CLINICAL PATHOLOGY

What is Clinical Pathology?

• Clinical pathology supports the diagnosis of disease using
laboratory tests (blood & other bodily fluids, tissues, and
microscopic evaluation of individual cells).
• Training in Clinical Pathology is accomplished through a
combination of practical experience, teaching sessions, and
one-on-one interaction with attending staff members
• Thus, clinical pathology is the study of scientific principles
of laboratory tests.
 The use of Laboratory test /clinical pathology

• Provision of conclusive diagnosis(remove guess work)

• Valid and concrete prognosis (hepatic enzyme ↑)

• Monitoring of disease (d+ progress & response of Rx)

• Screening (sub-clinical cases)

 Factors affecting validity of lab tests

• Appropriate inquiry of history, clinical examination and necropsy

findings
• Selection of donor animal (early & late cases)
• Appropriate collection and handling of sample
• Selecting a suitable laboratory test
• Technical proficiency of the lab personnel

• Selection culture vessel (need specific condition/surface)

• Ability to detect result (Mycoplasm Vs solid media)
• Interpretation of result (correlation)

• Reporting and documentation

 SPECIMEN COLLECTION, HANDLING, STORAGE AND
TRANSPORTATION
General consideration
• What is the purpose of sample collection?

- (Dx/screening/certification/monitoring )
• Source of specimen in veterinary practice (live/dead/Env’t)
• The sample should be representative of the case investigated
• Ethical method should be followed which important for animal welfare,
distress, risk of zoonosis, and biosecurity and health protection
 The request that is going to be sent to the laboratory with the
sample should contain all the information regarding to that sample
 Some of the common considerations affecting all types of specimen

• Failure to label a specimen correctly and to provide all pertinent

information required on the test request form.
• Insufficient quantity of specimen to run test or quantity not sufficient.

• Failure to use the correct container/tube for appropriate specimen

preservation.
• Inaccurate and incomplete patient instructions prior to collection.

• Failure to tighten specimen container lids, resulting in leakage

and/or contamination of specimens.
• Failure to maintain the specimen at the appropriate temperature
requirement.
 Specimen collection
During specimen collection, we have to be concerned about:
- purpose the sample
- site of collection
- material needed for collection
- procedure followed to collect sample
- amount to be collected
Samples to be collected are:
• Blood (whole Vs serum)
• Tissue fluid
• Swab
• Fecal
• Milk
• Urine
• Skin scrapping
• Cytological sample
 Sample collection at postmortem

• Sometimes it is difficult to get appropriate sample from live

animal, in which case it is necessary to do autopsy.

• Purpose: collection of sample from varies organ for:

- Pathological examination

- Microbiological analysis/ culture

- Chemical analysis

- Immuno-histo-chemistry: bone-immune response

- Genetic/ molecular analysis

 Information to be sent with samples
• Name and address of owner/occupier and geo-location where
disease occurred, with telephone and fax numbers.
• Name, postal and e-mail address, telephone and fax numbers of
the sender.
• Diseases suspected and tests requested.
• The species, breed, sex, age and identity of the animals sampled.
• Date of samples were collected and submitted.

• List of samples submitted with transport media used.

• A complete history would be beneficial for the laboratory and
should be included, if possible
 Packaging and transportation of sample

• The specimen should be sent faster as much as possible

• If stay beyond 24hrs better to put in refrigerator

• The shipper should ensure that the specimens are packed

• Very crucial using appropriate preservative and transport media

 What are the reasons that veins are preferred over arteries for

blood sample collection?

 BASIC PRINCIPLE OF CLINICAL HEMATOLOGY

Hematology is the study of cellular elements of blood, which

can be divided in to three categories:
• The erythrocytes/Red blood cells (RBCs)
• The thrombocytes/platelets
• The leukocytes/white blood cells (WBC)

1) Erythrocyte or Red Blood Cells (RBC)

• O2 and CO2 transport between the tissue and the lungs with
hemoglobin as a carrier.
• Erythropoiesis = red bone marrow (more extensive in young)
• Normally production and destruction of RBC is kept

balance = erythropoietin,

• So that total RBC number remains constant

• Mature RBCs are easily deformable (flexible) in order to

pass through small capillaries

• Mammalian RBC devoid of nucleus unlike birds and
reptiles

• The life span of RBC varies with species

 Abnormalities of RBCs
A) Polycythemia (AbH PCV)
Relative polycythemia = ↑PCV w/o ↑in an actual ♯ RBC.
- Dehydration

- Spleenic contraction
Absolute polycythemia = ↑ PCV with ↑ absolute ♯ RBC.
- Polyctthemia vera: Myeloproliferative (bone marrow tumor)

- Erthropoietin hormone producing tissue (kidney) neoplasm

- Secondary polycythemia: diseases that cause hypoxia and or
cyanosis like respiratory diseases
B) Anaemia (AbL PCV) ↓ in the proportion of RBC in the blood.

i. Hemorrhagic anemia:
 Trauma, rapture of neoplasm and poisoning = acute onset
anemia.
 Blood sucking ecto & endoparasites = chronic hemorrhagic
anemia.
ii. Hemolytic anemia: Low PCV with free hemoglobin/bilirubin in
the plasma, hemoglobinuria and jaundice.
• Infectious ( Babesia, and Bacillary hemoglobinuria),
• Toxic (copper poisoning) and

• Ag/Ab reaction (hemolytic anemia of new born).

