Prevalence of Antibody against Angiotensin II Type 1 Receptor (AT1R) Among Thai Kidney Transplant Patients.

BACKGROUND: Non-HLA antibodies specific to angiotensin II type 1 receptor (AT1R-Ab) are associated with antibody-mediated rejection after kidney transplantation. This study aimed to determine the prevalence of AT1R-Ab among pre-transplantation Thai patients and to compare the association of patient demographics with AT1R-Ab levels. METHODS: This cohort study enrolled nonsensitized kidney transplant patients with negative panel reactive antibodies at King Chulalongkorn Memorial Hospital, Bangkok, Thailand. AT1R-Ab level was measured with the use of an enzyme-linked immunosorbent assay kit in all pre-transplantation serum samples with the use of a cutoff of >17.0 U/mL to distinguish AT1R-Ab+ from AT1R-Ab at risk and AT1R-Ab- groups. RESULTS: In all, 70 patients were enrolled with a mean age of 45 (range, 17-68) years, of which 52 were male. Six patients (8.6%) were positive for AT1R-Ab (>17.0 U/mL). The mean age in the AT1R-Ab+ group was lower than in the AT1R-Ab at risk and AT1R-Ab- groups (P = .06). Moreover, the AT1R-Ab+ group had significantly more patients ≤30 years of age (P = .01). No differences were found regarding sex, body mass index, transplant characteristics, causes of end-stage renal disease, or kidney biopsy diagnosis. CONCLUSIONS: This study is the first to report the prevalence of AT1R-Ab among Thai kidney transplant patients. Further studies are suggested for the application of AT1R-Ab detection for routine testing.

Evaluation of Magnetized-Erythrocyte Group Antigens to Detect ABO Antibodies.

Screening for IgM titers of anti-A and anti-B is recommended when providing ABO incompatible platelet transfusion. The life-time of reagent cells depends upon the preservative diluents. We aimed to evaluate the IgM titers of anti-A and anti-B testing with magnetized-erythrocyte group antigens (MEGA) and fresh RBCs and study the relationship of ABO antibody titers between both techniques. Altogether, 100 serum samples from group O donors at the National Blood Centre, Thai Red Cross Society, Bangkok, Thailand were included. EDTA blood from three different A and B blood group individuals was prepared as fresh reagent RBCs and MEGA. Each serum sample was tested simultaneously for IgM anti-A and anti-B titers using fresh RBCs and MEGA by standard tube technique. Antibody titers were compared between both techniques. Test for reproducibility and stability of MEGA were performed. The IgM anti-A and anti-B titers using fresh RBCs yielded higher agglutination scores than MEGA (P < 0.001). However, a good correlation was obtained in the agglutination titers (anti-A, r = 0.838 and anti-B, r = 0.877). The mean and standard deviation of anti-A and anti-B titers using MEGA from five sera in triplicate showed no significant difference (P > 0.05). Moreover, the titer test results using MEGA after dilution remained stable up to 8 h. The MEGA can be used as a replacement for fresh RBCs to perform ABO serum grouping. It is simple to use, avoids centrifugation and provides good results in terms of stability and reproducibility.

Comparison of two commercial real-time PCR assays for detection of dengue virus in patient serum samples.

This study evaluated and compared the performance of two real-time PCR assays using nested reverse transcription (RT)-PCR as the reference method. Among 117 nested RT-PCR-positive cases, the abTES DEN 5 qPCR kit detected 97.4% of dengue virus (DENV) infections, while the innuDETECT Dengue TwoStep assay did so for 44.4%. Sensitivity varied by infecting serotype and the stage of infection. The abTES kit has the potential to replace nested RT-PCR for the rapid diagnosis of DENV infection.

