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CLINICAL SIGNIFICANCE &INTERPRETATION OF SERUM

1. SERUM GLUCOSE:-

Clinical Significance

 Glucose determination is mainly useful in the diagnosis of diabetes mellitus in which


blood glucose levels are elevated.
 Other diseases like hyperthyroidism, hyperpituitarism, severe nephritis,
pancreatitis, asphyxia, anesthesia, pneumonia, dehydration and certain hepatic disorders
also lead to hyperglycemia.
 Hypoglycemia occurs frequently as a result of over dosage of insulin or antidiabetic
treatment. Hypoglycemia may also be noticed in conditioned like hyperinsulinemia,
hypothyroidism, hypopitutarism and hypoadrenocorticism.
 Hypoglycemia may also occur in certain physiological conditions like pregnancy,
starvation, severe prolonged exercises and lactation.

2. LIPIDS:-

Clinical Significance

 High values of total cholesterol may be found in diabetes mellitus, hypothyroidism,


obstructive jaundice, nephrotic syndrome, biliary cirrhosis, atheroslerosis, etc.
 Low values of total cholesterol may be found in hyperthyroidism, malnutrition, gaucher’s
disease and acute hepatitis.
 Decreased level of HDL cholesterol lead to increased chance of coronary heart disease
(CHD) while increased levels reduce these chances.
 Lower values of HDL cholesterol and increased ratio of total cholesterol to HDL
cholesterol are taken as risk factor for CHD.

3. PROTEINS:-

Clinical Significance

 High albumin levels may be caused by


o Severe dehydration.
 Low albumin levels may be caused by
o A poor diet (malnutrition).
o Severe burns
o Kidney/Liver diseases

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o Autoimmune diseases, such as systemic lupus erythematosus (SLE) or rheumatoid
arthritis.
o Gastrointestinal malabsorption syndromes, such as sprue or Crohn's disease.
o Hodgkin's lymphoma; Uncontrolled diabetes; Hyperthyroidism; Heart failure.
o Decreased synthesis (malnutrition, malabsorption, liver disease, and other chronic
diseases),
o Increased loss (nephrotic syndrome, many GI conditions, thermal burns, etc.), and
o Increased catabolism (thyrotoxicosis, cancer chemotherapy, Cushing's disease,
familial hypoproteinemia).
 High globulin levels may be caused by
o Diseases of the blood, such as multiple myeloma, Hodgkin's lymphoma,
leukemia, macroglobulinemia, or hemolytic anemia.
o Autoimmune diseases, such as rheumatoid arthritis, lupus, autoimmune hepatitis,
or sarcoidosis.
o Kidney and Liver diseases; Tuberculosis.

 Low globulin may be seen in


o congenital or acquired hypogammaglobulinemic states.

4. BLOOD UREA NITROGEN:-

Normal Value

 Dog : 12-25 mg/dl


 Bovine : 20-30 mg/dl

Clinical Significance

 Decreased level of BUN in


o Protein malnutrition
o Hepatic insufficiency
 Increased level of BUN noticed in
o Physiological factors (Pre Renal )
 High protein diet, dry food and canned food.
 Catabolic break down of tissue as consequence of fever ,trauma, infection
and toxemia.
 Haemorrhage into gastrointestinal tract
 Iatrogenically by administration of certain drugs
 Anything that reduce renal blood flow
 Cardiovascular disease
 Shock (septic or traumatic)
 Factors that reduce net Glomerular filteration pressure
 Hypotension

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 Shock
 Adrenocortical insufficiency
 Dehydration
 Increased protein osmotic pressure
o Renal
 Elevation of BUN occur when approximately 70% of nephron are non
functional
o Post Renal Uremia
 It occurs with obstruction or rupture of excretory pathway of urine
( ureters, bladder and urethra

5. CREATININE:-
Clinical Significance

 The increased level of creatinine indicates functional impairment of kidney. The rate of
creatinine excretion is influenced by GFR and any other abnormality that decrease GFR
result in an increase in concentration of creatinine.
 In the earlier stages, Creatinine clearance test is a sensitive index of impaired renal
function.
 The prerenal factors that increase blood urea have less influences on creatinine
concentration. Hence for diagnosis of renal diseases, serum creatinine is preferred over
urea estimation.
 In addition to renal disease, elevated level of serum creatinine and creatinuria may be
observed in extensive muscle destruction.

6. URIC ACID:-
Clinical Significance

 In man and all primates, uric acid is the end product of purine metabolism.
 In plasma at pH 7.4, uric acid is in the ionized form of monosodium/potassium urate, and
only a minor portion as a free acid.
 Its determination help in differentiating gout from non-gout arthritis.

Increased levels are observed in gout , leukemia, broncho and lobar pneumonia and
polycythemia.

Physiologic changes in serum concentration of uric acid

 About 350 mg of uric acid are daily produced by endogenous synthesis, while about 300
mg/day are taken by food.

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o About 90% of uric acid filtered in glomeruli are normally reabsorbed in renal
tubules. Uric acid is normally excreted from the body via kidneys (80%) and
intestine (20%). About 4.76 mmol (800 mg) are excreted per day.
o Extreme physical exercise induces a significant increase in the concentration of
uric acid.
o Starvation and fat rich diet increase the concentration of uric acid in serum.

Pathologic changes in serum concentration of uric acid

 Increased values of uric acid in:

 coronary artery disease  diabetic keto-acidosis following intravenous


 Down’s syndrome (some cases) fructose
 gout  tissue destruction
 hyperlipoproteinemia  hemolytic anemias
 myeloid leukemia  lead poisoning
 pneumonia  pernicious anemia (especially after
 uremia treatment)primary and secondary polycythemia

 Decreased values of uric acid in


o acromegaly (some cases)
o administration of uricosuric drugs
o Fanconi syndrome
o hepatolenticular degeneration
o xanthiuria.

7. BILIRUBIN:-
Clinical Significance

Determination of bilirubin is of great importance in the differential diagnosis of jaundice.

The following types of jaundice are differentiated according to the concentration of


conjugated and unconjugated bilirubin in serum:

a. Hemolytic (prehepatic) jaundice characterized by enhanced hemoglobin


breakdown with normal liver function;
b. Hepatic (parenchymatous) jaundice due to hepatocyte lesion. Conjugated
bilirubin cannot be secreted to biliary capillaries but reenters the blood instead.
c. Obstructive (posthepatic) jaundice due to intra- or extrahepatic biliary duct
obstruction; and
d. Functional jaundice due to changes in the intracellular metabolism of bilirubin

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CLINICAL SIGNIFICANCE &INTERPRETATION OF EXAMINATION OF URINE

A. PHYSICAL EXAMINATION OF URINE


1. Colour

 The colour of the urine may be any one of the following: colourless, pale yellow, dark
yellow, yellowish-brown, greenish-yellow, red, reddish-brown, brown, green, blue and
milky. The yellow colour of the urine is due to urochrome pigment.
 Interpretation
o Cloudy - Pus in urine
o Dark yellow - Dehydration in vomition, diarrhoea, serous exudations, fever and
deficientintake of water
o Pale - Diabetes mellitus, diabetes insipidus, increased water intake, pyometra
and chronic interstitial nephritis in dogs
o Yellowish brown/Greenish yellow - Jaundice
o Red - Haemorrhage
o Faint pink - Congenital porphyria
o Green - Medication with methylene blue or diazan
o Brown/coffee coloured - Haemoglobinuria /Myoglobinuria

2. Transparency

 The transparency of the urine is tested by viewing it against light in a test tube. Normally,
it should be transparent and clear. But in the horse, normally, the urine is cloudy and
thick due to the presence of mucus and calcium carbonate crystals. So, the sample of
horse urine must be allowed for sometime to settle. The supernatant urine is then used for
other tests.

Interpretation

 The urine is cloudy if it contains epithelial cells in large numbers


o Leucocytes in large numbers as in pyuria
o Large numbers of bacteria
o Mucus in the urine of the horse
o Crystals-calcium carbonate or amorphous urates or amorphous phosphates
o When blood is present, it gives a red or brown smoky appearance
 In dogs, if the freshly voided urine is cloudy, then it is pathological due to the presence of
pus, blood or mucus.

3. Odour

 The odour of the urine is due to volatile acids; it becomes ammonical due to
decomposition.

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Interpretation

o Sweetish odour in diabetes mellitus


o In ketonuria, there is odour of acetone.
o In turpentine medication, the urine may have odour of violet

4. Foam

 Normal urine may foam if it is shaken. In albuminuria foam occurs in plenty.

