Proceeding2562

1536 การประชุมวิชาการระดับชาติมหาวิทยาลัยทักษิณ ครั้งที่ 29 ประจ�ำปี 2562 วิจัยและนวัตกรรมเพื่อการพัฒนาที่ยั่งยืน Statistical analysis Statistical analyses were performed using SPSS 23. Mann-Whitney U test and Kruskal-Wallis test were used to compare DNA concentration between EDTA and CPDA1 at different storage times. Data were considered statistical significant when p-value < 0.05. Graphs were generated with GraphPad Prism 6. Results Yields of gDNA from EDTA and CPDA1 blood EDTA and CPDA1 blood were collected from the same individual donors (n=40). DNA extraction of EDTA blood sample on day 3, 10 and 30 post-venipuncture showed average yield of DNA for 1,655.50, 1,828.00 and 1,403.50 ng, respectively. The average yield of DNA from CPDA1 blood sample on day 3, 10 and 30 were 1,584.50, 1,412.00 and 1,836.50 ng, respectively. Compare between the two anticoagulants, DNA concentration of EDTA and CPDA1 blood was similar after 3 days prior the extraction. However, the significances were observed after extending the storage time in the 4  C condition. At 10-day post-storage, hemolysis was observed in blood samples preserved in EDTA but not CPDA1 (data not shown), moreover, DNA concentration of EDTA blood samples was significantly higher than DNA from CPDA1 samples (p = 0.045). Interestingly, on 30 days post-storage, DNA in CPDA1 blood samples had significantly higher than those of EDTA samples (p = 0.037) (Figure 1). In addition, all EDTA blood samples showed hemolysis whereas, less than 10% of CPDA1 blood sample started to be lysed. DNA amplification from EDTA and CPDA1 blood A pair of EDTA and CPDA1 blood collected from the same donors (n=40), all individuals were RhD positive based on standard serological test, hence, RHD gene must be inherited. PCR-SSP amplified two amplicons from each sample, one control fragment at 1,200 bp, while the 600 bp fragment specific for RhD positive. RhD positive individual showed both amplicons, whereas, RhD negative individual showed only the 1200 bp control fragment. The 429 bp of human growth fragment was used as internal control (Figure 2). All DNA samples from EDTA and CPDA1 blood, extracted on day 3, 10, and 30 days showed two amplicon of RHD gene (Figure 3) indicated that no interference of CPDA1 during amplification process of PCR. Discussion and conclusion CPDA1 has been used to preserve donor red cell in blood bag which can prolong life-span of the cell and safe the transfusion patients. EDTA is anticoagulant widely used in hematological manner such as study of cell morphology or cell count, in addition, most of molecular study required DNA sample from EDTA blood. However, fresh EDTA blood is recommended for DNA extraction from buffy, otherwise the longer storage, the more necrosis and the loss of DNA from leukocyte (13). CPDA1 composes of dextrose and adenine which is ATP supplement for cell metabolism, hence, can prevent hemolysis or necrosis of