iii. Aplastic/hypoplastic anemia: inability of the bone marrow
to produce red cells.
 Nutritional deficiency: - protein, minerals and vitamins
results in mild to moderate anemia.
 Erthropoietin depression 2ry to other diseases (chronic
renal failure, hormone deficiency & chronic debilitating
disease)
 Neoplastic infiltration of the bone marrow
 True aplastic anemia (irradiation, bracken poisoning &
estrogen)
1.2. Terms to describe morphology and color of RBC
• Macrocyte: large RBCs (usually polychromatophilic)
• Microcytic: small RBC (sign of bone marrow problem)
• Anisocytosis: variation in the size of RBC
• Normocytic: RBCs with normal mature size
• Hypochromic: Very pale colored RBC (low level of Hb)
• Annulocyte: extreme form of hypochromic RBc
• Polychromatophilic: red – immarure RBCs (blue stain)
• Normochromic: Normal uniformily red colored RBCs
 1.3. Evaluation of erythrocyte (RBC)
I) Blood film preparation and morphological evaluation
• Prepared from whole blood or after centrifugation of blood
• Detection of cellular abnormality, evaluation of
reticulocytosis
-Wet film (motility of parasite)
-Thin film (differential WBC count, morphological
evaluation of RBC and estimation of platelets)
-Thick film
 The films are stained with one of the Romanovsky type
stain (Giemsa stain, Wright’s stain, Diff Quick stain etc.)
II) Determination of Hemoglobin (Hb) concentration

• Provides the direct indication of the O2 carrying capacity of blood

and approximately 1/3 of the PCV under normal condition.

a) Acid hematin method

- conversion of hemoglobin in to acid-hematin using HCl and

matched in the colorimeter (Sahil’s hemoglobinometer).
It is subjected to error because of
• the presence of non hemoglobin protein (myoglobin),
• difficulty to match with the glass standard

• the subjectivity of matching the colors,

b) Direct matching:
• Tallquist hemoglobin scale- In this case white absorbent
paper is used & paper with drop of blood is inserted in to a
serial color chart and the intensity or redness is matched.
This method is simple and inexpensive but less accurate.
• Dare hemoglobinometer. Drop of blood in capillary
chamber b/n small glass plates matched with permanent red
glass standard.
 c) Cyanomethemoglobin method-
• 5ml of diluents (cyanomethemoglobin reagent) is added in to
0.02 ml of blood in cuvette.
• After mixing the cuvette is placed in spectrophotometer for
reading.
• Percent transmission or optical density at 540 µm is recorded and
compared with reading obtained from cyanomethemoglobin.
• This method is more accurate than the other methods.

The concentration of hemoglobin decrease with anemic

condition and nutritional deficiencies such as iron, copper,
vitamin, protein deficiencies, etc.
 III) Hematocrit /Packed Cell Volume(PCV)

• the fraction of the blood volume which is occupied by the

RBC expressed in percent
• The centrifugal force will derive the blood to separate in to
three distinct layer based on their differential weight and
density
• These are the plasma, the buffy coat and PCV (packed cell
volume)
 IV) Erythrocyte (RBC) count
• Electronic counters (hematology analyser) provide more rapid and
accurate results.
• Direct visual counting is of limited value because of the large error
involved.
• For manual counting the red cell pipette gives a dilution of 1:200
when blood is drawn to the 0.5 marks and the diluents (hayem’s
solution) to the 101 mark.
• The diluted blood is then discharged to the hemocythometer chambers
and counted.
• The inherent error is about ±20%.
• The number of RBCs decrease in anemic condition and increase in
polycythemia.
V) Determination of erythrocytic (wintrobe) indices
• The decrease in one or more of the above three peripheral erythrocyte
parameters can tell us the presence of anemia but not the type of
anemia
i) Mean corpuscular volume (MCV)
• It represents the average volume of a single RBC in fimtoliter.
• determined most accurately by direct measurement using electronic
cell counters.
• indirectly by dividing the HCT by the total RBC count and
multiplying it by 10.
• varies with species; mammals have small RBC than birds, reptiles and
amphibians.
• Species with large RBC have lower RBC count but the same PCV,
due to this birds, reptiles and amphibians have higher MCV as
compared to mammals.
• Higher MCV in mammals is usually associated with
regenerative anemia (presence of large sized immature RBCs).

• Such anemia results from hemolytic disease (including

babesia, bacillary hemoglobinuria, trypanosomosis) and
myeloproliferetive disorders such as feline leukemia.

• Lower MCV occur under Fe and Cu deficiency which is

common in chronic hemorrhages (microcytic anemia) but it
takes more than one month for MCV to be lower than normal
(the body iron reserve).
 ii) Mean cell hemoglobin concentration (MCHC)

• It represents the average concentration of hemoglobin with

in erythrocytes.
MCHC=Hb Con. / HCT (PCV) X 100 in g/dl
• most accurate indices because RBC count is not included.

• It reflects the O2 carrying capacity of erythrocytes.

• MCHC decrease in regenerative anemia especially in stress

reticulocytosis and chronic iron deficiency.
• Such anemia is termed as hypochromic anemia.
 (iii)Mean corpuscular hemoglobin (MCH)

• The average weight of hemoglobin in a red blood cell is me
asured by the MCH. 
• The formula for this index is the sum of
the hemoglobin multiplied by 10 and divided by the RBC. 
• MCH values usually rise or fall as the MCV is increased or
decreased.
 So depending on the above indices
anemia can be classified as:
• Macrocytic hypochromic

• Macrocytic normochromic
• Microcytic normochromic

• Microcytic hypochromic
• Normocytic normochromic
D/t d⁺ that cause the d/t types of anemia are given as follows.