5. Specific gravity

 This is a measure of the relative amounts of solids in solution in the urine. It indicates the
capacity of the kidney to concentrate the urine by reabsorption. Generally, specific
gravity varies inversely with the quantity of urine. Exceptionally, in diabetes mellitus due
to the presence of sugar the specific gravity is high though the quantity is also high.
Specific gravity is determined by means of urinometer. This consists of a cylinder and
float at the stem of which is scale from top to the neck of the stem.

Interpretation
Increased specific gravity is seen in:

 Acute interstitial nephritis, the specific gravity is elevated due to the inability of the
kidney to excrete water
 Cystitis due to the presence of products of inflammation
 Diabetes mellitus due to glycosuria
 Dehydration, vomition, diarrhoea and reduced feed intake

Decreased specific gravity is seen in :

 Increased fluid intake


 Inability of the kidney to concentrate urine
 Advanced stages of uraemia
 Diabetes insipidus

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B.CHEMICAL EXAMINATION OF URINE

I. REACTION OF THE URINE

 It varies in different species and individuals, depending upon the diet and metabolism. In
herbivores, the reaction is alkaline, where as in carnivores and omnivores, it is acidic.
o Horse - alkaline (pH 8)
o Cattle - alkaline (pH 7.4 to 8.4)
o Sheep - alkaline
o Pig - acidic or alkaline
o Dog - acidic (pH 6 to 7)
o Cat - acidic (pH 6 to 7)
o Man - usually acidic (pH 4.8 to 7.5)
 Reaction is detected using a litmus paper. Acid urine turns blue litmus red. Alkaline urine
turns red litmus blue.

Interpretation

 Acidic urine
o Normal in carnivorous animals
o Nursing calves and foals that are on milk
o Diet with an excess of protein
o Starvation – catabolism of body proteins
o Fever
o Acidosis – both metabolic and respiratory
 Diabetes mellitus
 Uraemia
o Prolonged muscular activity
o Administration of acid salts
 Sodium acid phosphate
 Ammonium chloride
 Sodium chloride
 Calcium chloride
 Alkaline urine
o Normal in herbivorous animals
o Vegetable diet
o Cystitis
o Urine retention – decomposition of urea to ammonia
o Rapid absorption of transudates
o Alkalosis – both metabolic and respiratory
o Alkaline therapy
 Sodium bicarbonate
 Sodium and potassium citrate or acetate
 Sodium lactate
 Potassium nitrate

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o Urine becomes alkaline when the sample is kept at room temperature because of
ammonia formation as a result of decomposition of urea.

II. PROTEIN

Normal urine does not contain any protein

Interpretation

Physiological proteinuria

 It is transient and believed to be due to a temporary increased glomerular permeability as


a result of capillary congestion.
o Excessive muscular exertion
o Convulsion
o Emotional stress
o Ingestion of an excessive amount of protein

Pathological proteinuria

 Pre-renal: The protein originates from non-renal conditions and its loss in the urine is not
due to primary renal disease.
o Bence – Jone’s protein
o Haemoglobinuria
o Myoglobinuria
 Renal
o Increased permeability of glomerulus
o Impaired reabsorption of protein normally present in glomerular filtrate due to
tubular disease
o Blood or exudate of renal origin
 Post renal
o Protein gains entrance to urine after it leaves the renal tubules by contamination
with exudates or blood.
o Marked haematuria
o Inflammatory exudate
 Pyelitis
 Ureteritis
 Cystitis
 Urethritis
 Urolithiasis
 Extra urinary causes
o Blood or exudate from genital tract contaminates urine
o Chronic passive congestion of kidneys

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Neoplasms and emboli

III. GLUCOSE

Normal urine does not contain glucose. Although glucose passes through the
glomerulus, it should be reabsorbed by the proximal convoluted tubules.

Interpretation - Glycosuria

o Emotional stress
o Diabetes mellitus
o Hyperthyroidism
o Acute and chronic pancreatic necrosis
o Hyperpituitarism
o Overactivity of adrenal cortex
o Shock
o Chronic liver disease
o Enterotoxaemia in sheep
o Brain tumors
o Rabies
 False positive reaction for glucose can occur if other reducing agents like antibiotics,
lactose, ascorbic acid, salicylates, morphine, formaldehyde and uric acid are present.

IV. KETONE BODIES

 Ketone bodies are acetoacetic acid, beta-hydroxy butyric acid and acetone. They are not
normally present in the urine. But, when fat metabolism is impaired, ketone bodies
accumulate in the body, giving rise to ketonaemia and then ketonuria results.

Interpretation

Ketonuria

 Ketosis (acetonaemia)
 Diabetes mellitus associated with a hyperglycaemia
 Acidosis
 High fat diet
 Starvation or fasting
 Impaired liver function
 Milk fever
 Hyperfunction of the anterior pituitary or adrenal cortex, excess of female sex hormones

V. BLOOD

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Haemoglobin contains a peroxidase which can liberate oxygen from hydrogen peroxide. This
oxygen produces blue colour in the presence of benzidine

Interpretation

Haematuria

o Acute nephritis
o Nephrosis – marked degeneration
o Renal infarction
o Passive congestion of kidneys
o Neoplasms of kidney, bladder or prostate
o Urolithiasis – renal, cystic or urethral
o Abscess of kidney
o Pyelonephritis
o Ureteritis
o Cystitis
o Trauma to urethra – usually from improper catheterization
o During estrus or postpartum in female due to contamination by uterine or vaginal
discharge
o Severe infection – anthrax, leptospirosis, infectious canine hepatitis
o Chemical poisonings – Copper, Mercury, Sulfonamides and Phenol.
o Thrombocytopaenia
o Sweet clover poisoning
o Parasites - Dioctophymarenale and Dirofilariaimmitis in canines
o Acute vegetative endocarditis and congestive heart failure in canine
 If the blood is seen in the last drop of urine, then the source is bladder. If the urine is red
throughout, the source of blood is kidney. If the first portion of urine is red, then the
source of blood is some urethral lesions.

Haemoglobinuria

 Parturient haemoglobinuria
 Bacillary haemoglobinuria (Clostridium haemolyticum)
 Leptospirosis
 Piroplasmosis or Babesiosis
 Haemolytic disease of the newborn
 Photosensitization
 Severe burns
 Chemical haemolytic agents – sulfonamides, mercury and copper
 Incompatible blood transfusion
 Plant poisoning – Hellebose, Ranmunculus, Ash, Frosted turnips, Colchicum,
convolvulus and other roots
 Myoglobinuria

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VI. BILE PIGMENTS

Normally, bilirubin is present in freshly voided urine. On standing, it is


converted into biliverdin (green colour), bilicyanine (blue) and cholestelin
(violet, red, reddish yellow).

Interpretation

o Moderate to severe hepatocellular damage


o Obstruction of bile ducts, extrahepatic or intrahepatic
 In early hepatocellular damage and haemolysis urine bilirubin may be negative.

VII. UROBILINOGEN

Urobilinogen is normally present in urine. But, it will not be present in the


case of obstructive jaundice.

Interpretation

 Decreased amount or absence of urine urobilinogen


o Obstruction of biliary passages
o Decreased destruction of erythrocytes
o Impaired intestinal absorption
o Nephritis
 Increased amount of urine urobilinogen
o Hepatitis
o Cirrhosis of liver
o Haemolytic jaundice

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 Laboratory evaluation & diagnosis of samples for parasitic diseases
Practical no.1
 PARASITES / SEGMENTS OF HELMINTHS
 Collection
Collect the helminths during postmortem examination, sometimes immature amphistomes and
segments of tape worms can be collected from dung or faeces.
 Preservation
Trematodes: Fresh and live flukes are collected in normal saline. 70% or 90% ethyl alcohol used
as a preservative and fixative.
Nematodes: Nematodes are fixed in 5-10% hot formalin or 70-90% ethyl alcohol. Once the
nematodes are fixed they can be stored indefinitely in glycerin alcohol.
Cestodes: Tapeworms including the scolices (heads) should be placed in water at about 37ºC for
about one hour and then stored in a mixture of 5% glycerine and 70% alcohol or 5-10% formalin.

