Integrin Subunit Alpha M, ITGAM Nonsynonymous SNP Is Associated with Knee Osteoarthritis among Thais: A Case-Control Study.

Knee osteoarthritis (OA), which is one of the most common degenerative joint diseases, presents a multifactorial etiology, involving multiple causative factors including genetic and environmental determinants. Four human neutrophil antigen (HNA) systems can be determined using each HNA allele by single-nucleotide polymorphisms (SNPs). However, there are no data on HNA polymorphisms and knee OA in Thailand, so we investigated the association of HNA SNPs and knee OA in the Thai population. In a case-control study, detection of HNA-1, -3, -4, and -5 alleles by polymerase chain reaction with sequence-specific priming (PCR-SSP) was performed in participants with and without symptomatic knee OA. Logistic regression models were used to estimate the odds ratio (OR) and 95% confidence interval (CI) between cases and controls. Among 200 participants, 117 (58.5%) had knee OA; 83 (41.5%) did not and were included as controls in this study. An integrin subunit alpha M (ITGAM) nonsynonymous SNP, rs1143679, was markedly associated with symptomatic knee OA. The ITGAM*01*01 genotype was identified as an important increased risk factor for knee OA (adjusted OR = 5.645, 95% CI = 1.799-17.711, p = 0.003). These findings may contribute to our understanding of the application prospects for therapeutic approaches to knee OA.

World human neutrophil antigens investigation survey.

BACKGROUND AND OBJECTIVES: Human neutrophil antigens (HNAs) are categorized into five systems: HNA-1 to HNA-5. Given the importance of neutrophils in immunity, we sought to create awareness of the role of HNA diagnostic services in managing immune neutropenia and transfusion-related acute lung injury. To provide health communities all around the world with access to these services, we conducted a survey to create a directory of these HNA diagnostic services. MATERIALS AND METHODS: An Excel table-based survey was created to capture information on the laboratory's location and was emailed to 55 individuals with known or possible HNA investigation activity. The collected data were then summarized and analysed. RESULTS: Of contacted laboratories, the surveys were returned from 23 (38.2%) laboratories; 17 have already established HNA diagnostic (of them 12 were regular participants of the International Granulocyte Immunobiology Workshop [ISBT-IGIW]), 4 laboratories were in the process of establishing their HNA investigation and the remaining 2 responder laboratories, did not conduct HNA investigations. In established laboratories, investigation for autoimmune neutropenia (infancies and adults) was the most frequently requested, and antibodies against HNA-1a and HNA-1b were the most commonly detected. CONCLUSION: The directory of survey respondents provides a resource for health professionals wanting to access HNA diagnostic services. The present study offers a comprehensive picture of HNA diagnostics (typing and serology), identifying weak points and areas for improvement for the first time. Identifying more laboratories involved in HNA diagnostics with limited access to international societies in the field will globally improve HNA diagnostics.

Association between HLA class II Polymorphism and Knee Osteoarthritis in a Thai Population.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease commonly found among elderly populations. Multiple risk factors, including non-clinical and genetic factors, contribute to the etiology and pathogenesis of OA. This study aimed to investigate the association between the human leukocyte antigen (HLA) class II alleles and knee OA occurrence in a Thai population. METHODS: HLA-DRB1 and -DQB1 alleles in 117 patients with knee OA and 84 controls were determined using the PCR with sequence-specific primer (PCR-SSP) method. The association between knee OA and the presence of certain HLA class II alleles was investigated. RESULTS: DRB1*07 and DRB1*09 frequencies increased, while DRB1*14, DRB1*15, and DRB1*12 decreased among patients compared with controls. DQB1*03 (DQ9) and DQB1*02 frequencies increased, while DQB1*05 decreased among patients. Notably, the DRB1*14 allele significant decreased (5.6% vs. 11.3%, p = 0.039, OR = 0.461, 95% CI: 0.221 - 0.963), while the DQB1*03 (DQ9) allele significantly increased among patients compared with controls (14.1% vs. 7.1%, p = 0.032, OR = 2.134, 95% CI: 1.067 - 4.265). Moreover, the DRB1*14-DQB1*05 haplotype showed a significant protective effect on knee OA (p = 0.039, OR = 0.461, 95% CI: 0.221 - 0.963). A contrary effect of HLA-DQB1*03 (DQ9) and HLA-DRB1*14 was observed, wherein the presence of HLA-DQB1*03 (DQ9) seemed to promote disease susceptibility, whereas HLA-DRB1*14 appeared to protect against knee OA. CONCLUSIONS: Knee OA was more pronounced among females than males, especially those aged  60 years. In addition, a contrary effect was found regarding HLA-DQB1*03 (DQ9) and HLA-DRB1*14, in whom the presence of HLA-DQB1*03 (DQ9) seems to promote disease susceptibility, whereas HLA-DRB1*14 appears to be a protective factor against knee OA. However, further study with a larger sample size is suggested.