Normocytic normochromic

• acute hymolysis before reticulocyte response

• Acute hemorrhage before iron depletion - Chronic renal disease

• Chronic inflammation and neoplasia - Vitamin B12 deficiency

• Early iron deficiency -Aplastic & hypoplastic bone marrow

Macrocytic hypochromic - Regenerative anemia (hemolysis)

Microcytic normochromic- Regenerative anemia, Folate deficiency

Microcytic hypochromic/ normochromic-

• chronic Fe deficiency, anemia of chronic disease, Cu deficiency

Evaluation of Leukocyte and their Disorder
• The term leukocyte includes all WBCs & their precursors.

• Leukocytes are responsible for protecting the body against

foreign invaders.
• Unlike RBC, leukocytes are capable of independent movement.

• Their life span ranges from few hours to several years,

depending on the type of leukocyte.
• They are produced in several locations. The steam cells are
located in the bone marrow, where the most leukocytes are
produced
• however, lymphocyte steam cells colonize lymphoid tissue
2.2 leukocytes/ white blood cells (WBCs)

• Most f the WBCs complete their lives and die at the site of activity,

• lymphocytes are capable of recirculating, by moving in to the

lymphatic vessels and then entering back to the general circulation.

• The WBCs are categories in to granulocytes and agranulocytes

• neutrophils, eosinophils, basophils and monocytes are provide non-
specific immunity.

• A neutrophils and lymphocytes are the most numerous WBCs in body.

• In pets and equines about 70% or more of the WBcs are neutrophils

while about 65-75% of the WBCs in runimants are lymphocytes. But

they are nearly equal in swine.

• Evaluation of the leukocyte is rarely used for diagnosis of
specific disease, but the information gathered from the
leukogram is useful for:

- Narrowing the differential diagnosis

- Predicting the severity of the disease

- Monitoring the response to treatment

- Formulating prognosis
 Evaluation methods
1) Total leukocyte count

• WBCs count from stained blood smear

• Multiplying the result by 1500 to get the correct number of

cells per microliter.

• The most accurate method of enumerating leukocyte is the

Automated methods and Manual counting using

hemocytometer
 Interpretation

Leukocytosis is an increase in total leukocyte number.

A) Physiological: Such response usually occurs in frightened animals
as a result of release of epinephrine which acts on the spleen
smooth muscle, pulmonary and lymphatics.

B) Stress response: Under stress, animals release high concentration

of corticosteroids which cause

• ↓ adherence of neutrophils to the endothelium of capillaries

• ↑ mov’t of neutrphils from marginal pools to the central pool
• ↑ release of mature neutrophil from bone marrow to the peripheral
circulation
• These all conditions will culminate in neutrophilia ,
Monocytosis, eosinophilia and lymphopenia

C) Acute generalized infection: Leukocytosis marked by

netrophilia, monocytosis and change in other WBCs
D) Leukomoid response: characterized by marked (extreme)
elevation of leukocyte number. It is similar to leukemia.
• Leukomoid response occurs in disease conditions that provide
continuous stimulus to bone marrow precursor cells.
 Examples = suppurative inflammation in closed cavities,
acute coliform mastitis, etc.
Leucopenia: A decrease in the number of leukocyte.
This can also caused by a number of factors:

Leukocyte destroying viruses;

Overwhelming bacterial infection (foal salmonellosis,

septicemic pasteuroloiss, peracute black leg);
Shock;
Bone marrow hypoplasia (by drugs like CAF,
barbiturates).
 2. Differential leukocyte count

• used to determine the number of each WBC type in a specimen. 2

types
• The relative differential count is determined from stained smears on
which at least 100 cells are counted and classified. For greater
accuracy enumeration of 200 consecutive leukocytes can be counted.
• The absolute differential count provides a more consistent
evaluation of the number of each type of leukocytes present.
ADC = TLC * RDC which expressed as a decimal.
• It is the absolute differential count which is used for interpretation of
the result of count.
Differential count method in a stained blood smear
=Battleship method
1. Neutrophils (segmenters, polymorphoneuclear cells,
PMNs)
• They are responsible for short term phagocytosis especially
during initial stage of acute bacterial infection.
• Neutrophils are released from bone marrow in an age
related order.
• Segmentors are the first to be released from bone marrow
for phagocytosis
• When these are exhausted, cells from maturation pool
(band cells) are released.
• The intravascular phase has the circulating pool and the
marginal pool.
• The circulating pools are neutrophils that are found in the
main stream and are collected during vein puncture.

