1 Human Physiology-I (PHSL 205) Lab 2nd Lab Hakami,Hana A- 2010/2011 White blood cell count Prepared by; Hakami, Hana A Viewed by; Dr.Naseem Siddiqui 2 Human Physiology-I (PHSL 205) Lab 2nd Lab Hakami,Hana A- 2010/2011 White blood cell Blood contents are cells and plasma . Blood cells Include 3 main types are ; thrombocytes , erythrocytes and leukocytes . Blood plasma is the yellow liquid component of blood, in which the blood cells in whole blood would normally be suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid. It is mostly water (90% by volume) and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide (plasma being the main medium for excretory product transportation). Thrombocytes are cells that play a key role in blood clotting. In mammals, thrombocytes are anucleated cell fragments called platelets. The erythrocytes are the most numerous blood cells. They are also called red blood cells. In man and in all mammals, erythrocytes are devoid of a nucleus and have the shape of a biconcave lens. In the other vertebrates (e.g. fishes, amphibians, reptilians and birds), they have a nucleus. White blood cells (WBCs), or leukocytes (also spelled "leucocytes"), are cells of the immune system defending the body against both infectious disease and foreign materials. there are different and diverse types of leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a hematopoietic stem cell. Leukocytes are found throughout the body, including the blood and lymphatic system. 3 Human Physiology-I (PHSL 205) Lab 2nd Lab Hakami,Hana A- 2010/2011 WBC (Leukocyte) Manual Counting Principle Blood sample is mixed and diluted with weak concentration of hydrochloric acid (HCl), or acetic acid (in specified known volumes). Weak acids will lyse red blood cells, and will darken WBC’s to facilitate counting by the hemacytometer. Manual WBC counting is used in cases of very low WBC count (leukopenia) with automated hematology cell counters, and when automated cell counters are not available. Sample EDTA anticoagulated whole venous blood. Reagent and Supplies To Prepare Diluting Fluid 1- Volumetric Flask 100 cc. 2- Serological pipettes. 3- Concentrated HCL 4- Glacial Acetic Acid Preparation of Diluting Fluid Diluting fluid is either: 1% hydrochloric acid in distilled water ( 1 ml Conc. HCL + 99 ml Dist. water). 2% Acetic Acid in distilled water ( Turk’s solution) (2 ml glacial acetic acid + 98 ml distilled water). Glassware, Apparatus, Equipment 4 Human Physiology-I (PHSL 205) Lab 2nd Lab Hakami,Hana A- 2010/2011 1- Neubauer improved hemacytometer. 2- Clean cover slip slide (especially made for the hemacytometer). 3- Automatic micropipette (20 l, 380 l are the required volumes). 4- Gauze 10 x 10 cm 5- Glass/Plastic tubes- (12x75 mm). 6- Handy tally counter. 7- Conventional light microscope. Procedure 1- Mix the blood sample gently but thoroughly by inversion, manually or by mechanical rocking mixer. 2- Pipette 0.38 ml (380 l) of diluting fluid into a 12x75 mm tube. 3- Pipette 0.02 ml (20 l) of well mixed blood to be counted and wipe the tip with gauze into the tube containing diluting fluid and mix the tube. 4- Let the tube stand for 2-3 minutes to ensure complete RBC lyses, then mix well. 5- Prepare the clean hemacytometer and cover it with the designed coverslip. 6- Load one side of the hemacytometer with the aid of a capillary tube or micropipette, do not attempt to overload or underload the hemacytometer. 7- Allow the hemacytometer to sit for several minutes to allow the WBC’s to settle in the counting chamber, to avoid drying effect, place the loaded hemacytometer in a covered Petri dish with a moist gauze, until counting. 8- Place the hemacytometer in the microscope stage. 5 Human Physiology-I (PHSL 205) Lab 2nd Lab Hakami,Hana A- 2010/2011 9- Focus with x10 objective lens (low power), with lowering the condenser. 10-The WBC’s are counted in the 9 corner large squares, with the aid of hand tally counter. 11-Follow the counting pattern shown in the figure below. During counting, do not count cells that touch the right or bottom boundaries to ensure unduplicated counting. Begin 12- The total counted WBC’s in the 9 squares are added together. Fig. WBC’s are counted in the 9 hemacytometer squares If the number of cells in a square varies from any other square by more than 9 cells, the count must be repeated, because this represents an uneven distribution of cells, which is may be caused 6 Human Physiology-I (PHSL 205) Lab 2nd Lab by improper Hakami,Hana A- 2010/2011 mixing of the dilution or improperly filled hemacytometer. Calculations Total WBC count = N x D (mm) x DF A (mm2 ) = c/mm3 Where: N = Total WBC counted by the counting chamber. Depth factor in mm = 10 DF = Dilution Factor = 20 A (mm²) = Area counted = 3 mm x 3 mm = 9 mm² So, Total WBC count = N x 10 mm x 20 9 mm2 = c/mm3 Example 20 19 18 21 14 16 19 16 15 Hemacytometer Squares N= 20 +19 +18 +21 +14 +16 +19 +16 +15 = Tallied 158 Counted WBC Cell Total WBC count = 158 x 10 x 20 9 mm2 = 3500 c/mm3 = 3.5 x 109 /L