Identification of Lutheran Blood Groups and Genetic Variants within KLF1 among Thai Blood Donors.

Background: Lua and Lub are inherited as codominant allelic characters resulting from a single nucleotide variant (SNV) of the basal cell adhesion molecule (BCAM) gene. Red cells of the dominantly inherited suppressor of the Lutheran antigens In(Lu) phenotypically appear as Lu(a-b-) by the haemagglutination test. In(Lu) resulted from heterozygosity for mutations within the erythroid-specific Krüppel-like factor 1 (KLF1) gene. This study aimed to determine the frequency of the Lu(a) and Lu(b) phenotypes and genotypes and genetic variants of the distinct In(Lu) among Thai blood donors. Material and Methods: Samples from 334 Thai donors were phenotyped with anti-Lua and anti-Lub. These DNA samples and an additional 1,370 donor DNA samples with unknown Lu(a)/Lu(b) phenotypes were genotyped using an in-house PCR-SSP. In the case of the three Lu(a-b-) donors, the BCAM and KLF1 genes were analysed by PCR and sequencing. Results: A total of 331 of the 334 donors were Lu(a-b+), while the other observed phenotype, appearing as Lu(a-b-), was found among three donors. Of those three Lu(a-b-) donors with the LU*02/02 genotype, we identified KLF1 variant alleles, consisting of two variants: c.[304T>C, 1001C>G] and c.[304T>C, 519_525dupCGGCGCC], leading to the In(Lu) phenotype, and one homozygous variant (c.304T>C) mutation. Also, only one Thai donor was genotyped as LU*01/02, confirmed by serology test and DNA sequencing. Conclusion: In this study, we identified KLF1 variants to be included in Lutheran typing analysis in Thai populations. Therefore, the application of genotyping and phenotyping methods has simultaneously been in use to screen and confirm the rare Lu(a+) and In(Lu) phenotypes.

Comparative Analysis of a Developed Probe-Based Real-Time PCR and PCR-SSP for HLA-B*27 Detection among Thai Blood Donors.

BACKGROUND: The human leukocyte antigen (HLA) class I gene, the B locus, allele 27, HLA-B*27 is one of the most fascinating risk factors that is strongly associated with developing spondyloarthropathies (SpA). HLA-B27 testing has been routinely available in the diagnosis of those diseases. This study aimed to develop a fluorogenic real-time PCR and to compare it with PCR-SSP to detect the HLA-B*27 allele among Thai blood donors. METHODS: A total of 391 DNA samples were obtained from Thai blood donors at Thammasat University Hospital and tested for HLA-B*27 allele detection. A new real-time PCR was developed and validated to identify this allele and subsequently compared with those results tested with PCR-SSP. The sensitivity of detection was performed using known HLA-B*27-positive and -negative samples with concentrations ranging from 0.001 to 100 ng/µL. Additionally, HLA-B27 subtyping was performed by DNA sequencing containing second and third exons of this gene among all the HLA-B*27-positive donors. RESULTS: The validity of real-time PCR using known DNA controls and the results obtained by PCR-SSP techniques were in 100% concordance. The method was sensitive even at low DNA concentrations (1 ng/µL). Of 391 donors, 24 (6.14%; 95% CI, 3.97 - 9.00) were found to have the HLA-B*27 allele, while the remaining 367 (93.86%; 95% CI, 91.00 - 96.03) did not have this allele. Donors presented HLA-B*27-positive, HLA-B*27:06, the most common allele, followed by HLA-B*27:04, -B*27:05, and -B*27:07. CONCLUSIONS: HLA-B*27 using fluorogenic real-time qualitative PCR was found to be superior compared with that of PCR-SSP. The method is rapid, accurate, reliable, and sensitive for detection. In addition, this method provides convenience in the early treatment of SpA patients and relieves their suffering.