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. OBJECTIVES: The aim of this study is to develop an in-house multiplex PCR for HPA-1 to -7 and -15 genotyping in the Thai population. METHODS: One hundred DNA samples of known HPA genotyping by the PCR with sequence-specific primers (PCR-SSP), as previously described, were tested with the multiplex PCR. Additionally, 300 DNA samples of group O donors were tested for HPA-1 to -7 and -15 genotyping using multiplex PCR. RESULTS: The comparison of HPA-1 to -7 and -15 genotype results between multiplex PCR and PCR-SSP technique was in 100% concordance. Interestingly, HPA-2b2b genotype was found in two samples; however, other low-incidence genotypes such as HPA-1b1b, HPA-5b5b, HPA-6b6b and HPA-7b7b were not found in this study. Moreover, 30 samples were randomly tested twice for HPA genotyping using the multiplex PCR and demonstrated reproducible results. CONCLUSIONS: This study shows that the in-house multiplex PCR is simple, cost-effective and suitable for HPA genotyping for routine laboratories in other developing countries. Nevertheless, a large-scale evaluation of this technique through multicentre analysis is suggested.

HLA-A, -B, -DR haplotype frequencies in the Thai Stem Cell Donor Registry.

This study aimed to report antigen and haplotype frequencies (HFs) in the Thai Stem Cell Donor Registry (TSCDR). From 16,807 human leukocyte antigen (HLA)-A, -B, and -DR typed donors, 19 HLA-A, 41 HLA-B, and 13 HLA-DR antigens were found. HLA-A43, A80, B78, B82, B83, and DR18 were not observed. A total of 1921 haplotypes were estimated and 245 haplotypes were reliable and their cumulative HF was 75.74%. Similar to previous Thai subpopulation studies, the most common haplotypes were HLA-A33-B58-DR17 (4.54%), A2-B46-DR9 (4.11%), A33-B44-DR7 (2.85%), and A11-B75-DR12 (2.00%). To evaluate the probability in finding matched donors, from April 2002 to August 2008, 793 Thai patients were requested for unrelated stem cell donor searching. The number of patients with HLA-A, -B, -DR split matched donor found were 49%. Only 8% of the patients found matched donor within 1 month. However, high-resolution HLA-A, -B, -DR typing in TSCDR is suggested to accurately estimate HFs and evaluate the probability in finding a matched donor for patients in the future.

Detection of MNS hybrid molecules in the Thai population using PCR-SSP technique.

We developed a polymerase chain reaction-sequence-specific primer (PCR-SSP) technique to screen for hybrid molecules in the MNS blood group in the Thai population using two sets of newly designed primers specific for four GYP(B-A-B) hybrids, GP.Mur, GP.Hop, GP.Bun and GP.HF, and two GYP(A-B-A) hybrids, GP.Vw and GP.Hut. One thousand and forty-one blood samples were tested with human anti-Mi(a) by conventional tube technique, and 598 samples of these were tested by the PCR-SSP technique. Ninety-four samples (9.03%) were strongly positive with human antisera by conventional tube technique. For PCR-SSP test results, the GP.Hut, GP.Mur, GP.Hop, GP.Bun and GP.HF genotypes were amplified with the first set of primers, whereas GP.Vw genotype was amplified with a second set of primers. The GYP(A-B) hybrids (GP. Hil and GP.JL), GYP(A-B-A) hybrids (GP.Nob, GP.Joh and GP.Dane), GYPA, GYPB and GYPE were not amplified by either set of primers. Results of testing 94 Mi(a+) and 504 Mi(a-) by conventional tube technique and PCR-SSP were concordant. This study shows that analysis by PCR-SSP is simple and convenient; therefore, it can be used as an alternative to conventional tube technique for mass screening for MNS hybrids, especially when specific antisera are not available.

Differences in ABO antibody levels among blood donors: a comparison between past and present Japanese, Laotian, and Thai populations.

Passively transfused blood group antibodies cause clinical problems. High titers of anti-A and anti-B seem to be one reason for hemolytic transfusion reactions and for ABO HDN. In Japan, anti-A and anti-B titers notably decreased in the 15 years between 1986 and 2001. At present, titers of more than 100,as measured using the saline method, are rare. Differences in the level of anti-A and anti-B among ethnic populations have been reported; these differences were found to be the result of environmental factors rather than hereditary factors. In the present study, the anti-A and anti-B titers of random donors in three Asian populations are compared. In Thailand, the IgM anti-A and anti-B titers are low and are similar to the Japanese titers reported in 2001, but the IgG anti-A and anti-B titers are high and are similar to the Japanese titers reported in 1986. In the Lao People's Democratic Republic, both the IgM and the IgG anti-A and anti-B titers are high and are similar to those reported in Japan in 1986. In addition, anti-A and anti-B titers of different sex donors and of various age groups were also compared. High titers were found in 8.8 percent of the female donors in the younger than 30 age group and in 36.7 percent of the female donors in the older than 50 age group.

