manualwbc-austincommunitycollege.docVIP

MANUAL WBC LAB OBJECTIVE The student will be able to perform, within (20% accuracy as compared to the automated result, five manual white blood cell counts using the Unopette system. PRINCIPLE Free-flowing capillary or well-mixed anticoagulated (EDTA) venous blood is added to a diluent (ammonium oxalate) at a specific volume in the Unopette reservoir. The diluent lyses the erythrocytes but preserves leukocytes and platelets. The diluted blood is added to the hemacytometer chamber. Cells are allowed to settle for 10 minutes before leukocytes and platelets are counted. SPECIMEN EDTA-anticoagulated blood or capillary blood is preferred. REAGENTS, SUPPLIES AND EQUIPMENT Unopette reservoir no. 5854/5855 (1.98 ml of diluent + 0.02 ml blood = 1/100 dilution)) Ammonium oxalate 11.45 g Sorensen’s phosphate buffer 1.0 g Thimerosal 0.1 g Purified water qs to 1000 mL Unopette capillary pipet, 20-μL capacity Hemacytometer and coverslip Microscope Lint-free wipe (Kim Wipes) Alcohol pads Hand counter Petri dish with moist filter paper  Note on the Hemacytometer The hemacytometer counting chamber is used for cell counting. It is constructed so that the distance between the bottom of the coverslip and the surface of the counting area of the chamber is 0.1 mm. The surface of the chamber contains two square ruled areas separated by an H-shaped moat. These two squares are identical, allowing the technologist to duplicate the cell count. Each has a total area of 9 mm2 (1 mm on each side). These squares are divided into nine primary squares with an area of 1mm2. The four corner primary squares are used when counting leukocytes. (If the WBC is very low, you would count all 9 squares.) These 4 large corner squares contain 16 smaller secondary squares, each with an area of 0.04 mm2. All 25 secondary squares of the center primary square are used to count platelets, and each of these 25 squares is further divided into 16 s