Oytip Nathalang

Effects of heparin on polymerase chain reaction for blood white cells

Polymerase chain reaction (PCR) has been used with increasing frequency to diagnose infectious and genetic diseases. In this study, the effects of heparin on PCR were investigated, because heparinized blood may sometimes be used in PCR studies. HLA‐DQA1 gene amplification was used as a model. PCR was clearly interfered with when heparinized blood was used as a source of template DNA, and the degree of interference was affected by the following three factors; (1) type of Taq DNA polymerase; (2) leukocyte count in blood; and (3) concentration of heparin contained. When additional tests were conducted with additions of definite heparin concentrations to a PCR reaction mixture, specimens with large amounts of DNA tended to exhibit less interference by heparin. The addition of ≥ 0.1 to 0.0016 U of heparin per reaction mixture (50 μl) suppressed DNA amplification in a dose‐dependent fashion. We therefore concluded that much care should be taken when heparinized blood is used as a PCR material. J. Clin. Lab. Anal. 13:133–140, 1999.

Human neutrophil alloantigen genotype frequencies in Thai blood donors

BACKGROUND Antibodies to human neutrophil antigens (HNA) can cause transfusion reactions, as well as autoimmune and neonatal neutropenia. This study is the first to report the frequencies of human neutrophil antigen genotypes in the Thai population. MATERIALS AND METHODS Three hundred unrelated, healthy Thai blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok, Thailand were typed for HNA-1a, -1b, -1c, -3a, -3b and -4a using polymerase chain reaction with sequence-specific primers. Moreover, HNA-5a genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism. RESULTS The gene frequencies of HNA-1a, -1b and -1c were 0.470, 0.530 and 0.005, respectively. The frequencies of HNA-3a and -3b were 0.490 and 0.510, respectively. Additionally, the HNA-4a+/+ and HNA-4a+/- genotype frequencies were 0.947 and 0.053, respectively. The frequencies of HNA-5a+/+, HNA-5a+/- and HNA-5a-/- genotypes were 0.641, 0.297 and 0.062, respectively. Compared with other Asian populations, Thais have higher frequencies of HNA-1b (P<0.001). On the other hand, the frequency of HNA-5a observed in Thais is lower than that reported among Koreans (P<0.001). DISCUSSION These findings suggest that Thais would be more susceptible to HNA-1b alloimmunisation. Furthermore, our results could establish a useful human neutrophil antigen donor file to provide more effective transfusion of blood and blood components.

The Characteristics of ABO Antibodies in Group O Thai Blood Donors

This study aimed to characterize anti‐A and anti‐B hemolysins, IgM, and IgG titers in Thai blood donors. Altogether, 300 serum samples from group O donors at the National Blood Centre, Thai Red Cross Society, were screened for anti‐A and anti‐B hemolysins and treated with 0.01 M dithiothreitol to characterize IgM and IgG titers by standard tube technique. Antibody titers were compared with hemolysis grade. Male and female ratio = 1:1.3 and ages ranged from 17 to 60 years. The overall prevalence of anti‐A and anti‐B hemolysins was 69%. Anti‐A and anti‐B hemolysins comprised 18.3% and 16.7%, respectively and 34% had both antibodies. High titers of anti‐A hemolysins were associated with females (P< 0.05), and only anti‐B IgM titers were associated with age (P< 0.05). Interestingly, the association of anti‐A IgM titers, anti‐A IgG titers, and hemolysin grade was demonstrated (P< 0.05). A significant association between hemolysin grade and anti‐B IgM titers was found (P< 0.05). The prevalence of anti‐A and anti‐B hemolysins and high titers of IgM and IgG in Thais are high. Hemolysin grade showed significant associations with IgM titers; therefore, when providing ABO‐incompatible platelet transfusion, especially for female plateletpheresis donors, IgM high titers of anti‐A and anti‐B screening is suggested. J. Clin. Lab. Anal. 26:223‐226, 2012.

Risk Estimation of HNA-3 Incompatibility and Alloimmunization in Thai Populations.