HLA-B27 testing in Thai patients using the PCR-SSP technique.

We develop the HLA-B27 test kit using the PCR-SSP technique. Five hundred forty blood samples were tested for HLA-B27 by microlymphocytotoxicity test (LCT) and PCR-SSP. It was found that 127 (23.5%) and 134 (24.8%) of these samples were positive for HLA-B27 by LCT and PCR-SSP, respectively. The sensitivity and specificity of the PCR-SSP were 94.8 and 100%, respectively, when using LCT as the standard method. The PCR-SSP positive predictive value was 100%, negative predictive value was 98.3%, and a concordance rate of 98.7%. This study shows that the PCR-SSP is simple, convenient, and a more cost-effective in-house test kit.

External quality assessment scheme in red blood cell serology: a 5-year experience in Thailand.

From 2000 to 2004, 36, 58, 72, 78, and 86 laboratories participated in an external quality assessment scheme (EQAS) organized by the Department of Transfusion Medicine, Faculty of Medicine Siriraj Hospital. Each year the staff was requested to perform ABO grouping, D typing, antibody screening, antibody identification, and DATs on eight blood samples. Each participant received information on the correct test results and a coded summary. Regarding ABO grouping, the error rate ranged from 0.3 to 1.3 percent, mostly due to human errors. Error rates in D typing ranged from 0.7 to 5.7 percent, the most problematic being weak D phenotype interpretation. Although every sample was negative by the DAT, error rates due to false positive test results were determined to be 0.4 to 2.1 percent. Antibody screening errors were also found; however, errors steadily decreased from 4.2 percent in 2000 to 0.3 percent in 2004. Only 69.4 to 87.2 percent of laboratories performed antibody identification; however, correct results increased from 78.4 to 91.0 percent. In conclusion, an EQAS in RBC serology should be used to compare results from different laboratories and to identify those laboratories that need improvement in testing procedures.

Analysis of SERF in Thai blood donors.

The Cromer blood group system consists of nine high-prevalence and three low-prevalence antigens carried on decay-accelerating factor (DAF). We recently described one of these Cromer highprevalence antigens,SERF, the absence of which was found in a Thai woman. The lack of SERF antigen in this proband was associated with a substitution of nucleotide 647C>T in exon 5 of DAF, which is predicted to be a change of proline to leucine at amino acid position 182 in short consensus repeat (SCR) 3 of DAF. This study reports on PCR-RFLP analysis of the SERF allele with BstNI restriction endonuclease on more than one thousand Thai blood donor samples. One new donor homozygous (647T) and 21 donors heterozygous (647C/T) for the SERF allele were found. Among this cohort of random Thai blood donors, the SERF allele frequency was 1.1 percent. Thus, like other alleles in the Cromer blood group system, SERF is found in a certain ethnic group.

Gene frequencies of the HPA-1 to 6 and Gov human platelet antigens in Thai blood donors.

Human platelet alloantigens (HPA) are important in neonatal alloimmune thrombocytopenia (NAIT), posttransfusion purpura (PTP), platelet transfusion refractoriness, passive alloimmune thrombocytopenia, and transplantation-associated alloimmune thrombocytopenia. Thus, HPA genotyping is essential in diagnosis and treatment. We analyzed HPA-1 to 6 and Gov alleles, using PCR with sequence specific primers (PCR-SSP) in 500 Thai blood donors who had been HLA class I antigen typed. HPA-4a was present in all samples. HPA-1b, -2b, -5b, and -6b were rare, and HPA-4b was not found. HPA-3a and -3b showed frequencies of 56.0 percent and 44.0 percent, respectively. Gova and Govb showed frequencies of 49.1 percent and 50.9 percent, respectively. The prevalence rates of HPA-1 to 6 gene frequencies (GFs) were consistent with those of other Asian populations rather than those of Caucasians. We also report on the GFs of Gova and Govb, which also are comparable to those of Asian populations. Our results could establish a useful HPA- and HLA-matched plateletpheresis donor file and provide an improvement of platelet alloantibody detection in alloimmune thrombocytopenic patients, and, therefore, a more effective platelet transfusion program.

