Practical hematology

Blood sampling
Small amount Large amount

Puncture of the Wing vein

comb Inside the elbow joint

Cutting the tip of Tibial vein

comb Just above the knee joint at medial
aspect
Cutting of toe nail Shank vein (mainly in duck and
geese)

Jugular vein (right side is

larger vein)
 Anticoagulants
EDTA:
Dose:1-2 mg/ml blood
But after 15 min blood become dark & viscous
Although the blood does not clot but the cells
are disrupted enough to make sample
useless

Heparin:
Is the best for test requiring plasma
Dose: 25 Iu / ml blood
 Avian Haematology
Avian blood is a sticky bright red coloured fluid, it
represents about 6.5-8.5% of body weight.
Avian blood is composed of:
1-Plasma: Straw yellow in colour and slightly
turbid.
2-Cellular elements:
A-Rbcs
B-Wbcs
C-Thrombocytes
N.B All cellular elements are nucleated
 1-Plasma

1-It is a straw yellow in colour and slightly

turbid in appearance.
2-Water constitute about 80% of plasma.
3-It contains various of compounds as
proteins, hormones, vitamins ,glucose,
Minerals, fatty acids
4-These compounds differ according to age,
sex, egg production, type of foods,
condition of animals
5-Plasma of layers contain 2 folds increase in
calcium than non layers (9.3-12.3 mg%).

6-Plasma of layers contains 2-5 folds increase

in plasma lipids than nonlayers (due to
increased secretions of estrogen from ovary
in layers).
7- Glucose concentrations in birds range
from 126-204mg%

A- Glucose concentration drops with age.

B- Glucose concentration increase with
hatching.
C-In Stress glucose concentration increase.

So generally,plasma glucose concentration

in birds is higher if compared with
mammals.
8-Plasma proteins ranges from 2.3-6.1 gm%
A-Plasma proteins of male is lower than female (
for egg production).
B-The globulins are more concentrated in birds
than in mammals,so birds are good antibodies
producers.
9-Urates (uric acid):
A-Catabolic end products of protein and non
protein nitrogenous compounds.
B-It ranges about 40 mg%
C-It is a vehicle by which amino-nitrogen is
excreted.
Creatine & Creatinine
1-Creatine & creatinine are indicator for
muscle metabolism.
2-Increase creatine & creatinine with
increase uric acid indicates Muscle
catabolism.
3- Increase creatine & creatinine with
decrease uric acid indicate Muscle
Anabolism.
 Total RBCs and WBCs count
• Total RBCs and WBCs count can be
directly counted from the same blood
sample placed in the hemocytometer.
Apparatus
• Hemocytometer
• RBCs diluting pipette
• Diluting solution (Natt and Herrick)
 Natt and Herrick
Composition
Nacl 3.88 g
NaSo4 2.50 g
Na2HPo4.12H2O 2.91 g
KH2Po4 0.25 g
Formaline (37%) 7.50
Methyl violet 0.10g
Dissolved in Distilled water to a total volume =1000 c.c.
The solution has a pH =7.3
Advantages of Natt and Herrick solution
Have osmotic pressure as that of chicken blood(do not distort
blood cells)
Both total RBCs and WBCs count can be made from the same
blood sample
Procedure
• Blood till 1.0 mark in diluting pipette (dilution factor
1:100)
• Diluting solution till 101 mark (dilution factore 1:100)
• Allow the mixture to stand in the pipette 1-2 min
• Shake well for 1 min to obtain equal cell distribution
• Counting of RBCs and WBCs in the central primary
square.
Calculation
• RBCs/mm3 =N/80 X 400 X 100 X 10
• N= no of RBCs in five 2ry squares
• WBCs/mm3=N X 100 X10
• N= no WBCs in whole 1ry square
 Cellular elements

