Genotyping of human platelet antigens in ethnic Northeastern Thais by the polymerase chain reaction-sequence specific primer technique.

Human platelet antigens (HPA) are important in neonatal alloimmune thrombocytopenia (NAITP), post-transfusion purpura (PTP), refractoriness to platelet transfusion therapy and population genetics. The distribution of HPA in a Northeast Thai population was studied. 300 healthy, unrelated, and ethnic Northeastern Thais were randomly selected. Using the polymerase chain reaction-sequence specific primer technique (PCR-SSP), the frequency of HPA-1, -2, -3, -4, -5 and -6 were determined. The phenotype frequencies were 100 per cent for HPA-1a, 4a, 5a, and 6a. For HPA-1b, 2a, 2b, 3a, 3b, 5b and 6b, the frequencies were 5.7, 99.7, 12.3, 78.0, 71.3, 7.3 and 3.0 per cent, respectively. The HPA-4b was not found. The HPA frequencies in our subjects were quite similar to other Asian populations but were different from Caucasians. The distribution of HPA genotypes encountered in our study indicate that HPA-1a, -4a, -4b, -5a and -6a will not be involved in NAITP, PTP and refractoriness to platelet transfusion therapy in Northeastern Thais. Moreover, HPA-1b, -2a, -2b, -3a, -3b, -5b and -6b may induce alloantibodies in these patients.

DNA typing of the HLA-A, -B and -C genes: possible MHC class I haplotypes in the northeastern-Thais.

The phenotype and gene frequencies of HLA class I were studied in the Northeastern Thai population. Blood samples were collected from 100 unrelated healthy northeastern-Thais. HLA-A, -B and -Cw alleles were determined using the polymerase chain reaction- amplification refractory mutation system (PCR-ARMS). 12 HLA-A, 20 HLA-B and 14 HLA-Cw alleles were found. Linkage disequilibrium analysis indicated the existence of 7 HLA-A-B and 19 HLA-B-Cw haplotypes. A*0207-Cw*01-B*4601 was the most common possible haplotype in this population. These results provide regional basic information for further studies in anthropology, organ transplantation and MHC disease associations in the northeastern-Thais.

Production and evaluation of Taq DNA polymerase.

Taq DNA polymerase is an enzyme essential in performing Polymerase Chain Reaction (PCR) which has recently become a basic technology in research and diagnostic laboratories. In order to reduce the cost of research work in Thailand, recombinant Taq DNA polymerase was locally produced from pTaq cloned in E. coli. The enzyme was characterized and evaluated in comparison with the commercial Taq DNA polymerase produced by Perkin Elmer Cetus, U.S.A. The yield of enzyme was 6.72 mg/ml and the activity of 9,524 units/mg protein with the total of 448,000 units/litre of the bacterial culture. The preparation was free of DNase based upon its ability to degrade Lambda DNA evaluated by gel electrophoresis. Although the enzyme produced gave a high DNA polymerase activity, the preparation was not as pure as the enzyme produced by Perkin Elmer Cetus. Immunoblot analysis indicated that the enzyme preparation contained the products of enzyme degradation obtained during preparation and bacterial protein contaminations. In spite of the existence of bacterial proteins in the preparation, the Taq enzyme produced was proved to be applicable in performing PCR such as the PCR-SSP (Sequence Specific Primers) typing for HLA-DR. The cost of enzyme preparation was about 256 times less than that of the commercial enzyme. Economically, the locally produced Taq DNA polymerase can be used efficiently in the research laboratories performing PCR based typing of the HLA genes.

Distribution of HLA-A and B antigens in northeastern-Thais.

The phenotype and gene frequencies of HLA class I were studied in the Northeastern Thai population. Blood samples were collected from 100 unrelated healthy Northeastern Thais. HLA-A and B antigens were typed by using the standard microlymphocytotoxicity test. Twelve HLA-A and twenty-five HLA-B antigens were found in this population. HLA-A2, A24, A11 and the HLA-B46, B15, B22 antigens are commonly found in this group. Linkage disequilibrium analysis indicated the existence of fifteen haplotypes. The HLA-A2, B46 haplotype was the most common. These results will be useful for further studies in anthropology, organ transplantation and MHC associated disease in Northeastern Thais.

Immunoblot analysis to demonstrate antigenic variability of clinical isolated. Pseudomonas pseudomallei.

Pseudomonas pseudomallei (Ps.ps.) is the causative organism of melioidosis, and is widely distributed in Southeast Asia and Northern Australia. Clinical manifestations range from subclinical infection to fulminant septicemia. To demonstrate the antigenic variability of Ps.ps., 62 clinical isolates from 31 blood, 13 sputum, 9 pus, 3 urine and 6 body fluid culture specimens were studied by SDS-PAGE and immunoblotting. In SDS-PAGE, there were approximately 20 antigenic components with molecular weights ranging from 14 to 66 kilodaltons (KD) which suggested that there was antigenic variability among these 62 clinical isolates of Ps.ps. Attempts to correlate immunoblot profiles with clinical illness or sources of specimens were not successful but 6 common antigens were identified with molecular weight of 17.5, 21, 33, 34, 40 and 45 KD, respectively. Among these antigens, the 45 KD component was recognised by all patients' sera. Thus, the 45 KD protein antigen may be useful for the future approach in immunodiagnosis of melioidosis.