High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients.

Background: High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. Methods: Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of KEL*01 and KEL*02, JK*01 and JK*02, FY*01, FY*02, and FY*02 N.01, DI*01 and DI*02, GYPB*03 and GYPB*04, RHCE*E and RHCE*e, and DO*01 and DO*02. Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. Results: The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. Conclusions: We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

Development of DO*A and DO*B Allele Detections to Predict Transfusion-Induced Alloimmunization Risks in Thai Populations.

BACKGROUND: Two antithetical antigens, Doa and Dob of the Dombrock (DO) blood group system are implicated in acute to delayed hemolytic transfusion reactions among patients with anti-Doa or anti-Dob. Given the unavailability of specific antiserum, a polymerase chain reaction with sequence-specific primer (PCR-SSP) was developed to identify DO*A and DO*B alleles. This study aimed to determine DO*A and DO*B allele frequencies and to predict transfusion-induced alloimmunization risks in three Thai blood donor populations. METHODS: DNA samples obtained from 1,300, 300, and 400 blood donors from central, northern, and southern Thailand, respectively, were genotyped for the DO*A and DO*B allele detections using developed PCR-SSP. The results were confirmed by DNA sequencing. RESULTS: The validated genotyping results by PCR-SSP were in concordance with DNA sequencing. The DO*B/ DO*B was the most common genotype (77.0, 76.0, and 71.0%), followed by DO*A/DO*B (21.0, 22.7, and 25.2%) and DO*A/DO*A (2.0, 1.3, and 3.8%) among central, northern and southern Thais, respectively. The alleles found among central Thais showed significant differences from those found among southern Thais but not from those of northern Thais. The risk of anti-Doa production was higher than anti-Dob production among Thais. Concerning regional groups, the risk of Doa alloimmunization among southern Thais (0.2059) was higher than those among central (0.1771) and northern Thais (0.1824). CONCLUSIONS: This was the first study to distinguish DO*A and DO*B genotypes in Thai populations using in-house PCR-SSP. This would be useful to predict alloimmunization risks that might result from transfusion-induced reactions of undetermined red cell antigens among blood donors and in reagent red cells.

Blood group P1 prediction using multiplex PCR genotyping of A4GALT among Thai blood donors.

OBJECTIVES: This study aimed to investigate single-nucleotide variants (SNVs) associated with P1 expression among Thai blood donors and develop a genotyping method using multiplex polymerase chain reaction (PCR) to predict P1 blood group status. BACKGROUND: The α1,4-galactosyltransferase (A4GALT), also called Gb3/CD77 synthase or P1/Pk synthase enzyme, is encoded by the A4GALT gene and catalyses the transfer of galactose from uridine diphosphate-galactose to lactosylceramide, creating the Pk antigen (Gb3). The same enzyme synthesises the P1 antigen by adding terminal galactose to paragloboside. The A4GALT transcripts are elevated in P1 , and different SNVs in transcription factor-binding regions of A4GALT correlate with P1 and P2 phenotypes. MATERIAL AND METHODS: A total of 218 blood samples from Thai blood donors at the Thammasat University Hospital were tested for the P1 antigen using the conventional tube technique. Genomic DNA was extracted, and non-coding regions of A4GALT were sequenced and analysed. A multiplex PCR assay was developed and validated to identify P1-associated SNVs and was subsequently tested on 1022 Thai DNA samples of unknown P1 antigen status. RESULTS: In the tested cohort (n = 218), P1 and P2 phenotypes were found in 24.77% and 75.23% of donors, respectively. Moreover, three SNVs-rs8138197 (C/T), rs2143918 (T/G) and rs5751348 (G/T)-correlated 100% with both phenotypes. Finally, findings agreed with serological phenotyping and DNA sequencing results, confirming their validity for predicting P1 antigen positivity. CONCLUSIONS: This study confirmed that three SNVs also correlated with P1 /P2 phenotypes among Thais, as expected. A multiplex PCR found that SNVs rs2143918 (T) and rs5751348 (G) predicted blood group P1 and is an accurate, reproducible, cost-effective and less time-consuming alternative to traditional methods.