All cellular elements of birds are nucleated

A-Erythrocytes:
1-It is 10-15µm,larger than mammals(5-
7µm), smaller than reptiles( 18-23µm).
2-Elliptical in shape,biconvex,nucleated
,contain mitochondria ,its life span is about
one month.
3-Counted by haemocytometer
4-Their number varies from 1.5-
6.6millions\mm3.
5-Rbcs number depends upon :
Age: is higher in young animals
Sex: Male is higher than females
Hormones:
Anabolic hormones as thyroid hormones
increase number of Rbcs.
Estrogen interfere iron absorption so
decrease Rbcs count.
Seasons:
Winter stimulate thyroid activity so increase
Rbcs count .
Altitude:
High altitude increase Rbcs due to increase
Erythropoitin hormones.
Erythrocytes osmotic pressure :
O.P of birds is higher than in mammals (1.02
Nacl)
 B -Leukocytes

As in mammals there are Granular and

Agranular leukocytes.
Number of leukocytes:
Chicken: 19-30 thousands/mm3
Duck: 32 thousands/mm3
 Differential Leukocytic count

Preparation of blood smear

1-By using The Standard two-slides wedge
technique as in mammalian blood.
2-Uncoagulated blood or EDTA-blood should be
used and avoid heparin since it interferes with
the proper staining of the blood cells.
The commonly used stains Are:
A-Wright stain.
B-Wright –Leishman stain.
C-Wright – Giemsa stain.
Blood smear
 Leukocytes

Leukocytes are either granular or Agranular.

Granular leukocytes
1-Heterophils
2-Eosinophiles
3-Basophils
Agranular leukocytes
1-Lymphocytes
2-Monocytes
 1-Heterophils
1-It is analogue of mammalian neutrophils .
2-Its diameter about 10-15 µ.
3-The nucleus is polymorphic with varying
degree of lobulation.
4-The cytoplasm contains a characteristics
rod-like granules , pointed at their ends,
acidophilic bodies.
The percentage of heterophil in blood
ranges 20-30% (except in ostrich and
pheasants 60-70%)
Heterophils are phagocytic and use their enzyme-containing granules to lyse
ingested materials.
Heterophils are motile and can leave blood vessels to engulf foreign materials
 2-Eosinophils

1-The size of eosinophil is about the same

size as the heterophil.
2-Rounded cells , The cytoplasm contains
large, spherical, dull red coloured
granules.
3-The cytoplasm is Faint blue colour.
4-The nucleus is often bilobed and Darker
than that of heterophils.
Eosinophils make up about 2 to 3 % of the WBC population of
healthy birds.
 3-Basophils

1-The size of basophil is about the same

size as the heterophil.
2-Rounded cells , the cytoplasm is devoid of
colour and contains basophilic granules
which may mask the nucleus and
protruded from the surface of the cells
giving black berry appearance.
3-The nucleus is weakly Basophilic , may be
rounded or oval.
Basophils
 4-Lymphocytes

1-The lymphocytes constitute the majority of

the leukocytes in the blood of the fowl.
2-The cytoplasm is weakly basophilic , it may
forms a narrow rim around the rounded
nucleus in small lymphocyte.
3- The cytoplasm may constitute the major
portion of the cell, as in the large
lymphocytes. The nucleus is usually round
and has a fairly coarse pattern of chromatin.
The percentage of lymphocytes in blood ranges 60-70% (except in ostrich
and pheasants 20-30%)
Lymphocytes are either T-lymphocytes (formed in the thymus) or B-
lymphocytes (formed in the bursa of Fabricius).
B-lymphocytes produce antibodies; T-lymphocytes attack infected or
abnormal cells.
 5-Monocyte

1-Monocytes are large cells with

relatively more cytoplasm than the
large lymphocytes.
2-The cytoplasm of these cells has a
blue-gray colour and vaculated.
3-The nucleus is usually irregular in
outline
Monocytes %=5-10%
Monocytes are motile cells that can migrate using ameboid
movements.
Monocytes are also phagocytic.
 Thrombocytes

