‫جامعة عين شمس‬

‫كلية الطب البيطرى‬

‫قسم الفسيولوجيا البيطرية والكيمياء الحيوية‬

‫‪Practical Physiology‬‬
‫‪Code no. PAB212‬‬

‫‪2023/2024‬‬
Content:
• Blood sampling
• Packed cell volume (PCV)
• Hemoglobin estimation
• Erythrocyte sedimentation rate (ESR)
• Red blood cells count
• White blood cells count
• Differential leukocytic count
• Blood group
• Osmotic fragility test
• Blood pressure
• Muscle and nerve
• Cardiac muscle
Blood cells
count
Principle:
Estimation of the number of red blood cells and
white blood cells in a small volume of diluted
blood.
Equipments and Reagents:
 Microscope (with 4X, 10X and 40X objective
lenses)
Eye piece lens

40X lens

10X lens

4X lens

Coarse and fine

adjustement button Stage

Condenser adjustement Condenser

button
  Lancet and alcohol
 Diluting solutions:
The blood cells are so numerous that they must be
first diluted before they are placed in the
hemocytometer for counting. The diluting solutions
are :
1. Hayem's solution (isotonic, RBCs diluting solution)
2. Turk’s solution (WBCs diluting and staining solution).

Hayem's solution Turk's solution

lyses RBCs, dilutes and stains
lyses WBCs and dilutes RBCs
WBCs
Compositon:
Compositon:
Mercuric chloride 0.5 g Hayem's
Glacial acetic acid 2.0 ml
Turk's

Sodium sulfate 5.0 g Gentian violet 1.0 ml

Sodium chloride 1.0 g (1% aqueous solution)
Distelled water 200.0 ml Distelled water 100.0 ml
Hemocytometer
which consists of :
1. The diluting pipettes
Bulb
Bulb

Stem Stem
Red bead White bead

Rubber tube
Rubber tube

Red blood cell diluting White blood cell diluting

pipette pipette
 Hemocytometer
which consists of :
2. The counting chamber
 Thecounting chamber is a thick glass slide
with:
➢Two central platforms, containing the counting
scale, they were surrounded by two grooves.

➢On each side of the platform there are two

cross bars, on which the cover slip is placed.
The cross bars are raised by 1/10 mm above
the platforms.
 Cover slip
Platform

Cross bar Cross bar

Grooves

Cover slip

Cross bar Cross bar

Depth=1/10 mm

Platform
Under the screen lens of the microscope
(4X) the counting scale appears as:

1
1 m
m m
m

 One large square (of area =9 mm2), subdivided by triple lines into
9 primary squares (each of area = 1mm2)
 Each of the 4 corner primary squares (black dotted circles) is subdivided by
single lines into 16 secondary squares for counting of leukocyte.

WBCs WBCs

RBCs

WBCs WBCs

• The center primary square (red circle) is used for erythrocyte count and is
subdivided by triple lines into 25 secondary squares, each of which is
further subdivided by single line into 16 tertiary squares.
Upper left WBCs Upper right WBCs
primary squares primary squares

1 2 3 4
8 7 6 5
9 10 11 12
16 15 14 13

Lower left WBCs Lower right WBCs

primary squares primary squares
RBCs central primary
square

1 2 3 4 5

10 9 8 7 6

11 12 13 14 15

20 19 18 17 16

21 22 23 24 25
Red blood cells
count
 Procedure
Counting of RBCs
1. All equipment must be clean and dry
2. Sterilize the tip of the finger with 70% ethyl
alcohol and leave it to dry, then prick it sharply
to get a drop of blood or use coagulated blood.
3. Suck the blood into the stem of the diluting
pipette to the 0.5 mark, then wipe the tip of the
pipette and dip it into the diluting solution
(Hayem’s solution).
4. Suck the Hayem’s solution slowly up to the
mark 101 (avoid air bubbles).
4. Hold the pipette by placing the thumb over
the tip and the index or the middle finger
over the opposite end.
5. Shake the pipette using a back and froth
movement at right angles to its long axis.
6. Note that the capillary end of the pipette
stem contains only diluting solution. So
after diluting and mixing the blood, blow out
about 2-3 drops to remove the diluting fluid
(cell free fluid) from the stem of the diluting
pipette.
8. Touch with the tip of the pipette the space between the cover
slip and the counting chamber at an angle = 45oC to allow the
diluted blood to flow under the cover slip by capillarity
(bubbles should be avoided so that the cells become evenly
distributed).
9. Wait 2-3 minutes for the cells to settle and examine the scale
containing the RBCs by the low powers (4X and 10X objective
lenses), then count the cells by high power (40X objective
lens) in 4 corner and one central secondary squares.
9. To count RBCs, start at the
RBCs counting patterns
upper left square and
include in the count all cells
inside the counting square
and also the cells touching
the lines forming the left
and lower borders of the 5 3 4 4
counted square (L-shape)
and ignore cells touching
the upper and right border
5 3 4 5
line.
10. Move across the top row of 2 4 2 2
squares from left to right,
and then drop down to the
second row and proceed 3 3 2 4
from right to left (Zigzag
pattern). Repeat this
pattern in all RBCs
secondary squares.
 Calculation of RBCs number
• Substitute the count of erythrocytes in the 5 secondary
squares for N in the following equation:

