequence of the
infection (decreased production, increased margination and emigration into tissues).
c. Loss of lympthocytes – loss of lymph or loss into lymphocyte rich effusions eg chylothorax
d. Lymphocytolysis due to viral infection or high doses of corticosteroids
e. Primary or secondary immunodeficiency

Monocytes
- Large innate immune cells produced in bone marrow
- Circulate briefly in blood and migrate to tissues to differentiate further to become macrophages or
dendritic cells
- No storage pool in marrow
- Unevenly distributed between a marginated and circulating pool

Monocytosis
- Increase in absolute numbers of monocytes, due to
a. Stress response – common
b. Inflammation – common
c. Recovery from acute marrow injury eg secondary to chemotherapy – uncommon
d. Paraneoplastic response eg lymphoma due to cytokine secretion – uncommon
e. Monocytic/monoblastic leukemia - uncommon

Monocytopenia
- Difficult to document because reference intervals often go to zero and has no clinical significance

Eosinophils
- Produced in bone marrow
- Circulate in blood for a few hours and migrate into tissues where they survive for a few days
- Increased production is mediated by factors produced by some activated T lymphocytes and mast
cells, especially IL-5
- Involved in allergic reactions and immunity against parasites

Eosinophilia
- Increased absolute numbers of eosinophils due to
 a. Allergies or hypersensitivity reaction – common
b. Parasites eg Dirofilaria immitis in dogs – common
c. Paraneoplastic response eg mast cell tumours
d. Hypoadrenocortism – lack of corticosteroids, usually mild
e. Idiopathic hypereosinophilic syndrome
f. Chronic eosinophilic leukemia - rare

Eosinopenia
- Difficult to document because range may go down to zero

Basophils
- Produced in the bone marrow
- Normally the number is very small in all species, but usually a few can be found in smears from
healthy horses and ruminants
- Involved in immunity against parasites

Basophilia
- Rare finding, usually occurs in association with eosinophilia
- Can be due to chronic myeloproliferative disease eg chronic myeloid leukemia

Basopenia
- Range often down to zero, so is not a relevant finding

“Other WBC”
i. Histiocytes
ii. Mast cells
iii. Blasts

LEUKOGRAM
The leukogram includes all tests that evaluate WBC, including the following:
1. Assessment of leukocyte numbers:
a. Total WBC count (all cell types)
b. Relative (%) and absolute (cells/uL) differential leukocyte count (WBC separated by type)
2. WBC morphologic features: These can give clues as to underlying disease pathogenesis or can
identify the cause of the anemia, including parasites.

Assessment of leukocyte numbers

1. WBC count
- Total number of leukocytes in a volume of blood, expressed as thousands/µL.
- Count of the nuclei or total nucleated cell count, so if nucleated RBCs are circulating in
blood, they will be included in this count so this needs to be accounted for
- Corrected WBC = nucleated cell count x (100 ÷ [nRBC + 100])
2. Differential WBC count
- Done by counting 100 leukocytes in the monolayer of the smear
- Provides relative proportions of WBC normally found in blood

WBC morphology
1. Toxic change (immature features): Cytoplasmic basophilia, Döhle bodies, cytoplasmic vacuolation,
toxic granulation, immature chromatin. Semi-quantified as mild, moderate or marked.
2. Reactive lymphocytes (indicating antigenic stimulation): Semi-quantified as few, moderate or
many.
3. Smudged cells: These let you know how accurate the differential leukocyte count in. A few
smudged cells will not impact the count but moderate to many will. Smudged cells are more
frequently seen in “aged” or stored samples. Semi-quantified as few, moderate or many.
4. Abnormal cells: Presence of leukemic blasts, dysplastic changes in leukocytes, iron in leukocytes
(sideroleukocytes), histiocytes, mast cells.
5. Infectious agents: e.g. Anaplasma. Semi-quantified as few, moderate or many.
Leukogram patterns
Physiologic and pathologic processes cause certain alterations in the total and absolute differential WBC
counts that can be recognized. Identification of these leukogram patterns is key to interpretation of
changes in WBC in hemogram results. The usual patterns that we recognize from a hemogram are the
following:

1. Physiologic leukocytosis
- Due to epinephrine (fight-or-flight response).
- Characterized by a neutrophilia with no immature forms (no left shift) and lymphocytosis.
- An eosinophilia and basophilia may be seen in cats, in particular.
- Usually in young animals.
- Mechanisms:
- Neutrophilia – due to a shift from MGP to CGP
- Lymphocytosis due to release from the spleen

2. Stress leukogram
- Due to endogenous corticosteroid release.
- Characterized by a neutrophilia with no left shift and a lymphopenia and eosinopenia.
- In some species, a monocytosis may be present (e.g. dogs).
- Mechanisms for corticosteroid related leukogram changes are as follows:
- Neutrophilia – mainly due to a shift from MGP to CGP
- Lymphopenia – due to multiple mechanisms including decreased efflux from LN
and lymphotoxic effects
- Monocytosis – mechanisms unknown
- Eosinopenia – decreased release from BM
- Causes of a stress leukogram are
a. Endogenous stress – chronic or acute increases in corticosteroids – expected in
most ill animals
b.Hyperadrenocorticism
c. Exogenous administration of corticosteroids – may be accompanied by increased
liver enzymes in dogs (especially ALP)
- Other clinical pathologic results seen with a stress leukogram
- Hyperglycemia – due to corticosteroids
- Increased ALP

