Blood Examination
Blood : straw yellow colored alkaline fluid media (plasma) in which cellular components are suspended
(RBCs, WBCs and blood platelets) which produced from hematopoietic tissue (bone marrow, liver, spleen,
lymph nodes and reticular endothelial tissues).
Physical Characters :
pH :- 7.35 : 7.45 (average 7.4) slightly alkaline.
Volume :- 7 % : 8 % of animal body weight.
Distribution :- ¼ in heart and large blood vessels.
¼ in liver.
¼ in skeletal muscles.
¼ in other tissues.
Compnents :- Plasma 55 % (Serum and Fibrin)
Cells 45 % (RBCs, WBCs and Blood platelets)
Function :
1 . It supply o2, essential nutrient, enzymes, hormones, H2o, electrolytes & buffering system to tissues
cells.
2 . Remove metabolic waste products.
3 . Certain blood cells (WBCs) provide a defense mechanism against invasion of body by living pathogenic
agents as well as protection provided by immunoglobulin.
Indication of blood examination : It is the most important laboratory aids for diagnosis of diseases.
Generally blood examination is indicated in most of clinical cases characterized by fever :
1 . Hematological examination (estimation of RBCs, WBCs, Hb and others)
2 . Indicated in surgical condition.
3 . For differential WBCs counts :
a) eosinophilia : indicate allergy and parasitemia.
b) basophilia : indicate tumor.
c) monocytosis : indicate chronic diseases.
4 . Indicated in administration of fluid therapy.
5 . For blood grouping.
6 . For diagnosis of some viral diseases :
- By taking sample during viremia and examine under EM or T.C inoculation
7 . For determination of animal spp : (acc.to shape of RBCs)
a) oval non-nucleated : indicate bovine and ovine.
b) elliptical non-nucleated : indicate Camilidae.
c) circular non-nucleated : indicate equine and mammals.
d) oval nucleated : indicate poultry.
8 . For diagnosis of some bacterial diseases :
a) Pasteurella  polychrome methylene blue  short truncated bacilli with bipolarity.
b) Bacillus anthracis  “ “ “  pink sporulated bacilli against blue background.
c) Leptospira  fronton stain  zygote stage appear spiral shaped.
9 . For diagnosis of blood parasites :
a) Intracellular parasites :
1 – Babesia  B.bigemina (large 2:4 microns – pair pear shape – Located at periphery).
 B.bovis (small 1:2 microns – pair pear shape – Located at center)
2 - Theileria  Th.annulata (ring shaped – at center)
 Th.parva (comma shaped – at center)
3 – Anaplasma (A.centrale – A.marginale)  appear as bluish purple dots inside RBCs.
b) Intercellular (extracellular) parasites:
- present between RBCs such as (Trypanosome – microfilaria)
Blood Sampling :


Type - Site – Preparation – Precaution (Section 3 Sampling)
Common anticoagulant used :
1. EDTA (ethylene diamine tetra acetic acid) :
- Present in form of disodium or dipotassium EDTA
- Dose : 1 mg /1 ml blood (1 gm / 1 litre blood)
- Used in determination of urea, creatinine, uric acid, ph and glucose.
- Not used in determination of some electrolytes such as Ca & Cl because they bind with it
- The best anticoagulant used for hematological examination.
2. Heparin :
- Natural anticoagulant which prevent conversion of prothrombine to thrombine so fibrinogen
not converted to fibrin (No Clot)
- Dose : 0.1 ml / 1 ml blood
- Used in : blood transfusion & detection of serum biochemical alteration
3. Flouride :
- Present in form of sodium or potassium fluoride
- Dose : 2 drops as 40 % solution / 5 ml blood
- It interferes with enzymatic system of glycolysis so can be used in sugar determination.
- Not used in blood transfusion
4. Citrate :
- Present in form of trisodium citrate.
- Dose : 2 – 4 mg / 1 ml blood
- Used mainly in blood transfusion
5. Oxalates :
- Present in form of sodium, potassium or ammonium oxalates.
- Contraindicated to be used in blood transfusion why ?
a) combine with calcium forming calcium oxalates.
b) make shrinkage of RBCs.
c) make alteration of serum biochemical characters.
Blood Examination :

Microscopic examination of unstained blood films (wet blood film):
- Materials : glass slide(clean – dry – free from dirties – free from dusts – free from greases) –
cover slide – blood sample
- Procedures :
1 - Placing a very small drop of blood on the surface of very clean glass slide (Center).