RBCs count/mm3 = N/80 x 400 x10 x 200

N : No of RBCs in 5 secondary squares (5x16=80 tertiary

squares)
N/80 : The average no of RBCs in one tertiary square of area =1/400
mm2
400 : To estimate the count in area =1 instead of 1/400 mm2
10 : Height must be corrected from 1/10 to 1 mm to estimate the
count in volume = 1mm3 [Volume = area (1mm2) x height
(1mm)].
200 : To estimate the count in non diluted blood (dilution =1/200)
 RBCs indices
Erythrocyte indices are calculations for determining the size,
content and Hb concentration of a red blood cell. These indices
have been useful in classifying anemia according to RBCs
morphology and Hb content. These indices are:
Mean cell volume (MCV)
– MCV; the average volume of red blood cell is calculated (in
fl=10_15 L) as follows:

PCV
MCV = x10
RBCs count
Mean cell hemoglobin (MCH)
– MCH; the average Hb content of red blood cell is calculated
(in Pg=10_12 g) as follows:

Hb
MCH = x10
RBCs count

Mean cell hemoglobin concentration (MCHC)

– MCHC; the percentage of Hb in a given volume of PCV (in
%) is calculated as follows:

Hb
MCHC = x100
PCV
 Clinical significance
Normal values
Human 5-6 x106/mm3 (man) ♂
4-5 x106/mm3 (woman) ♀

Dog 6-8 x106/mm3 (intermediate count)

Goat 13-14 x106/mm3 (highest count)

Cow 7 x106/mm3

Horse 9 x106/mm3

Chicken 2.5-3.2 x106/mm3 (lowest count)

 Higher counts indicates:
Polycythemia
(increase in the no. of red blood cells in circulation)
It is classified into:
 Relative polycythemia
 Absolute polycythemia

Polycythemia
1.Relative polycythemia
Result from a primary reduction in plasma volume and
consequently a relative increase in RBCs mass which
occurs in:
• Dehydration
• Vomiting
• Diarrhea
• Hypertensive drugs (diuretics)
 2. Absolute polycythemia
Primary polycythemia
Polycythemia rubra vera
Result from an abnormality of the
hematopoietic stem cell characterized by
uncontrolled proliferation and
overproduction of erythroid,
granulocytic and megakaryocytic cells (a
clonal neoplastic disorder; bone marrow
cancer), despite normal or low
concentration of erythropoietin (EPO)
Secondary polycythemia
Result from inappropriate secretion of
erythropoietin hormone which occurs in:
• High altitudes
• Cardiac and pulmonary diseases
• Smoking
• Cobalt poisoning
• Renal diseases (renal cysts, renal
tumors, atherosclerotic narrowing
of renal artery)
• Tumors (uterine tumors, brain
tumors, hepatoma) which either
interfere mechanically with the
blood supply to the kidney as an
abdominal mass or secrete
inappropriate secretion of
erythropoietin hormone
 Lower counts indicates:
Anemia
(Reduction in the No. of circulating red blood cells and/or
hemoglobin concentration).
Classification of anemia depends on:
◦ The size and shape of RBCs (morphological or cytometric
classification)
◦ The rates of red blood cells production and destruction in
circulation (erythrokinetic classification).
◦ The etiology (biochemical/molecular classification)
The most common classification is the morphological or cytometric
classification, in which anemia is classified according to RBCs indices
into:
 Normocytic Microcytic Macrocytic
normochromic hypochromic normochromic
anemia anemia anemia
Normal MCV Low MCV High MCV
Normal MCH Low MCH Normal MCH
Normal MCHC Low MCHC Normal MCHC
Occurs in: Occurs in: Occurs in:
•Chronic disease •Iron deficiency •Vit B12 deficiency
•Hemolytic anemia •Thalessemia •Folic acid deficiency
•Acute hemorrhage •Chronic disease
•Aplastic anemia
Any questions???