HLA alloimmunization in patients receiving multitransfusions of red blood cells.

HLA antibodies were studied in 88 patients with chronic hemolytic anemia who received multitransfusions of red blood cells prepared by conventional (PRC-C), inverted centrifugation (LR-I) and leukocyte filter (LR-F) techniques. Their mean age was 8 years and 4 months with a duration of transfusion of 8 years. The patients were divided into five groups: group 1, receiving PRC-C (n=20); group 2, receiving LR-I (n=33); group 3, receiving LR-F (n=11); group 4, subsequently receiving LR-I and LR-F (n=10); and group 5, receiving PRC-C followed by LR-I and LR-F (n=14). The HLA class I antibodies were found in 30 out of 88 patients (34%). All were against HLA antigens commonly found in the Thai population. The patients receiving PRC-C exhibited HLA antibodies of 65%, which was significantly higher than those of patients receiving LR-I (24%) and LR-F (0%). Consequently, the transfusion reactions of fever, chill, rash and urticaria were also commonly found in patients receiving PRC-C (13.4%), which was significantly higher than patients receiving LR-I (0.4%) and LR-F (0%). The leukocyte filter technique has been shown to be effective in preventing HLA alloimmunization and transfusion reactions but the price is rather high. For the inverted centrifugation technique, only transfusion reactions were effectively prevented and the HLA alloimmunization continued to develop. A more effective and low-cost method for the removal of leukocytes should be investigated for these multitransfusion patients.

A preliminary study of the distribution of blood group systems in Thai blood donors determined by the gel test.

Two hundred blood samples obtained from volunteer blood donors at the Blood Bank, Army Institute of Pathology were studied for red cell groupings in the ABO, Rh, MNSs, Duffy, Lewis. P. Kell, Lutheran and Kidd Systems. Each sample was tested by the gel test using five cards; the ABO-Rh card, Diaclon Rh sub groups + K card, Antigen profile I card (P, Le(a), Le(b), Lu(a), Lu(b)), Antigen profile II card (k, Kp(a), Kp(b), Jk(a), Jk(b)) and Antigen profile III card (M, N, S, s, Fy(a), Fy(b)). For the ABO System, group O is the most common (40.5%) followed by group B (30.5%), group A (20.5%) and group AB (8.5%). The most common Rh gene complex was CCDee (51.5%), which was similar to other studies. The incidence of MMss and MNss gene complexes were the most common in the MNSs System. Fy(a) is very common as in other Asians. In the Lewis System, the incidence of Le (a-b-) was 23.5%, which is consistent with other findings in the Thai population. Sixty (30%) were positive with anti-P1. For the Kell System, only kk and Kp(b) positive types were observed in this study, as well as Lu (a-b+) in the Lutheran System. Jk (a-b-) was not found, which is considered a rare phenotype among Thai people. This study reveals the blood group distribution in 200 Thai volunteers using the gel test. Because of its simplicity and efficacy, this test is practical in population studies. Moreover, it is useful for mass screening and application in emergency situations.

Mutations at the activated protein C cleavage sites Arg336 and Arg562 of factor VIII in Thai patients with venous thrombosis.

Venous thrombosis is a multicausal disease, more than one genetic risk factor may cooperate to effect thrombotic risk. Factor V Leiden is found to be an important hereditary risk factor for venous thromboembolism. Analogous to factor V Leiden, a point mutation at amino acid positions Arg336 and Arg562 in factor VIII may predispose patients to thrombosis. Eighty-one Thai patients with venous thrombosis and 100 Thai healthy volunteers have been studied. Neither heterozygous nor homozygous mutations were detected both thrombosis patients or normal volunteers. However, further studies with larger samples of venous thrombosis patients are recommended.

