Collection of Blood Samples Determination of the Number of Platelets

C.3 Preparation of the Thrombocytopenic Group The method used by Crow and others 2003 was adapted in this study to induce thrombocytopenia to the white mice. Two µg of the 0.5 mgml rat anti-white mice antiplatelet antibody MWReg30 already in 200 µL Phosphate-buffered saline PBS of pH 7.2 from Genomax Technologies, Singapore was injected to the white mice intraperitoneally. The white mice were restrained properly refer to Appendix A and were held at a slightly downward angle. The surface of the area to be injected was disinfected first by rubbing cotton in 75 alcohol on the area. Then, the 26G ½‖ needle of the 1 mL syringe was injected on the left side of the white mice, halfway between the midline and the top of the hind leg, at approximately 0.5 cm deep into the abdominal cavity and at an angle of 15 -20. The solution was then injected slowly into the white mice at a constant rate. After that, the needle was removed and the white mice were put down gently. The white mice will have to become thrombocytopenic after 24 hours according to Crow and others 2001.

J. Collection of Blood Samples

Blood samples were collected using the tail-nicking method. The white mice were warmed under a lamp prior to blood collection. The lamp was placed over the cage for five minutes. The temperature at the level of the white mice did not exceed 85 - 90° F. The white mice were placed in a restrainer and the tail was prepped with 70 ethanol. The tail was stabilized with the thumb and forefinger of the hand that was not to be used to nick the tail. It was gently nicked using a 11 scalpel blade at the lateral tail vein in the general area around the midline of the tail. Blood was allowed to flow or drop into microcontainer with EDTA. The tail was not squeezed nor was not attempted to be milked to avoid tissue damage and contamination of the blood sample with tissue fluids. Good hemostasis was ensured using a sterile gauze pad after the appropriate volume of 0.25-0.50 mL of blood has been collected.

K. Determination of the Number of Platelets

Ten µL of blood from the blood sample was diluted in 1:100 dilution of 1 ammonium oxalate solution refer to section B.3. It was transferred to a reservoir using a capillary pipet. It was allowed to stand for 10 minutes to allow erythrocytes to hemolyze. The diluted blood was then charged on a clean hemacytometer by gently squeezing the sides of the reservoir to expel the contents until the chamber has been properly filled. The hemacytometer was put under a petri dish with a moistened filter paper together with it to prevent evaporation. It was allowed to stand there for 10 minutes to allow the cells to settle. The hemacytometer was mounted under a 40X light microscope and was photographed for the platelets to be counted. The number of platelets in all the 25 small center squares of the hemacytometer counting chamber was counted, wherein the first row, platelets will be counted from left to right, and right to left in the second row and so on. The number of platelets counted was averaged and was multiplied by 1000 to get the total platelet count of the white mouse. The data gathered was in ―number of platelets per μL‖. This method was based on the manual ―WBC Platelet Determination for Manual Methods‖ of Becton, Dickinson, and Company 1885. Refer to Appendix C for the laboratory manual of ―Manual Platelet Count‖ by Austin Community College District, TX 2010.

L. Infusion of Albumin to Test Subjects