A novel nonsense mutation found in the CD177 gene of Thai individuals with the HNA-2 null phenotype.

OBJECTIVES: The aim of this study is to explore the molecular basis and to develop a simple sequence-specific primer polymerase chain reaction (PCR-SSP) technique for screening genotypes associated with the human neutrophil antigen-2 (HNA-2) null phenotype among Thai blood donors. BACKGROUND: Single-nucleotide polymorphisms (SNPs) c.787A>T of the CD177 gene is well known to be primarily demonstrated as a genetic determinant for HNA-2 deficiency. METHODS: The SNPs in the CD177 gene (exons 7 and 9) of 49 Thai blood donors with the known percentage of CD177 expression by flow cytometry including 48 HNA-2 positive and 1 HNA-2 null individuals were identified by long-range PCR amplification and sequencing. Moreover, screening for the c.1254G>A mutation was developed using an in-house PCR-SSP technique and tested among 771 unrelated donor samples. RESULTS: A HNA-2 null sample from the first cohort was heterozygous for c.787A/T and homozygous for c.1291G/G, namely, a 787A-1291G/787T-1291G (AG/TG) genotype. Interestingly, we could identify SNP c.1254G>A (rs188387562, p. Trp418Ter) that caused a nonsense mutation of the CD177 gene in exon 9. This individual might have the 787A-1254A-1291G/787T-1254G-1291G genotype. From the second cohort (771 unrelated donors), the 1254GG homozygote was the most common (96.37%), followed by the 1254GA heterozygote (3.50%) and 1254AA homozygote (0.13%). Blood samples of two individuals with 787AT-1254GA-1291GG and 787AA-1254AA-1291GG genotypes were tested and the HNA-2 antigen expressions were 0.03% and 0.16% in rank. CONCLUSIONS: The c.787A>T is a primary genetic hallmark to determine the HNA-2 null phenotype. Additional screening of the novel c.1254G>A in combination with c.787A>T is a suitable, convenient and effective diagnosis among Thais.

Two Thai Burmese descendants with A4GALT*01N.21, p phenotype, and anti-PP1Pk.

CONCLUSIONS: Anti-PP1Pk is produced by p individuals without prior red blood cell alloimmunization. This antibody can react over a wide thermal amplitude, has the potential to bind complement, and has caused hemolytic transfusion reaction, hemolytic disease of the fetus and newborn, and a high rate of spontaneous abortions. This report of two cases describes the genetic basis of p phenotype underlying anti-PP1Pk production and the development of a semi-nested polymerase chain reaction (PCR) assay for screening this observed mutation among Thai blood donors. Antibody detection and confirmation were examined by serologic testing. Genomic DNA was extracted from two Thai Burmese descendants with the p phenotype and a history of spontaneous abortions caused by anti-PP1Pk; the entire coding region of the A4GALT gene of each was sequenced and analyzed. Additionally, a semi-nested PCR assay of the observed mutation was developed and used for screening the genomic DNA of 1502 Thai blood donors. Anti-PP1Pk was identified and the p phenotype was confirmed in the two Thai individuals of Burmese descent. A single-base duplication (c.201dupC in exon 3) in the A4GALT gene was detected in both p patients. The duplication is consistent with the A4GALT*01N.21 allele associated with the p phenotype and anti-PP1Pk production. A semi-nested PCR assay was developed and subsequently used for mass screening for this mutation. The mutation was not found among the 1502 Thai blood donors tested with this newly developed assay.

Establishment of KEL*01 and KEL*02 Genotyping to Recruit Uncommon, Kell-positive, Reagent Red Cells Among Thai Blood Donors.

