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.

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.

A nested sequence-specific primer-polymerase chain reaction for the detection of HLA-B*15:02.

In this study, we reported a new technique in detecting HLA-B*15:02 by using a nested sequence-specific primer-polymerase chain reaction (SSP-PCR) that can be used on genomic DNA and whole blood for carbamazepine hypersensitivity prediction. We tested a total of 200 blind samples with known human leukocyte antigen (HLA)-B allelic types (44 positive for HLA-B*15:02 and 156 negative for HLA-B*15:02) with this new nested SSP-PCR technique and compared its efficacy to that of commercial sequence-specific oligonucleotide probe-polymerase chain reaction (SSOP-PCR). Using starting materials from DNA and whole blood, we were able to detect HLA-B*15:02 in 44 of our samples correctly. The test is very sensitive and is highly reproducible.

Distribution of cytokine gene polymorphisms in Thai population.

The distribution of 21 cytokine polymorphisms within 13 cytokine and cytokine receptor genes was analyzed in 102 healthy Thai individuals using the LIFECODES Cytokine SNP Typing kit. The TGFB codon25 marker is monomorphic in the Thai population. The IL1B+3962, IL6-174, and TNFA-238 are very rare polymorphisms, with only 0.01-0.04 minor allele frequency (MAF). The IL4-1098, IL1A-889, and IL10-1082 are found only 0.06-0.08 in Thai. Other cytokine polymorphisms (IL1B-511, IL1R pst1 1970, IL1RN mspa1 11100, IL4RA+1902, IL12B-1188, IFNG+874, TGFB codon10, TNFA-308, IL2-330, IL2+166, IL4-590, IL4-33, IL10-819, and IL10-592) in Thai have MAFs more than 0.10, ranging between 0.13 and 0.47. When comparing the allele and genotype frequencies with public single nucleotide polymorphism (SNP) database, most cytokine polymorphisms in Thai show similar distribution to Han Chinese and Japanese, but significantly different from Caucasian and African populations. Only a few markers, including IL4A+1902, TNFA-308, IL1B+3962, and IL2+166 show statistically different distribution among Thai and other Asian populations especially with the Japanese.

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.

14th International HLA and Immunogenetics Workshop: report on the Prospective Chronic Rejection Project.

An international collaborative study of 45 transplant centers was undertaken at the 14th International HLA (human leukocyte antigen) and Immunogenetics Workshop to see if HLA antibodies detected posttransplant are predictive of chronic graft failure. With the newly developed assay, MICA (major histocompatibility complex class I-related chain A) antibodies were also measured and their effect analyzed. Total of 5219 sera from patients who were more than 6 months posttransplant with functioning graft were tested for HLA antibodies by enzyme-linked immunosorbent assay, flow cytometry, or Luminex. HLA antibodies were found in 27.2% of kidney patients, 23.6% in the liver, 52.7% in the heart, and 21.7% in the lung. The method of antibody testing did not have a marked influence on the frequency of antibodies detected. MICA antibodies were detected in 15% of kidney patients, 30% of heart patients, and 31% of liver patients. Among 948 kidney patients who had HLA antibodies, 7.3% had rejected their graft within 1 year of testing, compared with 1.7% in 2615 patients without HLA antibodies (P= 0.8 x 10(-17)). Death occurred in 1.4% of total kidney patients and did not correlate to the presence of antibodies. We conclude that patients with posttransplant HLA antibodies indeed have a higher rate of chronic graft failure and that posttransplant antibodies are predictive of chronic rejection.

14th International HLA and Immunogenetics Workshop Prospective Chronic Rejection Project: a three-year follow-up analysis.

The three-year follow-up of 4,144 patients of the 14th International Workshop Prospective Chronic Rejection study has reinforced the evidence that post-transplant HLA antibodies are predictive of long-term graft loss. Three years after a single testing for HLA antibodies, 10% of kidney recipients who were antibody-positive had lost their grafts, in contrast to only 5% of antibody-negative patients (p<0.0001). The adverse effect of post-transplant antibodies on graft survival was also observed in lung, heart, and liver transplants. Donor-specific antibodies and 'strong' non-DSA had stronger association with graft loss than 'moderate' non-DSA. Periodic antibody monitoring, combined with specificity and strength analysis, would help in the early identification of allograft recipients who are at high risk of graft failure.

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.