A. DIRECT EXAMINATION
1.Macroscopic Examination
Macroscopic examination of faeces is made for consistency, colour, present of blood or mucus,
presence of adult or larval parasites, spontaneously discharged tapeworm segments, immature
amphistomes and nematodes (roundworms, pinworms, Trichuris etc.).
They can be recognized by direct macroscopic inspection of the faeces, by decantation of faeces
in water in a glass tray or by pouring the faeces mixed with tap water through a sieve (mesh:
0.3mm), which will retain the worms.
These are then transferred into saline in a petridish and can be observed against a dark
background by the naked eye or by using a magnifying lens.
2.Microscopic examination
I. Unstained smear
Procedure
It is possible to demonstrate the eggs or larvae of helminths, by the examination of a thin smear
of emulsified faeces.
Place several drops of saline on a slide with an equal volume of feces
Mix the solution and feces together with a wooden applicator until the solution is homogenous

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Smear the solution over the slide into a thin film
Place a cover slip over the smear
Examine the area of the slide under cover slip with the compound microscope.
The slide is investigated using a magnification of 10x and 40x.
Merits
This method is quick.
Minimal equipment is needed.
Demerits
It is effective only when the concentration of parasite stages is high.
It is frequently difficult to identify them since they are partially covered by debris.
Quantitative results cannot be obtained.
II. Stained smear method
a. Iodine violet mount
b.Lactophenol cotton blue method
 Parasites found with the direct smear method:
Coccidia and helminth eggs (only when high numbers are present); Cestodes and trematode eggs
(mainly in birds).
For intestinal schistomiasis / portal schistosomiasis
Collect the dung with mucus.
Place 2-3 drops of 4% KOH or 4% NaOH solution on a slide.
Take a little quantity of mucoid dung and mix it with the alkaline solution on the slide.
Keep the slide for some time so as to allow mucolysis to take place.
Spread the suspension evenly.
Place a coverslip and examine under low power objective of the microscope.
Result
Egg of Schistosoma spindale will appear as spindle shaped / Napolean hat shaped with a terminal
spine at one end.
Egg of Schistosoma indicum appear as elliptical with a terminal spine at one end .
Note

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Be sure the density of the mount is correct. You should be able to read small print through it (but
not too clearly). If it is too thick or too thin, observation of the elements in the mount may be
difficult.

B.SEDIMENTATION TECHNIQUE

Procedure:-
Place a lump of faeces (1/2-1 tsp / 5-10 g/ 5-10 faecal pellets (of sheep or goats) in a cup or glass
container.
Add enough tap water and mix thoroughly with a spatula / glass rod until all the faecal material
is broken down.
The mixture is poured through a wire mesh sieve to remove coarse large lumps. Common tea
strainer will do. The strained fluid is collected in a bowl. The sieve is rinsed with water and the
debris left on the sieve is discarded.
Transfer the suspension to centrifuge tubes and centrifuge at 2000 rpm for 2 min.
Discard the supernatant.
Mix the sediment well and take a small quantity of it and mix it with a drop of water on a clean
slide.
Apply a cover slip and examine under low power objective of the microscope.
Thickness of the smear should be such that if the slide is placed on a newspaper, you should be
able to read the fine print through the smear.
Note
Select a fully representative sample of the stool for concentration.
Prepare well-mixed suspensions of faeces and water or saline.
Use the appropriate quantities of materials.
Use the correct centrifuge speed and time.
Prepare and examine mounts carefully as described for direct wet mounts.
Do not discard the tube containing the concentrated material until you have completed your
examination. You may need to make another mount.
Parasite stages detected with the sedimentation method
Eggs of trematodes
Larvae of lung worms

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Eimeria oocysts
Merits
This is one technique by which the trematode eggs can be recovered. Operculated eggs like that
of Diphyllobothrium latum also will be sedimented.
Demerits
Usually the lighter eggs of nematodes and oocysts of protozoa are missed while employing this
technique. Hence it is not considered by many people as suitable for these types of parasites.
The presence of debris which may pose a problem whie doing examination.

S. Species Host Description of


No eggs
.

1 Catttle,sheep,goat,Hors Oval shape,


e, thick walled,
presence of
operculum,larg
e in size

Amphistomes

2 Cattle, sheep Thick walled,


absence of
operculum,
yellow tinged,
large in size

Fasciola sp

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3 sheep, Barrel shaped
Trichuris sp goat, cattle,pig,dog with a
conspicuous
plug at both
ends,yellow or
brown colour

4 Similar
to Trichuris sp
but with
truncated plugs

Capillaria sp

Interpretations
No egg in the whole sample negative : (-)
0-2 eggs / field : (+)
2-4 eggs / field : (+ +)
4-6 eggs / field : (+++)
> 6 eggs / field : (++++)

C.FLOATATION TECHNIQUE
Separating the eggs from faecal debris by floating them on a variety of floatation solutions such
as
Saturated common salt solution
Sheather's Sugar solution
Zinc sulphate solution (32.5%) .
When faeces are emulsified in liquids of high specific gravity than that of eggs and protozoan
cysts, either centrifuged or allowed to stand, these float to the top while the heavy coarse debris
settles to the bottom.

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The top film can then be removed and examined. Many nematode and a few cestode eggs float in
a liquid with a specific gravity of between 1.10 and 1.20. Trematode eggs, which are much
heavier, require a specific gravity of 1.30-1.35.
I. SIMPLE FLOATATION TECHNIQUE
Simple floatation / Will’s technique
Small floatation tube with emulsion of faeces in the above solutions is filled to its full capacity
till a convex surface is formed and is allowed to stand for 20 to 30 minutes, by which time eggs
would have floated up.
Apply a coverslip or slide to the surface to remove the first drop of fluid, containing eggs and
then examine that drop on the slide.
It is worthwhile to remember that almost all cestodes, except the members of Cotyloda, do not
discharge eggs, but gravid segments containing numerous eggs are shed and passed out.
Hence it is not always possible to find cestodes eggs in faecal samples.
Parasite stages found with the floatation method
Eggs of cestodes and most of nematodes
Larvae of lungworms
Oocysts of coccidia
Trematode eggs are not satisfactorily detected with the flotation method.
MERITS AND DEMERITS OF FLOATATION TECHNIQUE
Merits
Lighter eggs of all nematodes, some cestodes and Oocyst of coccidia can be convinsingly
recovered.
Demerits

It is not possible to recover heavy eggs like trematode eggs using common floatation solution.
Hypersensitivity of floatation fluid tend to disfigure the eggs.so that identification becomes
difficult,hence period spent on floatation shall not exceed the prescribed time.
Examination of slide shall be completed as quickly as possible.

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Practical no.2
QUANTITATIVE FAECAL EXAMINATION

 The estimation of the worm burden or oocyst load can be done by quantitative methods
of faecal examination. This is useful in knowing the worm burden, epidemiology of an
infection and to note the effect of treatment.

Advantage

 Quantitative FE (faecal egg count) is essential for comparing the efficacy


of anthelmintics
 Determining the correct interval between anthelmintic treatments
 Contamination by faeces of grazing animals
 Assessing efficacy of control programmes, Anthelmintic resistance etc.

Disadvantages

 Epg (EPG = egg per gram of faeces) are less valuable in making judgments about the
clinical condition of an individual animal, since many factors affect the level of egg
production; the consistency of the faeces may affect the EPG markedly: the more watery
the faeces are, the more the eggs are diluted; thus, FE is not necessarily proportional with
the size and the extent of pathogenic effect of the actual worm intra-population. The EPG
of tapeworms has only a diagnostic value.
 Nevertheless, egg counts in excess of 1000 are generally considered indicative of heavy
infection. However a low epg is not necessarily indicative of very low infection. Since
one part of worm population may just, have started to reach patency but may already
cause disease.
 There is no simple solution to the problem, how to estimate the intensity of helminth
infection in an unbiased manner, group mean EPG, larvae per gram of faeces (Ipg) or
worm counts are often used as a parameter to express and compare levels and distribution
of helminth infections.

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MC MASTER METHOD

 This technique is used for counting of eggs, larvae or oocysts, which float in a saturated
solution of sodium chloride.

Procedure

 Weigh 1 g of faeces and add 14 ml tap water.


 Homogenize and pour suspension through a 250 µm aperture sieve, collecting the filtrate.
 Collect the filtrate, agitate and fill a test tube of 15 ml volume.
 Centrifuge at 2000 rpm for 2 min.
 Pour off the supernatant, agitate sediment and fill tube to previous level with flotation
solution.
 Invert tube 6 times and remove fluid with pipette to fill both chambers of Mc Master slide
quickly.
 Examine one chamber and multiply number of eggs or larvae under one etched area by
100 or two chambers and multiply by 50 to arrive at the number of eggs per gram of
faeces.

INTERPRETATION OF RESULTS

Interpretation of worm egg counts per g of faeces (EPG) in sheep and cattle (values for guidance
only)

EPG levels indicative of


Helminths intermediate degree of infection

Sheep Cattle

Mixed infection with unspeciated 1000-2000 200-700


gastrointestinal nematodes

Oesophagostomum sp. 1000-2000 200-700

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Bunostomum sp. 300-700 50-100

Haemonchus sp. 2500-8000 200-700

Ostertagia sp., Trichostrongylus sp. 250-2000 100-500

Nematodirus sp. 100-600 50-100

Fasciola sp. 200-500 10-25

 The data is circumstantial and subject to factors such as age, sex and breed of animals,
immunological status, feeding conditions, etc.