• The marginal pool neutrophils are those w/c are skimming

along the endothelial surface of the capillaries and they are
the first from the intravascular phase to tissue phase.
 Interpretation

Neutrophilia:
• Physiological neutrophilia: (release of epinephrine in frightened
animals)
• Inflammatory neutrophilia: (acute infection)
• Steroid-induced neutrophilia: (exogenous and endogenous
corticosteroids)
Neutropenia:
• Inflammatory neutropenia: as a result of overwhelming bacterial
infection
• Destruction (Canine distember, CAF)
• Shock due to movement of neutrophils from CNP to MNP
Qualitative disorders of neutrophils (left and right shift and toxic)

• Left shift indicates the presence of neutrophil precursors in

blood, typically increased numbers of band neutrophils and
rarely metamyelocytes and earlier precursors.
• The two types of neutrophilic left shift are:

1) Regenerative left shift

 the proportion of various immature neutrophils is orderly
( pyramidal distribution)
– with the most immature cells being the least numerous.
– Usually the mature neutrophils outnumber the immature cells.
They indicate acute disease with good prognosis. It
could also be
 slight left shift (neutrophilia that contain < 2% bands)
 moderate left shift (neutrophilia with some bands &
metamyelocytes)
 marked left shift -neutrophilia with band cells,
metamyelocytes and myelocytes).
2) Degenerative left shift
• young neutrophilic granulocytes exceeding mature ones.
• It reflects the inability of the bone marrow to respond to the situation.

3) Right shift
• increased numbers of hyper segmented neutrophils, with five or
more nuclear lobes.
• Hypersegmentation of neutrophils usually occurs as an aging
change, when neutrophils circulate in blood for a longer time.
– Glucocorticoid therapy or hyperadrenocorticism(inhibit the emigration ).

– vitamin B12 maladsorptive disorder, myelodysplasia , idiopathic neutrophil

hypersegmentation in horses
 4.) toxic neutrophils

 The presence of toxic neutrophils indicates toxaemia-

 the presence of toxins in the blood stream that cause morphologic

change to the cells.

Toxic neutrophils are characterized by

• Dohle body (gray mass in the cytoplasm),

• cytoplasmic basophilia,

• toxic granulation (large eosinophilic granules) and

• vacuolization.
 Monocytes
They are the only phagocytic cells of mononuclear cells.
Approximately they comprise about 5-10% of the blood leukocyte under
normal condition

Monocytes are immature cells of these series that are released from bone
marrow to the circulation.
When they move to tissue they become: Macrophages, Epitheloid cells
and multinucleated giant cells.
The macrophages form the part of phagocytic reticuloendothelial system.

they are phagosytic in chronic bacterial infection and against large

pathogens such as fungi.
 They also release important inflammatory mediators (interferons,
cytokines and prostaglandins).
As part of adaptive immune system they are responsible for antigen
presentation.
Monocytosis: Increased number of monocyte in the circulation. Such
response occurs when there is demand for phagocytosis
1) inflammation & infection (TB, Brucellosis, Listeriosis),
2) Tissue necrosis 3) monocytic leukemia.
Monocytopenia (decreased number of monocyte in the circulation)
• can occur sometimes but it is difficult to demonstrate because the
number of these cells in circulation is low.
Eosinophils

Eosinophils make up about 5-10% of the leukocyte in any species of

animal.

They are abundant mainly in tissues that have direct interface with the
exterior such as skin, lungs, GIT and urogenital tracts.

Their primary function is anti-inflammatory role and also mediate

resistance against large metazoan parasites such as helminths.
Eosinophilia : An increase in the number of eosinophil. It is seen in
animals with allergic conditions (food allergy, feline asthma, equine
bronchitis, etc), parasitic infection and in eosinophilic granuloma.

Eosinopenia is not a common clinical finding.

 Lymphocytes

Lymphocytes are the second most common leukocytes in small animals &
horses and the most abundant ones in ruminants.
The 10 function of lymphocytes is to provide adaptive immunity, by creating
a specific defense against specific pathogens.

They produce two broad types of adaptive immunity - B and T-lymphocytes

Lymphocytosis: A relative lymphocytosis can occur if something causes a

neutropenia.

Lymphocytosis may occur to an immune response (following vaccination &

in recovery stage of viral infections)

Marked lymphocytosis can occur in lymphosarcoma.

Lymphopenia: may occur due to acute viral infections, corticosteroids,

irradiation and relative decrease can also occur if there is neutrophilia.
Platelets/Thrombocytes and the coagulation factors
For a mechanically strong haemostatic blood, it is necessary to have
both a sufficient number of functional platelets and a complete set of
coagulation factors.

1 ) Normal running maintenance of the endothelium

In severe thrombocytopenia the endothelium becomes weak and red
cells may actually escape through the walls of intact uninjured
capillaries
Petechiation, ecchymoses and even spontaneous haemorrhage then
result.

2) Repair of damaged endothelium

 Platelet abnormalities
1) Thrombocytosis: platelet count greater than 500x109/l
Myeloproliferative disorders
Postsplenectomy
Reactive (postsurgical, post hemorrhage, anemias)
Inflammatory disorders
Malignancies (Megakaryocytic leukaemia)
2) Thrombocytopenia: platelet count less than about 200x109/l in most
species
Functional- early stage of heamorrhage
Thrombocytopenic purpura, DIC
Autoimmune
Primary bone marrow suppression
Lymphosarcoma,(bone marrow neoplastic)
CLINICAL ENZYMOLOGY / PLASMA ENZYMES IN
DIAGNOSIS
 1. INTRODUCTION
Diagnostic enzymology is concerned with the study & application of
plasma enzyme activity, & the evaluation of response to Rx or progress
of diseases.
Enzymes are biological catalysts responsible for supporting almost all of
the chemical rxns that maintain animal homeostasis
Key element in the clinical diagnosis and therapeutics
All enzymes are proteins except ribozymes which are ribonucleic acids.
Low levels of all enzymes normally appear in the plasma, reflecting the
balance b/n the release of enzymes during normal cell turnover, and their
catabolism or excretion.
This helps the utilization of enzymes for diagnosis purpose.
 2. ENZYME MEASUREMENT
Method of enzyme assay measure either:
 the rate of appearance of the product
 the rate of the disappearance of the substrate
 the rate of change in concentration of co-enzymes.
Plasma contains two types of enzymes: plasma specific enzymes and
non-plasma specific enzymes
The level of non-plasma specific enzymes increase in the plasma due
to varies factors while the level of plasma specific enzymes can
decrease or increase
a) Increased concentration in plasma enzyme
- rupture or necrosis of organs or tissues
- cellular proliferation (less extent)
- impaired enzyme excretion
The usefulness of serum enzymes as an aid to diagnosis is dependent
up on the organ specificity of the particular enzyme
b) Decreased concentration of plasma enzyme