BACKGROUND: The reagent red blood cells used to screen and identify antibodies have to include K+ cells in all batch productions. The data of K/k phenotypes among differing Thai blood donor populations remains unknown; hence, mass screening for uncommon K+ donors by serological test has some limitations. Implementing K/k genotyping may be useful to predict uncommon K+ donors to overcome this challenge. This study aimed to establish an in-house K/k genotyping technique and to report KEL*01 and KEL*02 allele frequencies among three Thai blood donor populations to increase the selection of K+ donors in rare blood group databases. METHODS: A total of 2,239 DNA samples obtained from 1,512 central, 427 southern, and 300 northern Thai blood donors were included. The KEL*01 and KEL*02 genotyping using PCR with sequence-specific primers (PCR-SSP) was developed and validated. All samples were genotyped using developed PCR-SSP. Moreover, the possibility of finding group O and predicted K+ phenotypes among Thai blood donor populations was calculated. RESULTS: The DNA controls were validated using two sets of primer combinations and the results of KEL*01 and KEL*02 genotyping were in agreement. The KEL*01 allele frequencies were 0.0007, 0.0047, and 0.0000, and KEL*02 allele frequencies were 0.9993, 0.9953, and 1.0000 among central, southern, and northern Thai donors, respectively. In addition, mass screening among 3,795 and 566 donors in central and southern Thai populations was required to find at least one group O and predicted K+ phenotypes. CONCLUSIONS: The in-house PCR-SSP for KEL*01 and KEL*02 genotyping provided reproducible and accurate results with cost effectiveness. Our results confirmed the low KEL*01 allele frequencies among Thais. PCR-SSP could be used as an alternative technique to simply increase the number of uncommon predicted K+ phenotypes for reagent red blood cell recruitments.

HNA-1, -3, -4, and -5 genotyping using multiplex PCR among southern Thais: Developing continual HNA-1 null detection.

BACKGROUND: Antibodies against human neutrophil antigens (HNAs) are involved in various clinical conditions including transfusion-related acute lung injury and auto/alloimmune neutropenia. We aimed to determine HNA-1, -3, -4, and -5 frequencies among southern Thais using multiplex PCR and to develop HNA-1 null detection. METHODS: Samples obtained from 427 southern Thai blood donors were genotyped HNA-1, -3, -4, and -5 using multiplex PCR and compared their allele frequencies with those previously reported in Thai populations. HNA-1 null samples were tested by newly developed PCR-SSP and PCR-RFLP and confirmed by DNA sequencing. RESULTS: The frequencies of FCGR3B*01, FCGR3B*02, FCGR3B*03, SLC44A2*01, SLC44A2*02, ITGAL*01, and ITGAL*02 among southern Thais differed from other Thai populations, except ITGAM*01 and ITGAM*02 frequencies. Two samples without specific fragments of FCGR3B*01, FCGR3B*02, and FCGR3B*03 tested by multiplex PCR were confirmed by PCR-RFLP to identify FCGR3B deficiency (HNA-1 null). Moreover, to reduce test steps, the newly developed PCR-SSP for FCGR3B deficiency was validated and tested in all samples and the results were in agreement with DNA sequencing. CONCLUSIONS: This was the first study to report HNA-1, -3, -4, and -5 frequencies among southern Thais. The indeterminate results of multiplex PCR for HNA-1 genotyping led to establish HNA-1 null detection using PCR-SSP, which is simple, convenient and cost-effective and can be used to identify FCGR3B deficiency.

Determining of JK*A and JK*B Allele Frequency Distribution among Muslim Blood Donors from Southern Thailand.

BACKGROUND: The Kidd (JK) blood group system is of clinical importance in transfusion medicine. JK*A and JK*B allele detections are useful in genetic anthropological studies. This study aimed to determine the frequencies of JK*A and JK*B alleles among Muslim blood donors from Southern Thailand and to compare how they differ from those of other populations that have been recently studied. METHODS: A cross-sectional study was used. Totally, 427 samples of dissimilar Thai-Muslim healthy blood donors living in three southern border provinces were selected via simple random sampling (aged 17-65 years old) and donors found to be positive for infectious markers were excluded. All samples were analysed for JK*A and JK*B alleles using PCR-SSP. The Pearson's chi-squared and Fisher exact tests were used to compare the JK frequencies among southern Thai-Muslim with those among other populations previously reported. RESULTS: A total of 427 donors-315 males and 112 females, with a median age of 29 years (interquartile range: 18 years)-were analysed. A JK*A/JK*B genotype was the most common, and the JK*A and JK*B allele frequencies among the southern Thai-Muslims were 55.2% and 44.8%, respectively. Their frequencies significantly differed from those of the central Thai, Korean, Japanese, Brazilian-Japanese, Chinese, Filipino, Africans and American Natives populations (P < 0.05). Predicted JK phenotypes were compared with different groups of Malaysians. The Jk(a+b+) phenotype frequency among southern Thai-Muslims was significantly higher than that of Malaysian Malays and Indians (P < 0.05). CONCLUSIONS: The JK*A and JK*B allele frequencies in a southern Thai-Muslim population were determined, which can be applied not only to solve problems in transfusion medicine but also to provide tools for genetic anthropology and population studies.