STOLL'S DILUTION METHOD ( FOR COUNTING NEMATODE AND TREMATODE


EGGS )

 Three grams of faeces is taken in a test tube (45 ml. graduated)


 Fill the tube upto 45 ml. with N/10 NaOH (4 gms of NaOH, 1 lit. of distilled water) and
add 10-12 glass beads. Close it with a stopper and homogenize the faecal material.
 0.15 ml. of the suspension is drawn with a pipette and placed on a slide and a cover slip is
applied.
 Count the total number of eggs.
 Multiply the number of eggs by 100, which give the eggs per 1 gram of faeces.

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Practical no.3

PASTURE LARVAL COUNTS&SKIN SCRAPPING EXAMINATION

 Introduction
 It is a useful technique for detecting the level of infective larvae (L3) of gastrointestinal
nematodes on pastures. It is not a clinical laboratory technique.
 Collection of materials
 Collect a reasonable number of samples of grass from the pasture and place in polythene
bags.
 Seal the bags and dispatch to the laboratory.
 Procedure
 Soak the grass thoroughly and wash.
 Pass the washings containing the larvae through a sieve(aperture 38u) to remove fine
debris.
 Collect the retained material and identify the infective larvae microscopically under
high power.
 Count the larvae.
 The numbers present are expressed as L3 per Kg of herbage.
 Interpretation
 Where count is in excess of 1000 L3 of gastrointetinal nematode / Kg of herbage -
moderately infective
 Larval count of over 5000 can be expected to produce clilinical disease in young cattle
during their first season at grass.
 Examination of skin scrapping
 Collection & processing
 It is important to remove hair coat by gentle clipping (the hair can be mounted and
examined separately).
 The surface of the skin can then be scraped using a blunt scalpel blade. The scrapings so
collected have to be transferred to a boiling tube, and heated with 10% potassium
hydroxide (KOH). Care must be taken to avoid vigorous boiling.

VLD-411 Page 21
 The fluid is then centrifuged at 1000 rpm for 5 minutes to concentrate mites, which can
be collected from the sediment.
 Discard the supernatent, collect a drop of the sediment, transfer to a slide and examine for
mites.
 Processing on slide if material is scanty
 The emulsion of scanty material (superficial epidermis) is spread over a microscope slide.
 2 or 3 drops of 10% KOH is added to it and mixed.
 It is then warmed for about 3 minutes.
 Covered with a glass coverslip and examined under the microscope.

 This technique can be used to identify surface mites and multiple scrapings should be
taken to increase the likelihood of ectoparasites detection.
 Deep skin scraping (deep epidermal examination)
 This is an extension of the superficial skin scraping technique and should be performed
following or during squeezing of the skin between the thumb and forefinger.
 After removing the superficial layers the procedure is repeated until capillary blood oozes
out. This technique is useful in the diagnosis of burrowing and deep follicular mites such
as Sarcoptes scabiei and Demodex spp. Multiple sites should be scraped to maximize
detection of ectoparasites.
 Results

S.NO Species Host Description

1 Demodex Dog, cattle, Elongated body(cigar shape), short stumpy legs, transversly
spp horse, goat, striated abdomen
sheep, cat and
pig.

2 Notoedres cat, rabbit Round ,smaller than Sarcoptes spp.spines and scales
sp absent.dorsal subterminal anal opening

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3 Sarcoptes Dog, cat rabbit, Body is globose and striate with scaly and spinose areas,
spp pig, cattle, posterior pairs of legs short, not extending beyond body
sheep, goat margin,pedicels bear sucker or bristle and not segmented,
and horse terminal anal opening.

4 Psoroptes Sheep, goat, Oval in shape, all legs extend beyond body margin, long
spp rabbit segmented pedicels with suckers on end of some legs. Anus is in
terminal. Dorsal surface devoid of scales and spines.

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Practical no.4
EXAMINATION OF BLOOD FOR PARASITES

1.Direct blood film (Wet film)


Procedure
Place one drop of blood on a slide, add a droplet of physiological saline, mix and cover with a
coverslip.
Examine directly under low power (10X) of a microscope for live microfilariae. Larvae can be
immobilized by placing a drop of 10% formalin at the edge of the coverslip. This can also be
used for detecting trypanosomes.
2.Indirect wet film Method
Drying and fixing of blood smears leads to some alteration of the morphology of intra
erythrocytic parasites. The present technique overcomes the problem of biconcavity of
erythrocytes so that intra erythrocytic parasites can be visualized because refraction of light
induced by the biconcavity of bovine erthrocytes makes it difficult to appreciate the morphology
of intracellular, unstained parasites. In a hypotonic environment, an erythrocyte swells to its
maximum and becomes a sphere and the parasites and associated structures become visible
within the cells. Stained haemoglobin appears to mask intracellular inclusions associated with
parasites (eg. Theileria orientalis), which are otherwise visible in the wet blood films.
Procedure
A small drop of blood enough to give a single layer distribution of erythrocytes is taken on a
glass slide and mixed with a smaller drop of water using a glass slide.
A clean coverslip is gently placed over the preparation avoiding the formation of air bubbles.
Excess blood is absorbed using a piece of blotting paper.
The smear is then to be examined under oil immersion.
Apart from Theileria, Babesia and Anaplasma also can be detected in a live condition using this
method.
Artefacts can be differentiated as follows
Howell jolly bodies are static and larger than Anaplasma and are seen in immature cells and in
less number of RBC’s.

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Chylomicrons and superimposed platelets appear as bright, translucent objects lodged above or
below RBC’s. They tend to get separated on constant observation due to Brownian movement in
wet medium and are extra cellular.
Erythrocytic pseudopodia appear as bright translucent bodies, lack characteristic movements and
the continuity of the erythrocyte membrane can be made out when the RBC’s move. Such
pseudopodia or crenations occur when samples are dehydrated.
Normoblasts are nucleated and appear as large spherical bodies. These are large and have an
entire margin. Static and wavy cytoplasmic movements of Babesia are absent.
 PREPARATION OF BLOOD SMEARS
Blood smears
It is essential that slides used in making smear preparations be unscratched, non-corroded and
meticulously clean, free from grease, dust, acid or alkali; that slides be handled by their edges;
that the blood be taken as it exudes; that the process be done rapidly so as to prevent coagulation;
and that smears be left to dry in a horizontal position away from flies and dust.
Mark necessary data with wax pencil on the end of each slide. Blood films should be stained as
soon as possible after drying to ensure proper staining.

Preparation of thin blood smear:-


A small drop of blood is applied to the slide approximately 20 mm from one end. A spreader (or
another clean slide) is placed on the slide at an angle of 20-30o and drawn back to make contact
with the blood.
The blood is allowed to run to each end of the spreader. The blood is spread along the slide in a
fairly rapid but smooth motion.
The spreader should be placed at an angle of less that 45o for making the smear. A thin and even
blood smear should result. Wave the slide in the air until it dries (a matter of few seconds if the
smear is thin enough).
If the smear is to be stained in Giemsa’s stain, fix it by dipping in absolute methyl alcohol.
If the smear is to be stained in Wright or Leishman’s stain, fixation is not necessary since it will
take place during the staining process.
If the smear is to be stored for more than a day or so before staining, it should be fixed in methyl
alcohol.

VLD-411 Page 25
While Leucocytozoon, microfilariae and sometimes Trypanosoma can be found with the lower
powers of the microscope, the stained smears should be examined with the oil immersion
objective for other protozoa.
The faster thin smears dry, the less distortion is produced; hence, the most natural appearing
protozoa are at the thin end and around the edges of the smear.
Preparation of thick blood smears:-
Use clean slides as for thin smears. Place a medium sized drop of blood or several tiny ones on
the slide and mix with a toothpick or the corner of another slide.
Allow to dry in air or in an incubator at 37o C; a hair dryer can be used to speed up this process.
Thick smears must be laked (i.e., the hemoglobin must be extracted) before staining. This can be
done by placing them in water until their color has disappeared.
If Giemsa’s stain is used and the smears are fresh, laking will take place during the staining
process. If the smears are to be stored for more than a day or so before staining, they should be
laked and then fixed with absolute methyl alcohol before storage, since it is often extremely
difficult to remove the hemoglobin from smears that have been stored for some time.
1.LEISHMAN'S STAINING TECHNIQUE
Dry the thin blood smear in air.
Cover the smear with a known quantity of the stain (normally, 8-10 drops) and allow to remain
for 1 minute.
Add double the quantity of neutral distilled water and mix it well with the stain by means of
blowing air through a pipette and allow to remain for 10-15 minutes.
Wash off the stain quickly and allow the slide to dry in a vertical position and then examine
under oil immersion objective.
Staining techniques
Leishman staining
Stain preparation
Leishman’s stain powder - 150mg
Absolute methonol (acetone free) - 100 ml
Grind the Leishman stain powder with a little methanol slowly in a pestle and mortar and then
add full quantity of methanol and filter it. Ripening period is about 3-4 weeks. Store it in amber
colored bottle.