Severe transfusion-related acute lung injury (TRALI) is often due to antibodies in blood components directed against human neutrophil antigen (HNA)-3a. This study aimed to report the genotype frequencies of the HNA-3 system and to estimate the potential risk of HNA-3 incompatibility and alloimmunization in two Thai populations. Eight hundred DNA samples obtained from 500 unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok and 300 samples from the Blood Bank, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand were included. HNA-3 genotyping was performed using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The observed frequencies of the HNA-3a/3a, HNA-3a/3b, and HNA-3b/3b genotypes were 0.528, 0.380, and 0.092 in central Thais and 0.600, 0.350, and 0.050 in northern Thais, respectively. The frequencies were used to estimate HNA-3 incompatibility and risk of HNA-3a alloimmunization. The HNA-3 incompatibility in central Thais (33.28%) was higher than northern Thais (28.75%), corresponding to a significantly higher probability of HNA-3a alloimmunization (P<0.05) similar to Japanese and Chinese populations. This study showed the high risk of HNA-3 incompatibility and alloimmunization, especially in central Thai blood donors. A molecular-based identification of the HNA-3 genotype of female donors is suggested to reduce the risk of TRALI following plasma and whole blood allogeneic transfusion.

Frequency of FCGR3B Alleles in Thai Blood Donors

Background Human neutrophil antigens (HNAs) are involved in autoimmune and alloimmune neutropenia and transfusion-related acute lung injury. The HNA-1 system is important in immunogenetics, and allele frequencies have been described in different populations. This study investigated the frequency of FCGR3B alleles encoding HNA-1a, HNA-1b, and HNA-1c among Thai blood donors and compared these frequencies with those previously reported for other populations. Methods Eight hundred DNA samples obtained from unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok, and the Blood Bank, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, were included. Samples were simultaneously typed for each FCGR3B allele using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. Results The frequencies of FCGR3B*1, FCGR3B*2, and FCGR3B*3 alleles in central Thai blood donors were 0.548, 0.452, and 0.004, respectively; only FCGR3B*1 and FCGR3B*2 alleles were found in northern Thai blood donors (0.68 and 0.32, respectively). Compared with other Asian populations, central Thais had higher frequencies of the FCGR3B*2 allele (P<0.001), while the frequencies of the FCGR3B*1 and FCGR3B*2 alleles in northern Thais were similar to those previously reported in Taiwanese and Japanese populations. In contrast, the frequencies of the FCGR3B*1 and FCGR3B*2 alleles in the northern Thai population were statistically different from those observed in central Thai, Korean, German, and Turkish populations. Conclusions FCGR3B allele frequencies were significantly different between central and northern Thai blood donors. Our in-house PCR-SSP method is a simple, cost-effective, and convenient method for FCGR3B allele detection.

Jk(a-b-) phenotype screening by the urea lysis test in Thai blood donors.

BACKGROUND The Jk(a-b-) phenotype is rare in most populations and often detected after transfusion or pregnancy. After immunisation, anti-Jk3 forms and it can be difficult to find compatible Jk(a-b-) donors. Using anti-Jk(a) and anti-Jk(b) in a conventional tube method is unsuitable for identifying Jk(a-b-) in mass screening of blood donors. Jk(a-b-) phenotypes are associated with the absence of urea transporters on erythrocytes, making red blood cells (RBC) resistant to lysis by 2M urea, while Jk(a+b-), Jk(a-b+) and Jk(a+b+) phenotypes are susceptible to lysis. MATERIALS AND METHODS We screened for Jk(a-b-) phenotypes in blood donors by the urea lysis test using a 96-well microplate. The Jk(a-b-) phenotypes were confirmed by the indirect antiglobulin test (IAT). RESULTS Altogether, 20,163 blood samples from Thai blood donors were tested and only RBC from five samples were resistant to lysis by 2M urea, while 20,158 samples were completely lysed within 5 min. In an IAT, both anti-Jk(a) and anti-Jk(b) failed to agglutinate RBC from all five samples. Using a micro-titre plate, the direct urea lysis test, costs * 0.01, about 480 times less than IAT. Moreover, the test time for each plate (94 samples) is about 18 times less than that for IAT. CONCLUSION Jk(a-b-) phenotype screening by the direct urea lysis test on samples in a micro-titre plate is simple, cost-effective and practical for mass screening of blood donors.