1-They are rounded to oval in shape and

usually present in clumps.
2-They have rounded very darkely stained
nucleus and a clear cytoplasm.
3-Thrombocyte are active participants in blood
coagulation.
In addition to blood clotting, these cells may
have the ability to phagocytize foreign
materials (e.g. bacteria) and they are able to
carry O2 like RBCs if an extreme anemic
condition exists.
The avian thrombocyte (upper cell) usually has a smaller, more densely-
stained nucleus compared to the lymphocyte (lower cell).
Thrombocyte cytoplasm tends to be paler in color, and more "ragged" or
vacuolated in character compared to that of the lymphocyte.
Some thrombocytes contain small reddish granules (arrows).
Leukocytes
 Total thrombocytic count (TTC):
an estimate of thrombocytes number can be obtained
using blood smears prepared as for differential
count
Calculation:
If the PCV is within the range of 40-50%:
Estimated TTC/ ul = number of thrombocytes in 5 fields
x 3500
If the PCV is outside the 40-50% range:
Corrected TTC/ ul = estimated TTC x (observer PCV/
normal PCV)
Comment:
Normally, there should be 2-3 thrombocytes
(10-15/ 1000 RBCs) per microscopic field
when viewed with the 100x objective under
oil immersion.
Increasd number of thrombocytes can
indicate a chronic disease condition.
 Haemoglobin Estimation

A-Modified Acid Haematin Method (Modified

Sahli 's method)
Characterized by
1-The volume of HCL N/10 was used up to the 8th
mark (% mark) instead of 10th mark (% mark).
2-The reading of Haemoglobinometer is multipied
by a factor 0.134 to obtain amount of Hb in gm%
For e.g.
85% (reading) x 0.134 = 11.4 gm%
 B-Cyanomethaemoglobin method
Characterized by
1-The ordinary Drabkin ‘s reagent is also used.
2-The Turbidity formed after addition of blood on
Drabkin ‘s reagents is removed either by:
A-Centrifugation of the tube containing the
Drabkin ‘s reagent and blood to ppt. turbidity.
B-Passing a beam of natural gas or Co-gas
through the tube.
3-Meaurement calorimetric at W.L 540nm.
4-Blank solution : drabkin ‘s reagent.
 Normal values of Hb

1-Chicken: 9.3 gm%

2-Ducks: 10.3 gm%
3-Turkey: 10.3 gm%
 Avian Haemoglobin
1- In most vertebrates, hemoglobin is made up of four
subunits, each one of which has its own binding
site for oxygen.

2- There are some significant differences between

avian hemoglobin and hemoglobin found in other
vertebrates:

A- In adult birds there are two different types of

hemoglobin, hemoglobin A and D, both of which
vary from each other in their affinity for oxygen.

B- Hemoglobin A is often the more prevalent form

and has a lower affinity for oxygen compared to
hemoglobin D.

C- A lower affinity means that oxygen is more readily

dissociated from hemoglobin as arterial blood
D- In general, avian hemoglobin shows more
cooperativity with oxygen than does hemoglobin in
other vertebrates.

E- Cooperativity is the phenomenon whereby the

binding of one molecule of oxygen with hemoglobin
facilitates the binding of the next molecule of oxygen
and so on up to the binding of four oxygen molecules
by a molecule of hemoglobin.

F- This cooperativity accounts for the sigmoidal shape

of the oxygen-hemoglobin binding curve.

G- The advantage of this high cooperativity is that it

increases the delivery of oxygen to tissues.
 H- Another feature of avian hemoglobin is its
interaction with inositol pentaphosphate and inositol
tetraphosphate.

I- In mammals the principal organic phosphate is 2,3-

biphosphoglycerate (2,3-BPG), formerly known as
2,3-diphosphoglycerate (2,3-DPG), which shifts the
oxygen-hemoglobin dissociation curve to the right
and decreases the affinity of oxygen for hemoglobin,
enhancing the delivery of oxygen to tissues.