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Leishman and Giemsa Cocktail stain
Leishman’s stain powder - 150 mg
Giemsa stain powder - 30 mg
Methanol - 100 ml
The stain solution is prepared as described above.
Staining procedure
Air dried film is placed on the staining rack and flooded with Leishman stain
Allow it to act for 30 seconds to one minute for fixation
The stain is diluted nearly by twice of its original volume with distilled water or buffer.The pH
of the buffer should be 7.2 for mammalian smear and 6.8 for avian blood
Blow the solution until it forms an uniform mixture
Allow it to act for 20 minutes
Wash carefully with tap water dry it and examine first at low power and then at oil immersion,
especially at the fringed edge and border
2.GIEMSA'S STAINING TECHNIQUE
Giemsa stain
Stain preparation
Giemsa powder - 1 G
Glycerol (AR) - 66 ml
Methanol (AR) - 66 ml
Azur II - 0.2 G
Take 54 ml of glycerol and place in a clean round bottom flask containing clean glass beads
Add 1.0 gram of Giemsa powder
Heat to 60°C in a water bath and hold at this temperature for one hour. Shake intermittently
Allow to cool at room temperature
Add 84.0 ml of methanol
Allow it to stand at room temperature for 2 days and shake regularly
Add 0.2 G of Azur II to every 100 ml prepared
Stand for an additional 2 days at room temperature, shake regularly
Filter and store in a dark place.
Procedure

VLD-411 Page 27
Fix the blood film in absolute methyl alcohol for 2 minutes. This can be done by dipping the
smear briefly in a coplin jar containing methyl alcohol.
Let the smear to dry.
Stain in a 1 in 10 dilution of Giemsa’s stain for 20 minutes. (Add 1 ml of stock Giemsa’s to 9 ml
of neutral buffered water)
Wash the smear , air dry it and examine under oil immersion objective for detecting the parasite.

BLOOD PROTOZOA IN LEISHMAN-GIEMSA STANIED SMEARS


Protozoa Specimen used Appearance in stained smear
for smear

Blood (thin Blue, pear-shaped organisms, singly or


smear) in pair in erythrocytes

Babesia canis
Blood (thin Rectangular blue-staining organisms (3
smear) x 6 µm) with reddish nuclei in
neutrophils - gelatin capsule like
organism

Hepatozoon canis

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Blood (thin Long, flattened, spindle shaped
smear) trypomastigote (15-20 m m in length)
Buffy coat (thick forms, with central nucleus, an
smears), lymph undulating membrane and a free
node aspirate or flagellum, free in the blood,
ascitic fluid extracellular

Trypanosoma spp
Thin blood smear Small rings, rods and commas many or
single found in RBCs organisms stain
bluish purple

Theileria annulata
Theileria spp Lymph node Schizonts (KBB) appear as roundish
aspirate smear multineucleate structure in the
cytoplasm of lymphoid cells organisms
stain bluish purple
Blood smear Morula stage (purple colour) in
monocytes and lymphocytes

Ehrlichia spp

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Blood smear Bright dots with a tendency to be
located centrally or marginally. They
may have a faint halo around them.

Anaplasma sp

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Estimation of Haemoglobin.
Principle :-

In this method haemoglobin is converted into acid hematin by dilute hydrochloric acid
and the brownish yellow color is matched with the standard in the comparator.

Matreial required :

1. Sahli’s haemoglobinometer (Haemometer)


2. N/10 HCL
3. Blood sample

There are various methods of haemoglobin estimation which are as follows :

1. Direct matching
2. Acid haematin
3. Alkaline hematin
4. Oxyhaemoglobin
5. Carboxyhaemoglobin
6. Pyridine haemochromogens

Among these various methods of the haemoglobin estimation, the acid hematin method the one
which is most commonly employed.

Sahli’s haemoglobinometer or haemometer is the one commonly used. This is equipped


with permanent glass standard and a graduated tube which permits reading of haemoglobin
values in gram per 100 ml of whole blood and also as percentage.

Procedure:

1) Place N/10 HCL solution in the graduated tube upto the mark 2 grams.
2) Mix the blood sample thoroughly in the vial and draw the blood in haemoglobinometer
pipette upto 20 cmm mark.
3) Wipe the tip of the pipette by clotting paper and pour the content of pipette into the
graduated tube containing N/10 HCL.

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4) Stir the mixture well with the help of glass rod provided with the instrument and keep the
tube for 1 - 2 minutes at rest.
5) Place the tube in comparator and match the color by adding distilled water or N/10 HCL
or normal saline, drop by drop until it matches the same color as that of comparator
standard.
6) Record the recording of the graduated tube expressed as gramper 100 ml of blood or
gram percent or gram per deciliter of blood.

Sources of error :

1. Not taking correct quantity of blood in pipette.


2. Not taking N/10 HCL upto the marks 2 grams in the graduated tube.
3. Errors in matching and forming bubbles during stirring.

Indication : Reading below normal is suggestive of anemia and anemia is typed as hypochromic.

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Estimation of RBC
Material and reagent required :

1. Thoma’s RBC diluting pipette (red glass bead),


2. Haemocytometer,
3. Blood sample
4. Microscope
5. Hand tally counter,
6. RBC diluting fluid,
7. Cover glass

Since the number of RBCs in the blood are more than 4 to 5 millions/cmm they appear too
crowded in a counting chanber therefore to make correct count the blood has to be diluted
sufficiently, so that RBCs may lie singly in counting chamber. For this a diluting fluid is
necessary. An ideal diluting fluid should have the following qualities

a. It should not haemolyze the RBCs.


b. It should promote their even distribution.
c. It should not bring about gross precipitate of proteins or other substances in blood.
d. It should act as a fixtive so that cells remain essentiallyat their original shape.
e. It should prevent the growth of bacteria, moulds and fungus.

Diluting fluids:

1) Ganti A. Satry’s Formula :


Contents
Iodine crystals 0.3 gm
Potassium iodine (KI) 0.4 gm
Sodium citrate 2.0 gm
Distilled water 100 ml
Note : keep the bottle in dry place.

Advantages :

VLD-411 Page 33
1) RBCs are stined brown and so it is easy to count them on faint yellow back ground.
2) Cells are not clumped nor their shape is altered.
3) Setting of cells in counting chamber is quick.

II) Hayem’s fluid ;

Contents :

Sodium sulphate 1 gm

(or Unhydrous 2.2 gm)

Mercuric chloride 0.5 gm

Distilled water 200 m

Disadvantages :

1) Fluid causes clumping of RBCs.


2) It precipitates the protein and so RBCs attach to such proteins and results in speedy
settling in diluting pipette.
3) It is difficult to count accuretly the red cells due to clumping.
4) RBCs also distorted in shape.

III) Gower’s fluid

Contents : Sodium sulphate (Naso4) 12.5

Glacial acetic acid 33.3 ml

Distilled water 200 ml

Disadvantages :

1) Solution casues clumping of RBCs.


2) Difficult to count due to clumping
3) Red stain in the pipette and it is rather difficult to wash the pipette.

IV) Physiological saline

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Content : Sodium chloride 0.85

Distilled water 100 ml

Disadvantages :

1) This does not fix the cells.


2) It does not inhibit the growth of bacteria.

V) Dacie’s formal – citrate solution

Contents : Tri – sodium citrate or sodium citrate 3.13 gm

Distilled water 100 ml

From this solution discard 1 ml solution and add 1 ml of 40% formaldehyde solution
concentrated formalin.

VI) Toisson’s fluid :

Contents Sodium chloride 1 gm

Sodium sulphate 8 gm

Glycerine 30 ml

Distilled water 160 ml

Principle and Indications :

Blood is diluted in a special pipette with whose osmotic pressure is great enough to prevent
haemolysis of erythrocytes. The diluted fluid is placed in the haemocytometer and the cells are
counted under the microscope. The puptte, haemocytometer and cover glass must be
scrupulously clean.

