Practical physiology

فسلجة عملي 17\10\2017

عدد الاوراق ( 3 ) المختبر 2
WBC manual count using hemocytometer
Introduction
Blood consists of 3 type of specialized cells:
Erythrocytes RBCs
Leukocytes WBCs
Platelets (thrombocytes)
These cells suspended in complex liquid plasma

WBCs count is the count of leukocytes in a volume of blood

Expressed as WBCs/mm³





Material
Blood sample (EDTA anticoagulated blood or capillary blood)
WBCs diluting pipette
Diluting solution(Turks solution)
Hemocytometer
microscope

Turk's solution

Türk's solution is a composed of a stain (Gentian violet) and 1-2% acetic acid. The acetic acid destroys red blood cells within a blood sample, and Gentian violet stains the nuclei of the white blood cells, making them easier to see and count. Turk's Solution is intended for use in counting leukocytes in a defined volume of blood. Erythrocytes are hemolyzed while leukocytes are stained for easy visualization.
Dr.Sajeda Al-Chalaby

The Hemacytometer

contains 2 Neubauer counting chamber
Each chamber contains:
*4 WBC counting squares
*Each contains 16 squares

Methodology

draw blood up to 0.5 marks on WBCs pipette and complete to 11 with WBCs diluting solution.
Mix for 2-3 minute.

Charge hemacytometer

Discard the first 4-5 drops
Place tip of the pipette at the edge central platform
Then let the hemacytometer to stand on the bench for 3-5 minute in order for the WBC to settle.

Count and calculate:

Calculation

Number of WBC/mm³=
counted cells in 1large square x
diluting factor x
volume(unit) correction factor.
It is better to count cells in 4 large squares then divide/4.


The dilution factor=
0.5/10 =1/20
Dilution factor=20
Counted volume =
The volume of (1) large square=
= (width x length x depth )x 4
= (1mm x 1mm x 1/10 mm)
=0.1mm³
Multiply by 10 to correct the unit to (1 mm³).
Number of cells/mm³=
counted cells in 1 large square(n) x
diluting factor x
volume correction factor.
Number of cells/mm³= n x20 x 10
= n x 200

Example:

If total number of WBCs in 4 squares is 120
The number of WBCs in 1 squares is 120/4=30
Then the number of WBCs in 1mm³=
30 x 200=6000 WBC/mm³


Normal values of WBCs:
Newborn 9000-30000 cell/mm³
Adult 4000-11000 cell/mm³
Neutrophil 62%
Eosinophil 2.3%
Basophil 0.4%
Monocytes 5.3%
Lymphocyte 30%

Significance of the test

Leukocytosis
Occur as an indicator of body defense against foreign materials (bacteria, parasites, toxins)
Metabolic disorders
Chemical and drug poisoning
Acute hemorrhage

Leukopenia

Result from X-ray therapy
Alcoholism
Antibiotic therapy
Typhoid infection
Measls
Infectious hepatitis
TB
And cirrhosis of the liver