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: Transport through the body O2 & CO2 Food molecules (Glucose, lipid, aa) Wastes (ex. Urea) Hormones heat 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 Blood sample (EDTA anticoagulated blood or capillary blood) WBCs diluting pipette Diluting solution (2% AA with methylene blue) Hemocytometer 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 Flooding of chamber with excess sample Failing to count all the cells in the squares or conversely including artifacts in the count.