RBCs count is indicated to know whether there is increase in number of RBCs.


(Polycythemia or haemoconcretion), decrease number of RBCs (Oligocythemia) or the RBC
count is within normal range.

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Procedure for loading Neubaur’s chamber :

1) Clean the counting chamber and adjust the cover slip.


2) Mix the oxalated blood thoroughly and draw the blood upto 0.5 mark in the RBC diluting
pipette and wipe the end of the pipette with cloth.
3) If blood has passed above 0.5 marks, bring it to the mark by touching the end of the
pipette on finger.
4) Do not use a filter paper or absorbant material since these being absorbant, plsma is
absorbed leaving cells.
5) Draw the diluting fluid upto 101 marl to get a dilution of 1:200 in the mixing chanber.
6) Shake the pipette for two minute by rolling on its longitudinal axis to bring about uniform
dispersion of erythrocytes in the diluting fluid and blow out 2 – 3 drops to remove the
cell free fluid from the stem.
7) Load the Neubaure’s chamber and count the RBCs under high power objective (i.e. 40 to
50 x) in 5 of the 25 small squares in the central area. Each of the 5 small squares are
divided into 16 small squares, hence, a total of 80 small square

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Estimation of Packed Cell Volume (PCV)

Material required :

1) Wintrobe tube (Length 10 cm, bore 3 mm, each division 1 mm)


2) Centrifuge machine
3) Long aspirator needle
4) Syringe

Principle :

PCV is the measurement of number of blood cells per unit volume of blood. The cells
packed at the bottom of Wintrobe hematocrit determine the quantity of packed RBCs, leucocytes
and platelets in given blood and plasma above gives the knowledge about icterus.

Methods :

1. Wintrobe method :

A wintrobe haematocrit is used. This is a tube with 3 mm bore and 10 cm length. It it is


caliberated in millimeters. The caliberation on right, start with zero from the bottom to 10 cm at
the top, subdivided into mms.

1) Take a 6 inches long aspirator needle or Pasture pipette with long nozel to fill the
wintrobe tube. Insert the needle to touch the bottom. Slowly fill the tube with oxalated
blood upto 10 marks.
2) See that no air bubble enters which can be achived by slow filling.
3) Out the tube into clinical centrifuge and rotate it at 3000 rpm for half an hour.
4) For better packing and accurate reading two consequent rotations at 3000 rpm for half an
hour is must.
5) After half an hour remove the tube to see the packed cell volume and the clear column of
plasma is separted by layer of buffy coat which consist of leucocytes.

Formula :

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Height of RBC in mm

PCV = ----------------------------------------------------- x 100

Total height of the column in mm

PCV is expressed in percentage.

2. Microhaematocrit method :
1. When the blood already contains an anticoagulant use plain capilery tubes (75 mm X
1.0 mm) and when the blood sample is taken directly from the ear tip, toe nail or
finger was the heparinized.
2. When the capillary tube is 2/3 full remove it and seal the vacant end of the tube with
special clay or plastic or over flame (taking care not to heat blood)
3. Apply one end of capillary tube to the surface of blood, which will rise in the tube
due to capillary action.
4. Place capillary tube into the groove of the centrifuge so that the sealed end is away
the center.
5. Since the tube are too small to be marked it is essential to keep track of their
indentification by noting the number of the groove of slot for respective tubes.
6. Fasten the cover of the centrifuge and centrifuge of 5 min at 10000 to 13000 rpm and
for 2 min at 16000 rpm and above.
7. Remove tubes and read percent of PCv by using any one of a variety of haematocrit
tube reader.
The simplest of the reader has a linear scale to compensate for the variable
amount of blood in all tubes except those caliberated for use with the clay-adams
Read it & centrifuge.
i) Using the linear chart of Graphic Reader requires that the haematocrit tube be
placed on the scale with the meniscus of plasma on the top line.
ii) Slide the tube intil the bottom of the erythrocyte corresponds with the zero
line.
iii) The line that intercept the top of the erythrocyte layer is followed along the
points where it can be real on the scale at the end of the line.

VLD-411 Page 38
8. The advantage of this methods over Wintrobe haematocrit are
1) Small quantity of blood is required
2) Shorter time is required for centrifugation
3) It is more accurate.

Interpretation:

1) Information derived from the examination of the haematocrit together with the
microscopic examination of the new methylene blue stained blood smear provides a
rapid, extremely useful haemogram of the patient. Not only the PCV is obtained but
also a rough estimate of the total leucocyte count as well as clues derived from the
characteristics of the plasma.
2) Because of the better accuracy of the PCV an indicator of the patients erythrocyte
status, the total erythrocyte count and Hb confined to conditions where computation
of erythrocyte indices are needed. The PCV has the best sensitivity and
reproducibility of the various techniques.

III) Changes in PCV :

1) Increased haematocrit : the haematocrit is increased haemoconcretion, as seen


in shock associated with surgery, trauma and burns and in polycythemia.
2) Decreased haematocrit : the haematocrit is decreased in anaemia and in
conditions asscociated with hydremia such as cardiac decompensation,
preganacy, exercise and administration of fluids. It may noted that in an
anaemic animal with haemoconcentration PCV may remain normal.

IV) changes in the thickness of the buffy coat :

These are largely attributable to changes in the number o leucocytes. Platelets do


not cpntribute significantly to the thickness of this layer.

1) From the buffy coat a rough estimation of leucocytes in the blood can be
obtained. According to Wintrobe, each 0.1 mm is equal to 1000 leucocytes
whereas each 0.1 mm beyond the first 1 mm is equal to 2000 leucocytes. For

VLD-411 Page 39
routine examination, a buffy coat of less than 0.5 mm would suggest
leucopenia while that above 1.5 mm indicates leucocytosis.
2) A rough approximation of total RBC space count can be obtained by dividing
PCV by 6 and te approximation of Hb % can be obtained by dividing PCV by
3.

Note :
This approximation should never used as subtitle for total RBC count,
total WBC count or haemoglobin estimation, since it is possible to be mislead
by a marked thrombocytosis or variability in cell size.

V) Changes in the color of the plasma :

The color of the plasma column in the haematocrit gives indication of presence or
absence of icterus. However, in cattle and horses carotene and carotinoid pigments may be borne
in mind as normal.

1) A yellow coloration, due to the presence of bilirubin (Jaundice) e.g following severe
liver damage, or obstruction of the bile duct.
2) A milky appearance due to the presence of fat droplets in the blood (lipaemia). This
usually occurs in animals which have been fed within the previous 3 hrs when found
in animals which have been fasted (as recommonded), it suggests the existence of
liver diseases, diabetes mellitus, acute pericarditis, hypothy`roidism,
hyperadrenalism.
3) A pink coloration due to haemolysis which has occurred either within the body (e.g
severe harmolytic anaemia) or more usually through faulty collection.

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Differential leucocyte count
Material required :

1. Glass slides (Size 75 mm x 25 mm thickness 1.2 mm)


2. Cover slips,
3. Spirit
4. Needle
5. Scissor
6. Cotton wool
7. Blood drop directly taken from the ear vein of the animal or collected from jugular vein
in anticoagulant vial.

Principle and Indication :

Blood film is prepared for the following purpose :

1. To determine differential leucocytic count (DLC)


2. To know the degree of variation in the size & shape of RBCs
3. To see the distribution of thrombocytes
4. To find out parasitic infection, if any.

Procedure :

There are two methods of preparation of blood smear.

1. Slide method 2. Coverglass methods

Slide method : the slide method is one which is most commonly employed.

a) Select several clean grase free slides with unbroken ends.


b) Place a medium size drop of blood at 0.75 inches from one end mid way between the
edges of the slides (leaving enough space for making with glass pencil)

VLD-411 Page 41
c) Another slide called spreader is held between the thumb anf index finger and it makes
contact with blood, the blood will spread along the edge of the slide by capillary action.

Wright stain :-

Contents : Wright powder : 0.1 gm

Methanol (acetone free) : 60 ml

Preparation :-

1) Take 0.1 gm powdered Wright’s stain in mortar.


2) Ad 60 ml acetone free methyl alcohol. A few ml at a time and grind the stain with a
pestle while adding the methyl alcohol.
3) Grind the stain for several minutes after all methyl alcohol added.
4) Transfer the stain to a tightly stoppered brown bottle and store in the dark for 2 to 4
weeks.
5) Filter through filter paper prior to use.

Method of staining and results :-

Similar to method described for Leishman’s stain.

Giemsa stain :-

Contents (Stock solution) 1. Giemsa powder 3.8 gm

2. Glycerine - 200 ml
3. Methanol – 312 ml

Working solution :- Dilute 1 ml stock Giemsa stain with 10 ml or 20 ml of distilled water.

