WBC manual count using Hemocytometer Introduction Cs B

WBC manual count using Hemocytometer

Introduction Cs B W f o r e b m u ate n u q e d a s i e r e h t ut. ether o h w d e e i r n r i a m c r s e i t t e n d ll cou • To e c e t i h w a , n o in the circulati r e b m u n e h t s a essed r p x e y l l a n o i t i re trad a s t n u o c od. l o l l e b c f o r e t i l l • Blood i ic m of cells per cub

• 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 • To accurately count WBC in Chamber Principle • 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 Hemacytometer and coverslip Thoma white pipette Microscope Alcohol pads

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.

WBCs Procedure Using the micropipette with a white bead. 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). White

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

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

Thoma Pipet WBCs RBC • Aspirate to 0. 5 • Dilute to 101 • DF = 202 WBC • Aspirate to 0. 5 • Dilute to 11 • DF = 22 RBCs

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. 1 = 0. 004 mm 3 • Volume of each 16 area = 0. 004 x 16 = 0. 064 mm 3

Calculation • Let X is the number of WBCs in 16 areas • So X • Y Y= X x 1 x 22 0. 064 mm 3 1 mm 3 WBCs / mm 3 * Normal range of WBCs in Women = (4, 000 to 11, 000 per cubic millimeter of blood)

w o n k to g n i t s Intere ! • A low white blood cell count is called leukopenia. A high white blood cell count is termed leukocytosis. Ø 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. Ø 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.