Much less frequently used for clinical interpretation

In many cases low enzyme findings means the sample has been
badly stored (most enzymes are fairly labile, especially without
refrigeration).
However, there are a few specific cases where low plasma
enzyme levels will indicate that the relevant organ is hypoplastic,
atrophied or destroyed

hence the normal evidence of cell turn- over is absent.

The commonest example of this is immunoreactive trypsin (IRT)
in exocrine pancreatic deficiency
3. LOCALIZATION OF DAMAGE
Very few enzymes are specific for only one cell type.
Many enzymes are actually present in nearly all cells, at different
levels,
most enzymes have 2 or 3 d/t tissues in w/c they are particularly
abundant.
Specificity can be improved in two ways:

(1) Isoenzyme determination

Where an enzyme is particularly prominent in more than one tissue it
is often the case that each tissue has its own particular isoenzyme
(2) Estimation of more than one enzyme
Relative concentrations of enzymes are seldom exactly the same in
two tissues and the damaged tissue can often be pinpointed by
estimation of several enzymes
4. ENZYMES OF CLINICAL IMPORTANCE

a) Creatinine Kinase (CK)

• Excreted from the body along with urine
• present only in muscle tissues and brain.
• Most commonly used for diagnosis of muscle dystrophy and
cerebral necrosis.
b) Alanine aminotransferase (ALT)
• present in high conc. in the liver and to lesser extent in muscles
and kidney.
• In dogs and cats it is virtually used for diagnosis of hepatic
damage.
c) Aspartate aminotransferase (AST)
• widely distributed in the liver, skeletal muscle and erythrocytes.

• The presence of AST in many tissues precludes the use of these enzymes as an
organ specific enzyme
• but it can be used in conjunction with other enzymes like LDH and CK.

d) Alkaline phosphotase (ALP)

• found in the liver, bone and intestinal wall.

• located on membrane of a various tissue but only two are diagnostically

important; hepatobiliary tissues and bones.
• Hence its activity increases in plasma when there obstruction of biliary tract,
liver damage and drug administration;
• under condition of rickets, osteomlacia, hyperparathyroidism etc. each issue has
its own iso-enzyme that can be identified by electrophoresis.
e) Gamma Glutmyltransferase (GGT) –

• most cells have some GGT activities ranging from kidney with highest
activity and muscle with the lowest activity while liver is between.

• Most GGT activities observed in serum originate from liver while GGT
from kidney do not appear in serum rather they are found in urine

• The greatest magnitude of increase in serum GGT is due to obstruction

of hepatobiliary tract and drug (in herbivores).

f) Pepsinogen

• found in abomasums /stomach(diagnosis and monitoring of gastric

damage due to parasite like Hemonchus, Ostertagia, etc).
g) Lipase
• pancreas specific enzyme.
• Its level in plasma increase when there is damage to pancreas as in
case of acute pancreatitis.
h) Alpha- Amylase
• pancreatic enzyme while some amount produced by salivary glands.
• execrated by kidney along with urine. Dx of acute necrotizing
pancreatitis.
i) Lactate Dehydrogenase (LDH)
• The highest activity of LDH is found in RBC and then followed by
plasma.
• It is also found in lungs, liver and muscle tissues.
• Its level can increase in hemolysis, liver damage, lung damage and
muscle dystrophy. 
 f x
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 Quiz ???????

Name _______________ IDNo._____________ Year

* Answer the following questions accordingly?

1. Write at least 3 enzymes used for clinical enzymology .

2. What is the difference between serum and plasma?

3. From serum and plasma, which one used for enzyme examination?

Why serum/plasma used over serum/plasma for enzyme evaluation?

 LABORATORY INVESTIGATION OF HEPATIC DISEASES

Pathophysiology of hepato-biliary system

• Liver performs a number of functions:

- Formation of animal starch (glycogen)

- Secretion of bile
- Detoxification of poisons

- Breakdown of uric acid and formation of urea

- Desaturation of fatty acids
• The liver is made up of; Hepatocytes (60%), Kupffer cells, Lipocyte,
Endothelial cells and Large granular lymphocyte
• Damage of both hepatocyte and other cells of liver can occur and cause
disturbed hepatic functions
• The damage of hepatocyte and biliary system is more common
• The hepatobiliary system is suited for accomplishment of Interediatery
metabolic role and clearance of toxin.
• The liver has 3 anatomical zones. Blood enters the liver through portal
traids and flows sequentially through zone 1, 2 and 3.
• There has been remarkable metabolic diversity among these hepatic zones.
• For example: Zone 1 Albumin synthesis, gluconeogenesis, cholesterol
synthesis, synthesis of coagulation factors
• Liver is an organ of urea cycle-conversion of intestinal derived ammonia
to urea nitrogen
• The other important function of liver is bilirubin excretion
 Bilirubin that obtained from hemoglobin during break down(senescent) of
RBCs is bound to albumin and transported to liver
• Whether it is due to hemolysis, hepatocellular damage or biliary
blockage, the level of bilirubin in the blood increases and causes
jaundice.
• Jaundice occurs mostly in tissues that have high amount of elastic fiber
such as scelera of the eye

All this function of the liver can be altered under disease conditions.

