LATTLE EXAMINATION Platelet properties: break easily, tend to stick to foreign surfaces, clot easily, difficult to distinguish from small feces. There are two ways to count platelets: 1) Direct Consisting of the Reeks Ecker method and the Breacher Cronkite method. In the Rees Ecker method The diluent solution used is BCB (Brilliant Cresyl Blue) which contains substances blue color so that platelets will be bright bluish under the microscope. BCB solution does not contains substances that can lyse erythrocytes so that erythrocytes will remain visible. Level error is about 16-25%. Cronkite's Breacher Method uses NH4 Oxalate 1% as diluent solution and functions to lyse erythrocytes, platelets will not appear color/clear under a microscope. Error rate 8-10%. 2) Indirect The Fonio method uses a peripheral blood smear and then stains I using Wright or Giemsa or May Grunwald. Platelets are counted in 1000 erythrocytes which is evenly distributed. The absolute number of platelets is calculated from the number of erythrocytes. The Fonio method of examination is rougher than the direct method. Practicum a. Tools and materials Materials: Capillary blood, cotton, 70% alcohol, Rees Ecker's solution, sterile lancet Equipment: Improved Neubauer Hemocytometer, Microscope b. Work procedures 1) Preparation of counting booth a) Prepare the counting chamber and cover glass in a clean and dry condition b) Moisten with a little water the two embankments of the counting chamber c) Place the cover glass on top of the counting chamber then slide it up and down simultaneously repeat until Newton's rings (rainbows) are formed on both embankments. 2) Dilution of blood using an erythrocyte thoma pipette a) Suction blood until the limit mark is 101 b) Clean the outside of the pipette tip from any remaining blood that is still attached c) Suction the Rees Ecker reagent to the 101 mark, avoiding any air bubbles d) Shake the thoma pipette for 2-3 minutes then discard the first 2-4 drops e) Put it in the counting chamber by pouring 1 drop on the edge cover glass f) Incubate 15 minutes to give the cells a chance to spread and settle down the platelets settle. 3) Counting Platelet cells a) Count the platelets under a microscope at 40x magnification. b) Count the platelets in 16 small boxes, with a size of 0.05 mm x 0.05 mm and count platelets in 25 medium boxes with a size of 0.2 mm x 0.2 mm. c) Cells that touch the left and top border lines Platelet count Platelet Count ( /µL blood) = nx Fp Vb Vb = 25 x L x W x H = 25 x 1/5 x 1/5 x 0.1 = 0.1 µL blood Fp = 100 volume of platelets taken n = Number of platelets counted in the counting room Fp = Dilution factor Vb = calculated plane volume Normal values: Women = Men (200000 – 500000/µL blood)