Predicted S and s phenotypes from genotyping results among Thai populations to prevent transfusion-induced alloimmunization risks.

BACKGROUND: S and s antigens of the MNS system are of clinical importance because alloanti-S and -s have usually caused delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Various red cell genotyping has been established to predict the phenotypes to solve serological test limitations. OBJECTIVES AND METHODS: This study aimed to determine S and s genotype frequencies and to estimate the alloimmunization risks among central, northern and southern Thai populations. Altogether, 1237 blood samples from Thai blood donors were included. Only 150 samples were tested with anti-S and anti-s by indirect antiglobulin test. All samples were genotyped for GYPB*S and GYPB*s alleles using inhouse PCR with sequence-specific primer. Additionally, the allele frequencies were used to estimate alloimmunization risks and compare with other populations. RESULTS: The phenotyping and genotyping results in 150 samples were in 100% concordance. The allele frequencies of GYPB*S in central, northern and southern Thais were 0.061, 0.040 and 0.097, and GYPB*s were 0.939, 0.960 and 0.903, respectively. The frequencies among central Thais were similar to those among northern Thai and Korean populations (P > 0.05) but significantly differed from those of Asian, Caucasian African American and Hispanic populations (P < 0.05). In addition, the risk of S alloimmunization among southern Thais (0.1566) was higher than those among central (0.1038) and northern Thais (0.0736). CONCLUSION: This was the first study to report S and s predicted phenotypes and estimate alloimmunization risks among Thais, which is beneficial to prevent transfusion-induced alloimmunization among donors and patients.

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.

Development of a Multiplex PCR for Human Neutrophil Antigens-1, -3, -4, and -5 Genotyping Among Thai Blood Donors.

BACKGROUND: Different polymerase chain reaction (PCR) techniques for human neutrophil antigens (HNA) genotyping have been implemented to diagnose the clinical conditions of patients with alloimmune neutropenia, febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury and to provide effective HNAmatched granulocyte transfusions. The present study aimed to develop an in-house multiplex-PCR for HNA-1, -3, -4, and -5 genotyping in the Thai population. METHODS: Altogether, 500 DNA samples obtained from unrelated, healthy Thai blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok, Thailand were included. Three hundred DNA samples of known HNA genotyping based on PCR with sequence specific primers (PCR-SSP), as previously described, were tested with the newly developed multiplex PCR. Additionally, 200 DNA samples of unknown HNA genotyped donors were tested for HNA-1, -3, -4, and -5 genotyping using multiplex-PCR. RESULTS: Validity of HNA-1, -3, -4, and -5 genotyping by multiplex PCR using known DNA controls and the comparison of the genotyping results between PCR-SSP and multiplex PCR were in agreement. Interestingly, the rare genotype HNA-4b4b was not found in this study, similar to previous studies in Thai and other populations. Moreover, 30 samples were randomly tested twice for HNA genotyping using the multiplex-PCR and demonstrated reproducible results and were confirmed by DNA sequencing. CONCLUSIONS: This study shows that the newly developed multiplex-PCR is cost effective and less time consuming compared with PCR-SSP. The multiplex PCR can be used as an alternative technique for HNA-1, -3, -4, and -5 genotyping for routine testing, especially in other developing countries due to its simplicity and accuracy.

Comparison of a simple-probe real-time PCR and multiplex PCR techniques for HPA-1 to HPA-6 and HPA-15 genotyping.