1. Primary hepatic diseases (those that directly cause injury to the liver
tissue or cell)

2. The rxn of liver to extra hepatic diseases(metabolic disturbances,

disorders in CVS, obstruction of bile flow )

The abnormalities can be either morphological or physiological that can

be assessed by various tests
 Terminology

• Jaundice (icterus)- yellowish discoloration of the tissues due to

deposition of bilirubin
• Cholestasis- bile without motion

• Hyperbilirubinemia- increased circulating levels of bilirubin in the blood

 In individual animal disease, initial laboratory evaluation of hepatic
disease depends on biochemical analysis.
 Biochemical tests, (liver "function" tests) can detect 3 basic processes that
can occur in liver disease:

- hepatocellular damage
-cholestasis
-reduced functional mass.
Liver function tests (Hepatic tests)

• Liver function tests are performed

a. to provide differential diagnosis of jaundice

b. to assess the prognosis /progress / response to therapy
c. to evaluate the involvement of the liver in disease condition

Serum hepatic enzyme assay

• Serum hepatic enzymes are grouped into 2, as leakage and over production
enzymes.
• Leakage enzymes are found in higher amount in the cytoplasm of the hepatocyte
and their presence in the serum indicates damage to hepatocytes,
• Over production enzymes are those which are found in the serum in small amount
under normal condition and their presence in the serum indicates excess production
1) Hepatocellular Damage (Degeneration and/or Necrosis)

a) ALT (Alanine amino transferase)-for hepatocellular damage in

dog and cat; combine with CK for certain muscle diseases

b) LD (lactate dehydrogense) and AST (Aspartate transaminase)-

They are not liver specific enzymes but can be used to detect
hepatocellular damage if other organ diseases can be ruled out
(especially muscle).
c) OCT (Ornithine carbamoyl transfarase) - Liver specific in most
species. It is rarely used except in swine.
2. Cholestasis

• reduced excretion of bile which may occur due to hepatocellular

damage or due to physical obstruction of intrahepatic or extrahepatic
bile canals.
• Most cases of hyperbilirubinemia due to cholestasis are accompanied
by high enzyme levels (ALP and GGT )
 Bilirubin metabolism and analysis
• When analyzing bilirubin metabolism; serum bilirubin, urine bilirubin, and faecal

bile pigments can be measured

• Serum bilirubin levels visually assessed by icterus index technique

• Accurate assessment of serum bilirubin requires the Van den Bergh's test.

Traditionally, icterus [hyperbilirubinemia] has been divided into 3 types:

– Haemolytic - excess production due to haemolysis of erythrocytes.

– Hepatocellular - is partly due to non-cholestatic mechanisms (reduced cell

conjugation) and partly due to intrahepatic cholestasis (reduced cell excretion due to

lack of energy and blocked bile canaliculi).

– Obstructive - principally refers to post-hepatic obstruction of bile flow or obstruction

to the larger bile ducts. The jaundice is completely due to cholestasis, at least in the

earlier stages
 Tests for reduced functional hepatic mass

a) Bile acids-levels of bile acids can be used to check for a wide variety of liver

related diseases

eg. hepatocellular damage, cholestasis, and reduced functional hepatic mass

b) Blood ammonia determination-Elevated blood NH3 may be due to:

i) Terminal hepatic insufficiency

ii) Circulatory bypass of the liver (intrahepatic or extrahepatic shunting)

iii) Urea cycle enzyme deficiency

iv) Shock

c) Serum protein estimation-useful in detecting a chronic hepatopathy.

-total serum protein and serum albumin, with a derived value for total globulins
 LABORATORY EVALUATION OF URINARY TRACT
DISEASES
• Composed of nephrons; basic structure involved in the filtration,
secretion, and re-absorption of materials in the body
• Each nephron consists of a glomerulus, proximal and distal tubules
and collecting duct

The main functions of the kidney

1. Filtration and Re-absorption
2. Synthetic functions (Erythropoetin)
3. Metabolic functions (Break down of several hormones )
4. Excretory Function (major function of kidney) - All toxic end
products of metabolism are eliminated through the kidney
• The rate of formation of the glomerular filtrate within a nephron (i.e.
the glomerular filtration rate - GFR) is dependent on 2 factors:
a) Effective filtration pressure in the glomerulus
b) Renal blood flow
• The renal function can be affected by:

1. Pre-renal – factors related to increased or decreased rate of blood

flow to the kidney
2. Renal- factors affecting the vascular system of the kidney (e.g.
Infection)
3. Post- renal –factors related to obstruction of urine flow (tumor,
renal calculi)
 Terminology