Detection of factor V Leiden in Thai patients with venous thrombosis.

The molecular defect underlying activated protein C resistance (APC-R) is caused by a G to A point mutation in the codon for arginine 506 in the factor V gene (factor V Leiden) which is a major risk factor for venous thrombosis, especially in Caucasian populations. This study is an analysis of the Thai population to determine the prevalence of the factor V Leiden mutation. Twenty-seven patients with apparent venous thrombosis were divided into two groups according to APC-R test. Thirteen patients were diagnosed as positive for n-APC-SR, ratio < 0.8 and fourteen patients were diagnosed as negative for n-APC-SR, ratio > 0.8. Two of thirteen APC-R positive patients and one of fourteen APC-R negative patients were found to have the heterozygous allele for the factor V Leiden mutation but the homozygous allele was not detected in these groups of patients. Neither the heterozygous nor homozygous Leiden mutation was detected in 200 healthy volunteer blood donors. In conclusion, our findings indicate that factor V Leiden mutation is related to venous thrombosis in Thai people. Moreover, a further study of other mutations at the activated protein C cleavage sites of factor V and factor VIII is recommended.

Comparison of the RPHA and EIA techniques for the detection of HBs antigen among pregnant Thai women.

Five hundred serum samples obtained from pregnant women attending an antenatal clinic in Bangkok were tested for HBsAg by reverse passive hemagglutination assay (RPHA) and enzyme immunoassay (EIA). It was found that 21 (4.2%) and 28 (5.6%) of the sera were positive by RPHA and EIA, respectively. The sensitivity and specificity of the RPHA were 75% and 100%, respectively, when using EIA as the standard method. The RPHA positive predictive value was 100% and the negative predictive value was 98.5%. Accuracy was 98.6%. This study showed that the RPHA was simple and required inexpensive equipment, making it suitable for mass screening. However, the possibility of false negative readings due to low levels of HBsAg should be kept in mind, especially in the blood transfusion practice.

HLA class II alleles in Japanese patients with non-Hodgkin's lymphoma.

The distribution of HLA-DRB1 alleles and DQB1 alleles in 30 Japanese patients with non-Hodgkin's lymphoma (NHL) was analyzed using polymerase chain reaction with the sequence-specific primer (PCR-SSP) method, and the association between the disease and the presence of certain HLA class II alleles was investigated. The frequencies of HLA-DRB1*0803, DRB1*0802 and DRB1*1502 were increased while those of DRB1*1501 and DRB1*0405 were decreased. On the other hand, the incidence of HLA-DQB1 alleles was similar to that in the normal population. However, none of these HLA class II alleles showed significant positive or negative associations with NHL. In addition, when allele frequencies of NHL Japanese patients were compared to Thai patients, only DRB1*0803 was significantly increased in Japanese patients. These results indicate that DRB1*0803 may not contribute to NHL susceptibility in the Japanese population. However, further studies with larger numbers of NHL Japanese patients are needed to confirm our preliminary findings.

HLA class II polymorphism in Thai patients with non-Hodgkin's lymphoma.

The distribution of HLA-DRB1 alleles and DQB1 alleles in 100 Thai patients with non-Hodgkin's lymphoma (NHL) was analysed using the polymerase chain reaction with sequence-specific primer (PCR-SSP) method, and the association between the disease and the presence of certain HLA class II alleles was investigated. The frequencies of HLA-DRB1*1502 and DRB1*09012 were increased while those of DRB1*0404, DRB1*0803 and DRB1*1106 were decreased. On the other hand, the incidence of HLA-DQB1 alleles was similar to that in the normal population. Interestingly, only HLA-DRB1*1502 showed a significant positive association with NHL, especially in patients < or / = 45 years and in male patients. It is concluded that the DRB1*1502 allele may contribute to NHL susceptibility in the Thai population. However, further studies on the functional roles of the HLA class II alleles are necessary to elucidate NHL susceptibility.