BACKGROUND: 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. METHODS: Five hundred DNA samples from the Thai National Stem Cell Donor Registry (TSCDR) of the National Blood Centre, Thai Red Cross Society were included. Human platelet antigen (HPA) genotyping was performed by simple-probe real-time PCR and multiplex PCR techniques. RESULTS: HPA-1, HPA-2, HPA-3, and HPA-4 genotyping results obtained by both techniques were in agreement. The misinterpretation of HPA-5, HPA-6, and HPA-15 genotypes was found in eight samples by simple-probe real-time PCR and HPA genotypes were confirmed by DNA sequencing. Two samples of HPA-5 were misinterpreted as HPA-5a5a instead of HPA-5a5b due to an NM_002203.3:c.1594A>C mutation (rs199808499) near the HPA-5 polymorphism (5' side). Five samples of HPA-6a6b were misinterpreted as HPA-6b6b because of an NM_000212.2:c.1545G>A mutation (rs4634) adjacent to the HPA-6 polymorphism (3' side). Interestingly, one sample of HPA-15a15b was misinterpreted as HPA-15b15b due to an NM_133493.1:c.2118C>A mutation near the HPA-15 polymorphism (3' side). CONCLUSIONS: HPA genotyping results by two PCR techniques were compared. Incorrect assignments were found due to genetic variations near each HPA single nucleotide polymorphism. Therefore, to avoid false assignation, the use of two genotyping techniques is recommended.

Kidd blood group allele frequencies in Thai blood donors.

BACKGROUND: We present Kidd blood group allele frequencies in Thai blood donors and compare them with other frequencies for other populations previously reported. METHODS: Eight hundred blood samples obtained from 500 central Thais and 300 northern Thais were genotyped by allele specific-PCR. RESULTS: It was found that JK*01 and JK*02 allele frequencies were 0.503 and 0.497 in central Thais, while the frequencies were 0.498 and 0.502 in northern Thais. The JK*01 and JK*02 allele frequencies in Thais were similar to those in Chinese populations, whereas the frequencies were significantly different from Japanese, French Basques, and African-Americans. CONCLUSIONS: This is the first to report of Kidd blood group allele frequencies in Thais, which is beneficial for the prevention of both alloimmunization and adverse transfusion reactions.

Genomic analysis of KEL*03 and KEL*04 alleles among Thai blood donors.

Background: The Kell blood group system is clinically important in transfusion medicine, particularly in patients with antibodies specific to Kell antigens. To date, genetic variations of the Kell metallo-endopeptidase (KEL) gene among Thai populations remain unknown. Objective: This study aimed to determine the frequencies of KEL*03 and KEL*04 alleles among Thai blood donors using an in-house polymerase chain reaction-sequence-specific primer (PCR-SSP) method. Methods: Blood samples obtained from 805 unrelated central Thai blood donors at a blood bank in Pathumthani, Thailand, from March 2023 to June 2023, were typed for Kpa and Kpb antigens using the column agglutination test, and the results for 400 samples were confirmed using DNA sequencing. A PCR-SSP method was developed to detect the KEL*03 and KEL*04 alleles, and genotyping results were validated using known DNA controls. DNA samples obtained from Thai donors in central (n = 2529), northern (n = 300), and southern (n = 427) Thailand were also genotyped using PCR-SSP for comparison. Results: All 805 (100%) donors had the Kp(a-b+) phenotype. The PCR-SSP genotyping results agreed with the column agglutination test and DNA sequencing. All 3256 Thai blood donors had the homozygous KEL*04/KEL*04 genotype. Frequencies of the KEL*03 and KEL*04 alleles among Thai donors differed significantly from those of Japanese, Native American, South African, Brazilian, Swiss, and German populations. Conclusion: This study found a 100% KEL*04 allele frequency in three Thai populations. These data could provide information on KEL*03 and KEL*04 allele frequencies to estimate the risk of alloimmunisation in Thai populations. What this study adds: This study demonstrates that in-house PCR-SSP can be used to determine KEL*03 and KEL*04 alleles to predict Kpa and Kpb antigens. Even though only homozygous KEL*04/KEL*04 genotypes were found among Thai donor populations, the established PCR-SSP method may be useful for estimating the risk of alloimmunisation in other populations.