WBC manual count using Hemocytometer

Introduction To determine whether there is adequate number of WBCs in the circulation, a white cell count is carried out. Blood cell counts are traditionally expressed as the number of cells per cubic milliliter of blood.

The hemocytometer was invented by Louis-Charles Malassez. The hemocytometer is a device used to count cells. It was originally designed for the counting of blood cells. The hemocytometer was invented by Louis-Charles Malassez. Consists of a thick glass microscope slide.

Objective Principle To accurately count WBC in Chamber WBCs are counted using hemocytometer with a white bead, after rupture of RBCs and staining of WBCs in order to be more easily spotted. The used stain is gentian violet dissolved in 3% acetic acid, which is used to disrupt RBCs.

Hemocytometer

haemocytometer chamber Thoma white pipette Rubber sucking tube

Requirements White blood cells count diluting fluid WBCs RBCs White blood cells count diluting fluid Hemacytometer and coverslip Thoma white pipette Alcohol pads Microscope

Applications Blood counts: for patients with abnormal blood cells. Sperm counts. Cell culture: when subculturing or recording cell growth over time. Cell processing for downstream analysis: accurate cell numbers are needed in many tests (PCR, flow cytometry). Measurments of cell size: in a microphage, the real cell size can be inferred by scaling it to the width of a hemocytometer square, which is known.

Procedure Using the micropipette with a white bead. White WBCs Procedure Using the micropipette with a white bead. White Suck capillary blood up to the mark 0.5. Suck up gentian violet solution up to 11 marks (DF= 20). Count the WBCs in the sixteen big areas (each of 16 squares = one large square).

Count the WBCs in the sixteen big areas (each of 16 squares = one large square).

Do not count cells touching Counting Rule Bottom line Right line Do not count cells touching

Thoma Pipet WBC RBC Aspirate to 0.5 Dilute to 11 DF = 22 WBCs RBCs RBC Aspirate to 0.5 Dilute to 101 DF = 202 WBC Aspirate to 0.5 Dilute to 11 DF = 22

Hemocytometer Chamber Grids RBC use 5 small squares in the center large square WBC use 4 corner large squares

Calculation Depth = 0.1 mm Length=0.2 mm Width= 0.2 mm Volume of each area= 0.2 x 0.2 x 0.1 = 0.004 mm3 Volume of each 16 area = 0.004 x 16 = 0.064 mm3

Calculation Let X is the number of WBCs in 16 areas So X 0.064 mm3 Y 1 mm3 Y= X x 1 x 22 WBCs / mm3 0.064 * Normal range of WBCs in Women = (4,000 to 11,000 per cubic millimeter of blood)

Interesting to know ! A low white blood cell count is called leukopenia. A high white blood cell count is termed leukocytosis. Leukocytosis is white blood cells (the leukocyte count) above the normal range in the blood. It is frequently a sign of an inflammatory response, most commonly the result of infection, but may also occur following certain parasitic infections or bone tumors. It may also occur after strenuous exercise, convulsions such as epilepsy, emotional stress, pregnancy and labour, anesthesia, and epinephrine administration. Low white cell count may be due to acute viral infections, such as with a cold or influenza. It can be associated with chemotherapy, radiation therapy, myelofibrosis, aplastic anemia (failure of white cell, red cell and platelet production). and AIDS is also a threat to white cells.