Monitoring of T-cell subsets in patients treated with anti-CD 25 antibody.

Daclizumab and basiliximab, the antibodies to the interleukin-2 receptor (anti-IL-2R), decrease the incidence of acute rejection in renal transplantation. However, prolonged blockade of IL-2 receptor (IL-2R:CD25) may hamper apoptosis of reactive T-cell clones and thus may obstruct tolerance induction. We determined the effect of varying doses of anti-IL-2R on the number of CD3+CD25+ cells as an index of CD 25 blockade. The number of CD3+CD25+ cells was determined in four groups of induction therapies: no antibody induction; two doses of 50 or 25 mg daclizumab on day 0 and day 14; and two doses of 20 mg basiliximab at day 0 and day 4 (n=10, 24, 10, and 10, respectively). The number of CD3+CD25+ cells were monitored in whole blood before antibody infusion as well as 24 hours thereafter and weekly after transplantation. With two doses of 50 mg daclizumab, two doses of 25 mg daclizumab, and two doses of 20 mg basiliximab, the expression of CD3+CD25+ cells was completely suppressed for 12, 10, and 12 weeks posttransplantation, respectively. The reappearance of CD3+CD25+ cells above the baseline for each induction regimen was: 17 weeks for two doses of 50 mg daclizumab, 11 weeks for two doses of 25 mg daclizumab, and 13 weeks for two doses of 20 mg basiliximab. Monitoring of CD3+CD25+ cells may be utilized to tailor anti-IL-2R administration at a minimal dosage, yet retaining adequate IL-2R blockade for at least 3 months posttransplantation.

Neonatal alloimmune thrombocytopenia due to anti-Nak(a).

BACKGROUND: The accurate diagnosis of neonatal alloimmune thrombocytopenia is essential in the effective treatment of potentially serious bleeding in neonates. CASE REPORT: Reported here is a case of a full-term female baby who was delivered by vacuum extraction from a gravida 1 para 1 healthy mother. She presented with generalized petechiae and bilateral cephalhematoma, which she had had since birth. At 7 hours of life, she had an upper gastrointestinal hemorrhage and was found to have severe anemia and marked thrombo-cytopenia. Coagulation screening tests were normal. The diagnosis of neonatal alloimmune thrombocytopenia was suspected, and maternal serum was collected for further study. The baby was treated with a single dose of hydrocortisone (10 mg/kg) and IVIG (400 mg/kg) while waiting for irradiated platelets from her mother. After 30 mL of a transfusion of maternal platelets, the baby's platelet count rose dramatically, from 15,000 to 162,000 per microL, and it remained stable at that level. She was discharged on the 10th hospital day in good condition. During the follow-up period of 8 months, her growth and development were satisfactorily normal, as well as her platelet count. A high-titered platelet antibody was detected in the maternal serum by use of a solid phase platelet adherence technique. RESULTS: The specificity of the platelet antibody was identified as anti-Nak(a) by the mixed passive hemagglutination test method. CONCLUSION: These findings suggested a diagnosis of NAIT caused by anti-Nak(a).

The effects of cigarette smoking on peripheral blood leukocytes and lymphocyte subpopulations: an urban population-based study in Thailand.

Preliminary studies for peripheral blood leukocytes and lymphocyte subsets were done in smokers and non-smokers. There were 20 smokers (smoked more than 10 cigarettes per day) for more than a year and 20 non-smokers (smoked less than 20 cigarettes/20 years). Ages of smokers and non-smokers were respectively 21-57, and 18-55 years. Cigarette smoking was associated with a statistically significant increase in the number of neutrophils, activated lymphocytes, CD25 and CD19; but a statistical decrease in the percentage of CD7 and CD3. (P < 0.05)

Comparative analysis of serological and molecular results for HLA-DR typing in 120 Thai subjects.

In this study, serological HLA-DR typing results were compared to typing results obtained with sequence-specific primers in the polymerase chain reaction (PCR-SSP). HLA-DR typing was performed on 120 random Thai individuals. Differences in HLA-DR typing results were found in 18 out of 120, which were due to cross reactive antibodies and the lack of potent antisera to define proper HLA-DR splits by serology. Furthermore, PCR-SSP is fast and easy to perform as HLA-DR typing results can be obtained within 2 hours. From the results of this study it can be concluded that PCR-SSP is a reliable and promising technique for HLA-DR typing which can replace the serological technique in routine clinical practice.