• Azotemia: the presence of excessive amount of nitrogenous compounds in the

blood
• Uraemia: a clinical syndrome occurring in kidney failure characterized by a
variety of systemic and neurological signs.
• Polyuria: the passage of an excessive quantity of urine
• Oliguria: a reduction in the quantity of urine produced
• Anuria: suppression or arrest of urinary output
• Isosthenuria: Continued inabilities of the kidney to produce either concentrated
or dilute urine
• Hyposthenuria: the constant secretion of urine of low specific gravity
• -uria: a combining form denoting either condition of urine or the presence of a
substance in urine e.g. hematuria, crystalluria, bacteriuria
• Cylindruria: the presence of casts in the urine
• Pyouria: the presence of increased leukocytes (’pus’) in the urine
 Some of the laboratory tests used to detect renal
dysfunction
1) Assessment of glomerular function
a) Clearance tests
b) Blood concentration levels
-Blood urea
-Blood creatinine
c) Urinalysis
2) Assessment of tubular function
i) Blood urea level
ii) Tests based on water elimination and reabsorption (urine
concentration tests)
iii) Aberrations of acid/base balance
iv) Urinalysis (best to assess tubular function)
 Urinalysis involves the following procedures and should be done
completely in the first instance.
i) Observation of physical properties(volume, color & odur )
ii)Estimation of solute concentration (specific gravity )-refractometer

iii) Chemical analysis (Protein (reagent strips and Robert’s test),

Glucose (reagent strips and benedict test), Ketones (Ross test and
reagent strips), Bilirubin (foam test or Ictotest / the bilirubin strip ),
pH & Blood (haematuria, haemoglobinuria or myoglobinuria) )
• iv) Sediment examination (Epithelial Cells, ) Erythrocytes,
Leukocytes, Casts, Lipid droplets, Spermatozoa, Fungi, Bacteria and
Crystals
 LABORATORY INVESTIGATION OF SOME DIGESTIVE
TRACT DISORDERS
• Malassimilation: Collective term for maldigestion and malabsorption
• Maldigestion: the failure of normal digestive processes
• Malabsorption: the failure of normal absorptive processes
• Diarrhea: increased frequency of defecation and volume/fluid
consistency of the feces
• Melena: the black colored feces altered by blood. Usually indicate
small intestinal bleeding
• Steatorrhea: increased amounts of fat in the feces
• Creatorrhea: increased amounts of nitrogen (protein) in the feces. In
dogs and cats it is usually detected by increased amounts of undigested
muscle in the feces
• Amylorrhea: increased amounts of starch in the feces
• Hyperlipidemia: is an increase in serum or plasma lipids
(triglycerides, cholesterol or phospholipids)
 Rumen function
• In Rts carbohydrate are source of energy. Sugar, starch, and fiber are
digested by rumen microbes, converting them to VFA
• Fiber digesting bacteria growth is favored with a pH from 6.0-6.8
while starch digesting bacteria growth is favored by a pH from 5.5-
6.0
• Several factors impact change in rumen pH:-
-The type of diet can shift pH
-Physical form of feed
-Level of feed in take
-Wet ration can reduce rumen pH
-Adding unsaturated fats and oil can reduce rumen pH
 Rumen disorders

 Indigestion: dysfunction of the reticulorumen which can be due to:

• Abnormal motor function of the reticulorumen (TRP, bloat, ruminitis)

• Fermentative disorder (biochemical /microbial)

• Incorrect diet, prolonged starvation, or inappetence (secondary to

systemic infection) and hyper acidity; all impair microbial digestion.

• The bacteria, yeasts, and protozoa also may be adversely affected by

the oral administration of drug that are antimicrobial or alter the pH.
 Rumen function test
a) Physical examination of the rumen fluid
-Color (varies depending on the nature of the feed)
-Consistency (Normal rumen fluid has slightly viscous
consistency)
-Odor (The rumen fluid has typical odor = aromatic under
normal condition)
-PH (Physiologic rumen fluid range b/n 6 & 7 in animal mostly
on forage diet but lower in animals kept on grain diet (5.5-6.6))
b) Sedimentation test= The sedimentation activity time provide a
rapid evaluation of micro-flora activity (4-8 minutes)
c) Titrable acidity (measurement of rumen pH; 0.1 N NaOH
and phenophtaline is used).
 Under normal condition it ranges from 8-25 clinical units

d) Cellulose digestion time (to assess the proper function of

cellulytic bacterial populations)
e) Microscopic examination (normal number is 6-7 per field)
f) Evaluation of VFA pattern (When evaluating VFA
patterns, the ratio of acetate to propionate reflects the
rumen fermentation pattern (> 2.2 to 1)
 Tests for exocrine pancreatic disorders

• Pancreatic disorders can be divided into acute pancreatic necrosis

(necrotizing pancreatitis) and pancreatic insufficiency
• Pancreatic necrosis commonly presents as an acute abdominal
problem while pancreatic insufficiency is a major cause of
maldigestion
 To confirm acute bouts of pancreatic necrosis

a) Elevation of serum enzymes = elevations of amylase and lipase

often occur in pancreatic necrosis
b) Other supportive laboratory findings for acute pancreatic necrosis
-Hematology, hyperlipidemia and pre-renal azotemia
 Investigation of diarrheas