Genotyping of HPA-1 to -7 and -15 in the Thai population using multiplex PCR

Background: Different polymerase chain reaction (PCR) techniques for human platelet antigens (HPA) genotyping have been implemented, in order to diagnose the clinical syndromes of patients with thrombocytopaenia and provide effective HPA‐matched platelet donors.

Comparison of a Simple‐Probe Real‐Time PCR and Multiplex PCR Techniques for HPA‐1 to HPA‐6 and HPA‐15 Genotyping

We aimed to compare HPA‐1 to HPA‐6 and HPA‐15 genotyping results obtained by a simple‐probe real‐time polymerase chain reaction (PCR) technique with the multiplex PCR technique.

Distribution of DI*A and DI*B Allele Frequencies and Comparisons among Central Thai and Other Populations

Alloantibodies to the Diego (DI) blood group system, anti-Dia and anti-Dib are clinically significant in causing hemolytic transfusion reactions (HTRs) and hemolytic disease of the fetus and newborn (HDFN), especially in Asian populations with Mongolian ancestry. This study aimed to report the frequency of the DI*A and DI*B alleles in a Central Thai population and to compare them with those of other populations previously published. Altogether, 1,011 blood samples from unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok were included. Only 391 samples were tested with anti-Dia by conventional tube technique. All samples were genotyped for DI*A and DI*B alleles using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The DI phenotyping and genotyping results were in 100% concordance. The DI*A and DI*B allele frequencies among 1,011 Central Thais were 0.0183 (37/2,022) and 0.9817 (1,985/2,022), respectively. Allele frequencies were compared between Central Thai and other populations. Our data shows that DI*A and DI*B allele frequencies are similar to Southeast Asian, Brazilian, Southern Brazilian and American Native populations; whereas, these frequencies significantly differ from those reported in East Asian, Italian, Alaska Native/Aleut, Hawaiian/Pacific Islander and Filipino populations (P<0.05), corresponding to the results of a matrix of geometric genetic distances. This study confirms that the prevalence of DI*A and DI*B alleles among Central Thais is similar to Southeast Asians and different to others populations of the world. A PCR-based identification of DI genotyping should overcome some of the serological limitations in transfusion medicine and provides a complementary tool for further population-genetic studies.

Red Cell Genotyping by Multiplex PCR Identifies Antigen-Matched Blood Units for Transfusion-Dependent Thai Patients

Background: Antigen-negative red cell transfusion is required for transfusion-dependent patients. We developed multiplex PCR for red cell genotyping and calculated the possibility of finding compatible predicted phenotypes in Thai blood donor populations according to red cell alloantibodies found among Thai patients. Methods: 600 DNA samples obtained from unrelated healthy central and northern Thai blood donors were tested with the newly developed multiplex PCR for FY*A, FY*B, JK*A, JK*B, RHCE*e, RHCE*E, DI*A and GYP*Hut, GYP*Mur, GYP*Hop, GYP*Bun, and GYP*HF allele detections. Additionally, the possibility of finding compatible predicted phenotypes in two Thai blood donor populations was calculated to estimate the minimal number of tests needed to provide compatible blood. Results: The validity of multiplex PCR using known DNA controls and the phenotyping and genotyping results obtained by serological and PCR-SSP techniques were in agreement. The possibility of finding at least one compatible blood unit for patients with multiple antibodies was comparable in Thai populations. Conclusions: The multiplex PCR for red cell genotyping simultaneously interprets 7 alleles and 1 hybrid GP group. Similar strategies can be applied in other populations depending on alloantibody frequencies in transfusion-dependent patients, especially in a country with limited resources.