J- The presence of two hemoglobins with different

oxygen affinities, means that the erythrocytes have a
greater range of oxygen partial pressures over which
oxygen can be bound and released, favoring the
uptake of oxygen where there is little oxygen
available in the environment (Fig. 8).
K- Carbon dioxide also affects the binding of oxygen to
hemoglobin.

L- In mammals, the binding of carbon dioxide with

hemoglobin is more so with deoxyhemoglobin than
oxyhemoglobin, to form carbamino compounds,
which result in small increases in P50.

N- It appears that in birds the strong binding of the

organic phosphates to hemoglobin prevents this
carbon dioxide effect.
Fig. (8): Oxygen dissociation curves of adult fowls'
blood at 40oC. Effects of variation of pCO2.
 Erythrocyte sedimentation rate

Applied by a Westergreen ‘s method

Characterized by
A-The tube should be tilted at 45 0 angle ,as
the vertical position is unsatisfactory for
avian blood because avian blood cells
settle very slowly.
B-Normal values:
ESR= 1-4 mm\hr
 Packed cell volume (PCV)
A-Macrohaematocrite ( Van -Allen Method)
1-Using Van-Allen Haematocrite tube
2-Centrifugation at 2000 r.p.m for 20 min.
( the oxalated-blood sample is filled into Van-
Allen haematocrite tube till the mark 100%
and then centrifuged at 2000 r.p.m\20 min.
3-The opened end of the tube is firmly closed
by spring clip cap.
B -Microhaematocrite
By using Heparinized capillary tube
Tube is filled with blood then close with clay
and centrifuge.

Normal values of PCV

Chicken 32%
Ducks 33%
Rabbits 41%
 Coagulation screening tests for avian
blood
1-Whole blood clotting time (CT)
Blood is collected into non-siliconized tube
(2ml). Tilted every 15 seconds until a firm
clot is observed
N.B.
Contamination of blood sample with tissue
thromboplastin may shorten clotting time
2-Prothrombin time (PT)
Avian origin thromboplastin source is
required for PT assay. Acetone-
dehydrated brain powder have more
thromboplastic activity.

Principle and procedure

Citrated plasma (tested sample) +
thromboplastin (of avian origin)
(37-38.5oC)
Fibrin clot
(PT: time required for tissue thromboplastin
to cause clotting of tested plasma/sec)
Normal for chicken: 9-11 sec
Recalcification time (RT)
Intrinsic pathway is evaluated with
recalcification time
Principle and procedure
Citrated plasma (total plasma) + crushed
glass (contact activator; XII XIIa)

(37-38.5oC)
(1-3 min)
+CaCl2
(37-38.5oC)
Fibrin clot
(RT: time required for fibrin clot formation
after additon of CaCl2)
Activated partial thromboplastin time (APTT)
1-Intrinsic pathway is also evaluated with
APTT, where platelet factor substitue
prepared from chicken brain can be used
as cephalin source.
2-Plasma must be preincubated with kaolin for
3 minutes before CaCl2 is added.
Principle and procedure
Citrated plasma + cephalin of avian origin
+kaolin

(37-38.5oC)
(1-3 min)
+CaCl2
(37-38.5oC)
Fibrin clot
Comment
1-PT is a screening test used to monitor the
extrinsic pathway of blood clotting.

2-RT and APTT are used for monitoring the

intrinsic pathway of blood clotting.

3-Coagulation tests are prolonged in liver

diseases, ingestion of vitamin K antagonists
(e.g coumarin or warfarin), ingestion of ration
contaminated with mycotoxins
(aflatoxicosis).

4-Individual clotting factor assay.

5-Individual clotting factor assay
1-Individual clotting factor activity (e.g factor
II, VII, IX, X and XI) can be measured.

2- The use of tissue thrompoblastin of avian

source gives an accurate results.