1. Fecal examination (coprology)

i) Gross characteristics (odor, color, consistency, volume and presence of
unusual material)
ii) Microscopic examination

iii) Measurement of Trypsin in feces and Trypsin-like Immunoreactivity

(TLI) in serum for exocrine pancreatic insufficiency (EPI).
iv) Fecal culture
2. Peritoneal fluid analysis – abdominocentesis
3. Absorption tests
4. General haematological and biochemical testing
5. Endoscopy, exploratory laparotomy, intestinal washings and biopsy
BODY FLUID ANALYSIS AND DIAGNOSTIC
CYTOLOGY
 Body Fluid Analysis
1. Body cavity effusions
• Mainly referring to abdominal and thoracic fluids but there is
applicability to pericardial and other cavities
• Effusions is a pouring out of any fluid into a body cavity or
tissue. i.e excess fluid
Effusions can be divided into:
i) Pure transudates
• hypoproteinemic states due to lowered plasma osmotic pressure
and in some forms of chronic liver disease
• body fluid in excess (therefore low protein and low cells).
ii) Modified transudates
• These are still non-inflammatory in origin

• They are modified by the addition of protein or cells (limited compared

to exudate)
• Chronic heart failure → thoracic & abdominal effusions that quickly
pass from pure transudates to modified transudates(RBC are common)

iii) Chylous effusions

• Most commonly occur in the thorax and are assumed to be due to
leakage from major or minor lymphatics.
• Chylous effusions are Xized by many small lymphocytes and high
triglycerides.
iv) Exudates (high protein and total nucleated cell numbers)

1) Non-septic exudate
• either develops from a modified transudate or due to direct
inflammation caused by irritants (minor toxic to neutrophils)

2) Septic exudate
• The product of inflammation caused by a wide variety of
microbes that are toxic to neutrophils
• Values for protein and total nucleated cells are high compared to
the other categories.
v) Hemorrhagic effusion (recent or long standing)
• Recent hemorrhage will be Xized by clear supernatant on
centrifugation and platelet clumps on examination of the smear
• the supernatant = red-brown or yellow due to degradation of
erythrocytes.
 smear will show erythrophagocytosis (haemosiderin in
macrophages).
It is important to remember that the basis for the classification of
body fluid effusions depends upon protein estimation, total
nucleated cell count and differential cell count on a smear.
2. Synovial fluid analysis
 Except for septic arthritis (like hygroma), synovial fluid analysis
rarely provides a specific diagnosis
Laboratory evaluation of synovial fluid
i) Normal gross characteristics
- volume ,color, transparency, viscosity, mucin content
ii) Normal cytological characteristics
-A total nucleated cell count is determined from synovial fluid
collected in EDTA.
-A differential cell count is determined from a smear of synovial fluid
(preferably made directly from withdrawn synovial fluid).
iii) Other characteristics (investigated when indicated by disease)
e.g. Protein levels, glucose levels and certain enzymes (e.g. AST,
ALP).
3. Cerebrospinal fluid evaluation
• CSF is produced partly by diffusion from plasma and partly by active secretion from
the choroid plexus and ependymal linings
• In the dog and cat, CSF is collected usually from the subarachnoid space at the
atlanto-occipital articulation. In the horse and dog the subarachnoid space at the
lumbosacral articulation can be used.
i) Physical Examination
1) Color; CSF = colourless. Xanthochromia = jaundice
Red CSF may indicate recent haemorrhage
grey-green CSF may indicate suppuration
2) Turbidity; Normal CSF = clear. A turbid CSF suggests excess cells/ perhaps
protein /bacteria.
3) Coagulation; Normal CSF doesn't clot. Clotting may be due to inflammation or
haemorrhage
ii) Total nucleated cell count
• CSF is collected in an EDTA tube ( risk of clotting) and in a plain
serum tube ( protein estimation).
iii) Differential cell counts

iv) Protein estimation

4. Analysis of airway and pulmonary lesions by respiratory
washes
Respiratory washes (transtracheal aspirates and bronchoalveolar
lavages) for airway and pulmonary disease can be performed in any
animal species, but is commonly done in the dog, cat and horse
8.2. Diagnostic Cytology (Exfoliative Cytology; Cytopathology)

• Diagnostic cytology is the examination of individual cell details for

the purpose of diagnosis and prognosis.
• Although more information can usually be gained from the
examination of tissues (histopathology) rather than individual cells,
Cytology does have distinct advantages over histopathology:

a) Cytological samples are cheaper and easier to obtain

b) Cytological samples are simpler to process and the result quickly
obtained.
c) Agents of disease are sometimes’ easier to detect because of better
resolution at 100 x magnification
 Histopathological examination usually needs to be done to support
the cytological findings (by biopsy or at necropsy), and is the only
way that tissue architecture (including margins of a lesion) can be
examined.

a) Solid tissue cytology

b) Diagnostic cytology as a component of body fluid analysis

 Diagnosing neoplasia by cytology

1. Is the lesion primarily proliferative (growth disorder ); benign or

malignant?

2. Are the predominant cells round, spindle, or epithelial?

3. Can the cell types be subcategorised?

 Cytology sample collection and preparation

1. Cytology

Tissue imprints – from crusted and ulceration skin lesion/impression of

deeper surgical biopsies

Scrapings – scalpel blade or edge of slide is used to gently scrape across

lesion or tissue biopsy

Swaps – for sampling of fistulous tracts, ear canals, and exudates and

for vaginal cytology

Fine needle biopsy (FNAB) – the best and most commonly used method

for sampling proliferative lesions and masses

 Aspiration Vs non-aspiration

• A minuimu amount of sample with in the hub of needle is

adequate
Non-aspiration for sampling vascular masses to reduce
blood contamination
 Preparation of slide

• The aim of slide preparation for cytological evaluation into

achieve a monolayer of well-preserved cells
A. squash preparation – used for FNAB (best)
B. needle/starfish preparation
C. blood smear technique
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