3. Inflammatory leukogram
- Due to inflammatory cytokines (interleukin-1, interleukin-6, tumor necrosis factor-α)
- Characterized by a left shift (regardless of total leukocyte numbers) or the presence of
immature neutrophils in blood (band neutrophils typically; even earlier forms may be seen
with severe inflammation).
- Toxic change is frequently seen as part of an inflammatory leukogram (but is not always
present).
- Inflammation may result in a concurrent lymphopenia and eosinopenia, although this also
could be due to a concurrent stress leukogram.
- BM senses an increasd demand for neutrophils through cytokine stimulation and initially
responds by releasing the storage pool (results in neutrophilia with a left shift). Cytokines
also kick in and stimulate granulopoiesis within 3-5 days
- Mechanisms
- Neutrophila – mild to very severe
- Left shift in neutrophils – the more severe the left shift, the more severe the
inflammatory stimulus
- Toxic change – indicates immaturity and accelerated release. May not always
occur in an inflammatory leukogram
- Monocytosis – usually seen with inflammation that is more long-standing or
resolving
 - Concurrent lymphopenia (and eopsinophilia) – could be due to concurrent
endogenous stress
- Causes of inflammation are many and include
- Infectious agents
- Immune mediated conditions eg vasculitis
- Cancer – cytokine production by tumour cells
- Necrosis – hypoxic injury from anemia, infarct, tissue burns
- Foreign body
- Other clinical pathologic changes seen with inflammatory leukogram are:
- Thrombocytosis – due to inflammatory cytokines
- Changes in albumins and globulins
- Changes in iron panel
- Changes in glucose

Stress versus inflammation?

Both stress and inflammation can manifest with similar leukogram patterns, i.e. a mature neutrophilia (no
left shift or toxic change), monocytosis and lymphopenia and eosinopenia. Under these situations, it is
unclear from a single hemogram, if the changes are due to stress or inflammation. In these cases, you
should use other laboratory data (e.g. hyperglobulinemia, hypoferremia, hypoalbuminemia) and clinical
data (e.g. fever) to decide which you think is more likely (or possibly both may be occurring). Measurement
of sequential hemograms may also be helpful (to document improving or worsening inflammation or an
absence of stress).

Pattern differentiation
The following table may help with differentiating between leukogram patterns. Note, that clinical
information, results of other testing (e.g. imaging) and other clinical pathologic results will also assist with
leukogram pattern differentiation.

Leukogram patterns
Evidence of
Pattern Neutrophils Left shift? Lymphocytes Monocytes Toxic change
inflammation
↑ (dogs, cats,
Stress ↑ No ↓ No ±*
especially)
Physiologic ↑ No ↑ No change No Not usually
Mild/chronic ↑ (if chronic,
↑ ± N or ↓ No Hopefully
inflammation especially)
Acute inflammation** ↑ ↑ ↓ N or ↑ Usually Yes
Overwhelming
↓ ↑ to ↑↑ ↓ N Yes Yes
inflammation
Sequestered
↑↑ ± N or ↓ N or ↑ ± May be difficult to find
inflammation
* A stressed animal may have localized, chronic or mild inflammation.
** Acute inflammation in cattle will result in a neutropenia with a left shift as indicated above.

4. Leukemia
- The presence of neoplastic cells in circulation and/or the BM
- Classified by
- Stage of maturation – acute or chronic
- Lineage – myeloid or lymphoid
- Leukaemia is a progressive malignant disease of the bone marrow, characterized by
abnormal proliferation and development of haematopoietic cells and their precursors.
- In most instances, excessive numbers of abnormal neoplastic cells are present in both the
peripheral blood and the bone marrow.
 - This may be accompanied by a reduction in the uumber of normal blood cells (cytopenia),
as the bone marrow becomes overwhelmed by the neoplastic cells.
- Aleukaemic leukaemia (smouldering leukaemia)
- On occasion, the lieoplastic process is contained within the bone marrow and is not
accompanied by excessive numbers of abnormal circulating cells.
- The haemogram will reflect the ongoing disease process in the marrow in the form of
non-regenerative cytopenias.

a. Myeloproliferative Disease
- Myeloproliferative disease (MPD) is a general term used to describe all the non-lymphoid
neoplastic and dysplastic conditions of haematopoietic cells.
- The term was introduced because the myeloid leukaemias represent a constantly
changing spectrum of diseases, which may progress from dysplastic marrow conditions to
aleukaemic leukaemias and finally to overt leukaemia.
- In addition, although one particular cell lineage may predominate at any time, a second
cell lineage is often affected and, as the disease progresses, transitions to other cell
lineages may occur.
- Thus, depending on the time of sampling, the diagnosis of the specific cell line involved
may differ.

b. Lymphoproliferative Disease
- Lymphoproliferative disease (LPD) is the term used to describe all the neoplastic (and
dysplastic) conditions arising from lymphoid cells.
- Because only one cell lineage is involved there is not the same spectrum of disease as
seen with the myeloid leukaemias, and dysplastic conditions occur rarely, if at all.
- In addition to acute lymphoblastic leukaemia (ALL) and chronic lymphocytic leukaemia
(CLL), LPD includes lymphoma and multiple myeloma