Preparation :-

1) Take 3.8 gm of Giemsa powder in a mortar.


2) Add 200 ml glycerine slowly and grind the stain with a pestle.
3) After this, add slowly 312 ml of methanol and grind the stain for several minutes.
4) Transfer the stain to tightly stoppered brown bottle and store in dark.

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5) Filter through filter paper prior to use.

Methods of staining ;-

1) Cover the blood smear by methanol foe 3 – 5 minutes and dry.


2) Prepare the working solution of stain in proportion of 1 : 10.
3) Then pour on film working solution of Giemsa stain and allow it to remain for 15 –
20 minutes.
4) Wash the smear with neutral distilled water or buffer solution. Heinz bodies are
clearly demonstrated.
5) Chlomicra of lipomic blood appear as tiny refractile bodies surrounding the RBC.
6) The nuclei or WBC are stained intensely with some practice one can use this method
as a routine for differential count.

Causes and correction of poor staining :

a) Washed out appearance of the stain entire film


1) Water was allowed to act too long in washing the slide or it was not drained
dry quickly enough.
2) Insufficient number of drop of buffer to the stain after fixation.
b) Nuclie stain faintly but erythrocytes and granules of eosinophil take intense eosin
stain.
1) Buffer was too acidic.
2) Smear was washed with acid water.
c) Nuclei intense blue, erythrocytes also blue, cytoplasm of WBC not stained
1) Buffer was alkaline.
2) Smear was washed with alkaline water.
d) Blood film covered with precipitate.
1) Failure of flow ooff the stain from the slide.
2) Not enough stain placed in slide during fixation so that alcohol evapourated
leaving a precipitate.
e) Over staining

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1) Remove stain by dipping the film in 95 % ethyl alcohol and restain reducing
the staining time.

Examamination of stained blood smear

The examination of well stained blood smear can provide more valuable information that
any other simple laboratory tests.

1) The blood smear of examined under oil immersion objective and it includes observation
of the abnormal forms of RBCs, WBCs and parasite if any.
2) The erythrocyte should be inspected in regards to
a) Size – Variation (Anisocytosis) normocytic, macrocytic or microcytic.
b) Shape – variation (Poikilocytosis) target cells, spherocytes, acanthocytes
c) Color – normochromic, Hypochromic and hyperchromic.
d) Abnormal condition – Polychromasia, reticulocyte (New methylene blue stain),
nucleated, Howell jolly bodies, Heinz bodies, carbos ring, basophilic strippling,
intercellular parasites.
3) Parsites – trypanosomes, babesia, Theileria, Anaplasma, etc.
4) To study distribution of various types of leucocytes (DLC)

Differential leucocytic count (DLC) :

DLC is determination of percentage of the various type leucocytes occur in the blood.
When a smear is made there is tendancy of neutrophil to reach to the edges. The lymphocytes
to remain mainly in the body of the smear, while the monocytes and eosinophils tend to
become evenly distributed through.

1) The DLC is done by counting and classify atleast 100 WBC or better 200 WBC’s under
oil immersion.
2) There are three methods for making tile differential count in the blood film.
a) The straight edge method which follows the edge of the film.
b) Cross sectional method which consist of going back and forth across the side. This
method is used for differential count.

VLD-411 Page 44
c) Battlement method which consist of three horizontal, three vertical fields from the
edge (As shown in figure) and is used for examination of blood parasites.

Fig. Slide methods

A Straight edge method.

B Cross sectional method

C Battlement method

3) For exanination of blood parasite, examine the margin of

ABSOLUTE DIFFERNTIAL LEUCOCYTE COUNT :

When differential count is expressed in percent it is necessary to scrutinize each


percentage figure in the light of total leucocytic count to determine whether a normal number of
cells exist or an absolute decrease or increase has occurred.

PROCEDURE :

If the total leucocyte count of a given sample is 8000/- cumm of blood and the
differential leucocyte count is N : 28, E : 2 – 9. B : 0 to 1, L : 58, M : 4. Then the differential
absolute count for various leucocytes will be –

800 x 27

Among 100 leucocytes 28 are neutrophil = ----------------------- = 2240/cumm of blood

1000

Then among 8000 leucocyte __________________ ?

Likewise for E : 720, B : 80, L : 4640, M : 320

Classification of the total and differential leucocytic response :

a) Regenerative left shift : it is characterized by leucocytosis due to neutrophilia and with


the appearance of immature neutrophils. A slight shift to the left is limited to the occrance

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of band cells. A moderate left shift includes band cells and few juveniles while a market
left shift will bring myelocytes and promyelocytes into peripheral blood.
b) Degenerative left shift : the total leucocytic count remain with in the normal range or is
only slightly elevated in dogs or a decreases in the total count with the occurance of
immature neutrophils. A degenerative left shift is common in most septicaemic
conditions.
c) Leukemoid blood picture : it is similar to regenerative left shift. The haemogram
stugnates granulocytes leukemic because there is significantly elevated total counts with
left shift. A leukemoid haemogram can be anticipated in pyometra, abscess formation,
peritonitis and also following severe haemorrhage.

Leucocyte response in relation to the type of disease process ;

1) In septicaemic condition there is often no leucocytic response but more commonly a


degenerative left shift.
2) Bacterial infection with localization and pus formation stimulates a marked neutrophilia
leading to leucocytosis.
3) Neutrophillia is also seen in variety of non infectious conditions which stimulates the
stress reaction, such as malignancy, uremia and other intoxication, post operative state,
blood loss and haemolyte crisis.
4) Eosinophilia is most commonly associated with antigen antibody reactions and the
release of histamine from decomposition of tissue.
5) Monocytosis is evidence of chronicity. In the dogs it is also as expression of stress
reaction due to release corticosteroids.
6) Leukepenia is common in the viral diseases.

Interpretation of leucocyte picture:

1) Bacterial infection with localization and pus formation stimulates marked neutrophillia
leading to leucocytosis.
2) Neutrophillia is also seen in variety of non infectious conditions which stimulates stress
reaction sich as malignancy, uaremia and other onwards reaction, post operative state, blood
loss and haemolytic crisis.

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3) Eosinophillia indicates an allergic condition or parasitic infection.
4) Monocytosis represents chronicity of infection.
5) Lymphocytosis is usually countered in chronic infection.
Red Blood cell in anemias

Anisocytosis Poikilocytosis

Macrocyte Howell Jolly bodies

Microcyte Basophillic stirppling

Target cell

Reticulocyte

Nucleated red blood cell

Leucocytes

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LEUCOCYTE COUNT
Material and reagent required :

1. Thoma’s WBC diluting pipette (White glass bead),


2. Haemocytometer,
3. Blood sample
4. Microscope
5. Hand tally counter,
6. RBC diluting fluid,
7. Cover glass

Diluting fluid :

1. WBC diluting fluid consist of


a. Glacial acetic acid 2 ml
b. 1 % Aqueous solution of Gention violet – 0.1 gm
c. Distilled water 100 ml
2. Turk’s fluid :
a. Glacial acetic acid : 2 ml
b. Mercuric chloride 0.1 gm
c. Distilled water 100 ml
Or

One person solution of acetic acid in Distilled water.

Add one drop of methyl violet solution to give a color to the solution.

Principle and Indication :

Blood is diluted in a specific pipette with WBC diluting fluid which haemolizes
erythrocytes but does not alter the leucocytes. The diluted fluid is placed in the haemocytometer

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and the leucocytes are countered under the microscope. The pipette, haemocytometer and cover
glass must be clean.

Meucocyte count is indicated to know whether there is leucocytosis, leucopenia or the


leucocyte count is within the normal range.

PROCEDURE FOR LOADING THE NEUBAUER’S CHAMBER :

1) Clean the counting chamber and adjust the cover slip.


2) Mix the blood in the thoroughly and with WBC diluting fluid pipette draw the blood upto
0.5 mark on the stain. The tip is wiped clean.
3) Draw the WBC diluting fluid upto 11 mark to get a dilution of 1:20 in the mixing chamber.
4) Shake the pipette for two minutes by rolling on its longitudinal axis to bring about a uniform
dispersion of blood cells in the diluting fluid.
5) Discard 2 to 3 drops from the pipette and load the counting chamber.
6) After loading, allow the cells to settle and count number of white cells from the corner four
primary squares which contain 64 small squares, under low power objectives i.e. 10 x
7) For the leucocyte to be detectable as dark uniform objects it is necessary to reduce
illumination as much as possible.