2 - Put cover slide on the drop.
3 - If the cover slide and glass slide are clean and blood drop is very small, blood spread
out into a thin blood film.
4 - examine under microscope.
- Indication :
1 – cytological examination to detect type of anemia acc.to shape and size of RBCs.
2 – Diagnosis of extracellular blood parasites such as trypanosome and microfilaria which
lead to change in RBCs shape by their movement and lashing .
-
N.B In case of microfilaria we can add one drop of acridine 1/5000 as a stain giving the
microfilaria a yellow color.
 Microscopic examination of stained blood films (dry blood film)
 Materials : 2 glass slides(clean – dry – free from dirties – free from dusts – free from greases) –
stain – blood sample
 Procedures (preparation of blood film) :
1. Put drop of blood at one end of one glass slide (clean – dry Free from dirties – free
from dusts – free from greases)
2. By second glass slide (spreader: edges of the spreader is free from notches) make
backward movement toward blood drop by an angle 30◦ : 45◦ (according to the
thickness wanted) till touch blood drop which will spread along the line of contact
between two slides
3. The spreader is then pushed forward spreading the blood behind it into a film.
4. Air dryness by movement of slide in air for 3 : 5 minutes.
5. After drying, the film should be identified by writing the date, number of slide, the
species of the animal, and the name of the examiner by pencil.
6. Or staining of blood film and examination under microscope.
 Types (Consistency) of blood film :
Items
Thin Film
Thick Film
Size of drop
small
large
Angle between spreader and slide
narrow
wide
Speed of spreading
rapid
slow
indication
Intracellular parasites
Extracellular parasites
 Care of blood film :
1.
2.
It must be stained immediately as soon as possible.
The blood film prepared at the farm should be protected from fly.
 Common faults of blood film :
1. Too thick :
- Too large blood film.
- Too large angle of spreading.
- Too slow spread speed.
2. Too Thin :
- Too small drop.
- Too small angle of spreading.
- Too fast spread speed.
3. Streaks throughout the film :
- Irregular or notched edges of spreader.
- Presence of dried blood at edges of spreader.
4. Presence of spots on blood film :
 Presence of greasy material on slide that absorb blood
5. Alternative thick and thin bands on slide :
 Jerky movement (hesitation) of the hand during spreading.
6. Too narrow and thick blood film :
 Raise edges of spreader from surface of slide during spreading.
 When spreading occurs before blood had run along the edges of the spreader.
 Blood Staining :
1. Leishman’s stain :
 A) Preparation of blood film ( 1 to 4)
 B) After air dryness immerse slide in stock solution of leishman’s stain and leave
for 2-3 minutes (fixation period).
 C) Dilute with equal quantity of distilled water and leave for 5-10 minutes with
thoroughly mixing by pipette (staining period)
 D) Decant the satin and wash the slide several times with tap water (1 minute).
 E) Dry blood film by filter paper and leave to dry in air.
 F) Cover the film with coverslip and examine under the oil immersion lens after
adding one drop of cedar oil.
2. Giemsa Stain :
 A) Preparation of blood film (1 to 4).
 B) After air dryness immerse slide in methyl alcohol 70 % and leave for 3 : 5
minutes (fixation period) why?
- If immerse slide in giemsa stock solution directly the blood films will be
removed.
 C) leave blood film to dry in air
 D) immerse slide in giemsa stain solution (1 part of stock solution + 9 part of
D.W) and leave for 30 : 45 minutes (Staining period).
 E) Decant the satin and wash the slide several times with tap water (1 minute).
 F) Dry blood film by filter paper and leave to dry in air.
 G) Cover the film with coverslip and examine under the oil immersion lens after
adding one drop of cedar oil.
Blood examination for diagnosis of some bacterial disease :
1.
2.
3.
4.
5.
6.
7.
collection of blood during septicemia
prepare thick blood film
put slide in staining jar
flood with 1 % aqueous solution of methylene blue
allow stain to act for 2 minutes
wash by D.W to remove excess stain.
Dry blood film and examine under microscope.
Blood examination for diagnosis of some viral disease :
1. collection of blood during viremia.
2. Obtain buffy coat or plasma (as virus present attached to WBCs or free in plasma).
3. T.C inoculation to detect cytopathic effect (CPE).