Proceeding2562

1534 การประชุมวิชาการระดับชาติมหาวิทยาลัยทักษิณ ครั้งที่ 29 ประจ�ำปี 2562 วิจัยและนวัตกรรมเพื่อการพัฒนาที่ยั่งยืน Introduction Detection of blood group in hospital laboratory routinely uses specific antibody to detect the antigen on the red cell, so called serology test. Advantages of this technique are convenient and not expensive, however, some certain antibodies are not commercially available or highly expensive causing disadvantage of serological test. In addition, when the serological result is discrepancy, molecular technique is needed to confirm blood group in some patients. Due to highly polymorphism of human blood group antigen, several PCR methods have been modified to determine blood group specific alleles such as real time PCR (1), multiplex PCR (2,3) sequence specific primer PCR (PCR-SSP) (4-7). PCR-SSP are widely used in detection of single nucleotide polymorphisms (SNPs) on different blood group as well as human leukocyte antigen (HLA) and human platelet antigen (HPA) (4-6). Initiation of PCR requires DNA template and source of DNA is the extraction from white blood cell in whole blood. Sample of whole blood was stored in anticoagulants. Anticoagulants are the chemical agents which prevent the coagulation of blood such as EDTA (ethylene diamine tetraacetic acid), sodium citrate, heparin, or ACD (acid citrate dextrose). EDTA is commonly used in laboratory. It binds calcium in blood therefore deplete calcium lead to prevent coagulation. Sodium citrate binds calcium in blood as well but does not stimulate platelet function therefore it is suitable for coagulation and platelet function tests (8-9). For molecular purpose, blood sample should be collected in ACD or EDTA because stability and yield of cellular DNA are higher than those of sample collected in heparin (10). Heparin is antithrombin and antithromboplastin. It prevents conversion of prothrombin to thrombin resulting in anticoagulation. Heparin cannot be used as anticoagulant for cell counts because heparin causes clumping of cells resulting in erroneous counts and moreover inhibit gene amplification of PCR when sample were collected in heparinized tube (10-11). In blood bank, donor blood in blood bag unit is stored in ACD or CPDA1 (citrate phosphate dextrose adenine-1) because they are inexpensive, easy to prepare, and have good properties for blood preservation. ACD and CPDA1 are acidic with a pH of 5 to 5.8 so that the dextrose does not caramelize when the solutions are autoclaved (13). CPDA1 can preserve 2,3-diphosphoglycerate (2,3-DPG) therefore, preserves red blood cells longer than ACD. Meanwhile, side tubes of blood donor have been collected during blood donation for blood group phenotyping, infectious serological and molecular testing. Most laboratories used EDTA as common coagulation for those purposes for the convenience. However, particular blood bank laboratory as well as in Regional Blood Centre XII Songkhla, Thailand using different blood coagulation types for specific purpose; CPDA1 blood for blood group phenotyping, clot blood for serological test, and EDTA blood for molecular test. Even though CPDA1 can preserve cells longer than EDTA blood, but its effect on molecular processes has not been demonstrated. So, this study aimed to investigate the effect of CPDA1 as anticoagulation for blood group detection by PCR-SSP. Yield of DNA at various storage times and RHD gene amplification of CPDA1 and EDTA blood samples were determined and compared.