The cell on left and upper border lines of the square are to be included in the four square.
Cells on lower and right border are ignoed.

Calculation :

Dilution factor X Inverse of area of smallest square counted


Total number of = Total number of cells counted X -----------------------------------------------
-------------------
Of WBC Total number of small squares counted

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1) The depth of the chamber, when cover slip in position is 1/10 mm and each side of small
square is ¼ mm. therefore area of smallest square counted is ¼ X ¼ X 1/10 = 1/160 mm
2) Dilution factor is 1:20 ().5 ml of blood + 9.5 ml WBC diluting blood)
3) Total number of small squares counted = 4 x 16 = 64
4) Let theumber of cells in 64 small squares be ‘W’ then

20 X 160

Total number of WBCs = ------------------------------------------------

As per formula 64

= W X 50.cumm of blood.

Sources of error :

1) Glassware not being dry.


2) Using bark cork for specimen tube/ vial.
3) Using absorbant material to reduce the column of blood to 0.5 mark in pipette.
4) Not wiping the end of the pipette.
5) Using pipette with end.
6) Not discarding first two drops (fluid in the stem is only diluting fluid withpout any blood
and this must be discarded).
7) Blowing with the mouth for discarding 2- 3 drops.
8) Greasy chamber or chamber containing duct particles.
9) Air bubbles in counting chamber.
10) Drying of fliud in chamber before and during counting.
11) Not allowing sufficient time for setting of the cells in the chamber.
12) Errors in counting.
13) Inadequate shaking the mixture in the pipette.
14) Contamination of diluting fluid with yeast cells.

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15) See that the blood samples is not too old. As far as possible all the experiment should be
done within 6 hours of collection.

Cleaning the haemocytometer and blood diluting pipettes :

Haemocytometer and coverglass ;

1) Immedietly after use rinse in cold or leuke warm water and dry cleansing tissue or lens
paper.
2) If diluted blood has dried on the chamber, it may be washed off with soap and water,
using a gentle action.
3) Before use, the haemocytometer must be clean and free from lint, water marks and figer
prints.
4) After it ahs been cleaned the chamber of cover glass should never be touched except at
the edges.

Pipettes :

1) Throw the contents of the pipettes.


2) Aspirate distilled water into the pipette and again throw it.
3) If stain or precipitated protein remain in the bulb then rinse the pipette with blench or
cleaning solution.
4) Aspirate acetone into the pipette, then hold the pipette in air until dry.
5) If the pipette is properly cleaned and dried, the bead in the bulb should move freely.
6) A small style may be used to remove clotted blood from the care is taken to prevent
damage to the tip of the pipette.
7) The slightest chip that eners the bore will render the pipette unsuitable because of
dilution inaccuracy.

Factor that influence leucocyte count :

1) Age : at birth total WBC count is high in calf and dog , pig count is low.
2) Exercise : Leucocytosis

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3) Emotional stat : In fever there is higher leucocyte count
4) Pregnancy : in case of cow increased counted in latter stages gestation are seen
5) Oestrus : Count higher on the day of the oestrus
6) Stage of digestion : Dog an hour after eating, count is higher.
Pig – ¾ hours after feeding, count is higher.

Interpretation :

There may be decreased (leucocpenia) or increased (leucocytosis) intotal number of


leucocytes which can be interpreted as below –

1) Leucopenia is common in many viral diseases like Canine Distemper, Blue tongue, rinder
pest, and Hog cholera etc.
2) Bone marrow abnormalities due to ionizing radiation, chemical agents, toxic metabolic
products.
3) Neoplastic conditions causing dysplasia aplasia of bone marrow.

Leucocytosis:

It is brought about by an increase in either neutrophils, eosinphil, lymphocytes and


monocytes. Leucocytosis may be physiological or pathological.

a) Physiological leucocytosis : in this there is transitory increase which may be due to


muscular exercise, exposure to cold and haemorrhages.
b) Pathological leucocutosis : Seen in the following conditions i) Pyogenic conditions,
Pyometra and Pyelonephritis.
1) All sorts of bacterial infections may be localized or generalized
2) Allergic conditions : Asthama, Hay fever
3) Parasitic infection : Helminthiasis, Trichinellosis
4) Chronic diseases like TB, JD etc.
5) In stress due to increase adrenal activity.

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Clinicopathological examination of blood in various disease conditions (Erythrocytic
indices)

Anaemia is a decrease below normal in the erythrocytes, the haemoglobin value and the packed
cell volume (PCV).

Anaemia is not disease but a sign of underlying disease,

1. The objective of the laboratory test is to determine the type of anaemia as and
discovering the cause. A search for the underlying cause is essential so that it died or
removed and can serve as a guide for proper therapy.
2. Erythrocyte indicates define the size and haemoglobin content of the erythrocyte from
values obatained for the erythrocyte count, the haemoglobin concentration.

A) Mean volume (MCV):


The MCV expresses the average volume of the individual erythrocyte and is
calculated from the formula.
PCV X 10
MCV = --------------------------------------------------------
Erythrocyte count (Million/cmm of blood)
Result expresses the mean volume of the erythrocyte in cubic microns (cu) or fl.

Interpretation :

1. Normal MCV – Normocytic


a. Acute haemorrhage
b. Haemolysis
c. Lack of blood formation
2. Increased PCV – macrocytic
a. Increased activity of the bone marrow in some conditions usually
associated with normocytic anaemia such as acute haemorrhage and
haemolysis.
b. Some deficiencies of haemopoietic factors.

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3. Decrease MCV – microcytic
a. Iron deficiency
b. Copper deficiency in some animals
c. Some deficiency of haemopietic factors.

B. Mean Corpuscular Haemoglobin concentration (MCHC):

1. The MCHC is the concentration of haemoglobin in the average erythrocyte or the ratio
of weight of haemoglobin volume in which it is contained and is calculated from the formula.

Haemoglobin (g/100ml of blood)

MCHC = ---------------------------------------------- X 100

PCV

And the result expressed as percent haemoglobin per cell or g/dl.

3. Example : haemoglobin 14 m/100ml of blood and PCV 45%

14
MCHC = ---------- X 100 = 31 percent haemoglobin per cell

15

c) Interpretation :
1) Normal MCHC – normochromic
a) In many of anaemia an increase or decrease in the average size of the cell
is accompanied by a corresponding increases or decreases in the average
haemoglobin content, so that the MCHC remains within the normal range.
2) Decreased MCHC – hypochromic
a) In the true hypochromic anaemia the reduction in haemoglobin is
relatively greater than the average decrease in erythrocyte volume.
3) Increased MCHC
a) There are no condition in which the MCHC in increased above the normal
range, since the erythrocyte can not be supersaturated with haemoglobin.

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b) In so called hyperchromic anaemia there is an increase in the weight of
haemoglobin in the average erythrocyte, but the concentration of
haemoglobin per unit volume is not increased.
4) Mean corpuscular haemoglobin (MCH) :
a) The MCH is the amount of haemoglobin weight in the average erythrocyte
and is calculated from the formula.

Haemoglobin (g/100 ml of blod)


MCH = -------------------------------------------------------
Erythrocyte Count (Million/cmm of blood)
The result is expressed pico gram (pg)
b) Example – haemoglobin 13 -15 g/100ml of blood and total erythrocyte
count 6.5 millions/cmm of blood.
c) Interpretation :

The MCHC is more valuable meaurment than MCH because in


anaemia in which the MCH is not affected proportionately with the lack of
correlation is indicated by a change in the MCHC.

II) Normal values for erythrocyte indices :

Species MCV MCHC MCH


(fl) (% Hb/cell) (pg)
Horse, through bred 42 (37 - 50) 33 (31 - 35) 13.3
Horse, Graft 44 (39 - 52) 33 (31 – 35) 15.2 - 18.6
Cattle 50 (40 - 60) 30 (26 – 34) 14.4 - 18.6
Sheep 32 (23 - 48) 32 (29 -35) 9.0 - 13.0
Goat 20 (18 -24) 32 (30 - 35) 5.0 - 8.4
Pig 63 (50 - 68) 32 (30 - 34) 16.6 - 22.0
Dog 70 (60 - 77) 23 (31 - 35) 19.0 - 23.0
Cat 45 (40 - 55) 23 (31 - 35) 13.0 - 17.0
Human 87 (82 - 92) 34 (32 - 36) 29.0 – 27.31

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III) Morphological classification of anaemia :

Classification MCHC normal MCHC decreased


MCV normal Normocytic Normocytic
Normochromic Hypochromic
MCV Increased Normocytic Macrocytic
Normochromic Hypochromic
MCV Decreased Microcytic Microcytic
Normochromic Hypochromic

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