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Physiology Lab 1, WBC
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WBC manual count using hemocytometer
Introduction
Blood represent about 8% of total body weight
It consist of 3 type of specialized cellular elements:
Erythrocytes RBCs Leukocytes WBCs Platelets (thrombocytes)
These cells suspended in complex liquid plasma
Introduction Blood perform two major
function:1. Transport through the body
O2 & CO2 Food molecules (Glucose, lipid, aa) Wastes (ex. Urea) Hormones heat
2. Defense of the body against infections & other foreign materials, all WBCs participate in these defenses
Introduction
All various types of blood cells are produced in the bone marrow
Introduction
WBCs (leukocytes) Have nuclei Consist of:
lymphocytes with relatively clear cytoplasm
And 3 types of granulocytes whose cytoplasm contain granules
Introduction
WBCs count is the count of leukocytes in a volume of blood
Expressed as WBCs/mm³
Principle
The # of WBCs is very large, so its practical to dilute a sample with diluting solution (2% Glacial acetic acid with methylene blue)
This solution will lyses cytoplasmic membrane, and leave the nuclei of WBCs.
Material
1. Blood sample (EDTA anticoagulated blood or capillary blood)
2. WBCs diluting pipette3. Diluting solution (2% AA with
methylene blue)4. Hemocytometer5. microscope
The Hemacytometer
contains 2 Neubauer counting chamber
Each chamber contains: *4 WBC counting
squares *Each contains 16
squares
The Hemacytometer
The Hemacytometer
]0.25mm
]0.20mm
Methodology
With a safety bulb 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.
Count and calculate:
Count and calculate:
Calculation
Number of cells/mm³= counted cells in 4 large square x diluting factor x volume correction factor.
Calculation
The dilution factor= total volume/sample volume = 11-1/0.5 =20 Volume correction factor= Desired volume/counted volume 1 mm³/ counted volume
Calculation
counted volume =The total volume of the 4 large squares== Volume x number of large squares= (width x length x depth )x 4= (1mm x 1mm x 1/10 mm) x 4
=0.4mm³ Volume correction factor= 1 mm³/ counted volume = 1 /0.4 = 2.5
Calculation
Number of cells/mm³= counted cells in 4 large square x diluting factor x volume correction factor.
Number of cells/mm³= n x20 x 2.5 = n x 50
Example:
If total # of WBCs in 4 squares is 120 Then the # of WBCs in 1mm³= 120 x 50 =6000
Normal values of WBCs:
Newborn 9000-30000 cell/mm³ Adult 4000-11000 cell/mm³
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
Significance of the test
Leukopenia Result from X-ray therapy Alcoholism Antibiotic therapy Typhoid infection Measls Infectious hepatitis TB And cirrhosis of the liver
Sources of error
1. Flooding of chamber with excess sample
2. Failing to count all the cells in the squares or conversely including artifacts in the count.