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.

Characterization of GYP(B-A-B) hybrid glycophorins among Thai blood donors with Mia-positive phenotypes.

BACKGROUND: GYPA and GYPB genes encode the antigens of the MNS blood group system carried on glycophorin A (GPA) and glycophorin B (GPB), or on a hybrid molecule of GPA and GPB. GP hybrid variants are created through unequal crossing over and gene conversion, typically from the parent genes GYPA and GYPB. In the present study, we characterized the GYP(B-A-B) hybrid variants among Thai blood donors with Mia-positive phenotypes using PCR-based coupled to DNA sequencing techniques. MATERIALS AND METHODS: Altogether, 1,020 samples from Thai blood donors were tested with anti-Mia by conventional tube technique (CTT). Polymerase chain reaction with sequence-specific primer (PCR-SSP) was initially used to differentiate normal GYPB, GYP*Vw and groups of GYP*Hut, GYP*Mur, GYP*Hop, GYP*Bun and GYP*HF alleles. Subsequently, GYP(B-A-B) hybrid variants were investigated using DNA sequencing. RESULTS: Among 1,020 blood donors, 127 (12.45%) were Mi(a+) phenotypes. The comparison Mia typing results between CTT and PCR-SSP were concordant. All Mi(a+) samples were positive with only group of GYP*Hut, GYP*Mur, GYP*Hop, GYP*Bun and GYP*HF alleles by PCR-SSP. Regarding the sequencing results, 115/1,020 (11.27%) donors carried the GYP*Mur, of which 111/1,020 (10.88%) were GYP*Mur/GYPB heterozygotes and the other 4/1,020 (0.39%) donors were GYP*Mur/GYP*Mur homozygotes. The remaining 12 donors included different GYP*Bun-like alleles; 11 of them (1.08%) were GYP*Thai/GYPB heterozygotes, and one (0.10%) was GYP*Thai II/GYPB heterozygotes. With 5.83% (119/2,040) of the total hybrid alleles, GYP*Mur was the predominant allele. The GYP*HF, GYP*Bun, GYP*Hop and GYP*Kip alleles were not observed in this study. DISCUSSION: Regarding the hybrid GP variants, a consensus of observed prevalent GYP*Mur and GYP*Bun-like alleles, respectively, was identified in the Thai population. The introduction of our strategy has allowed us to identify the zygosity for GYP hybrid variants, particularly GYP(B-A-B) hybrid genes, when antisera are unavailable and lacking adequate phenotypic features to determine GP variants.

Agonistic Bivalent Human scFvs-Fcγ Fusion Antibodies to OX40 Ectodomain Enhance T Cell Activities against Cancer.

(1) Background: Understanding how advanced cancers evade host innate and adaptive immune opponents has led to cancer immunotherapy. Among several immunotherapeutic strategies, the reversal of immunosuppression mediated by regulatory T cells in the tumor microenvironment (TME) using blockers of immune-checkpoint signaling in effector T cells is the most successful treatment measure. Furthermore, agonists of T cell costimulatory molecules (CD40, 4-1BB, OX40) play an additional anti-cancer role to that of checkpoint blocking in combined therapy and serve also as adjuvant/neoadjuvant/induction therapy to conventional cancer treatments, such as tumor resection and radio- and chemo- therapies. (2) Methods and Results: In this study, novel agonistic antibodies to the OX40/CD134 ectodomain (EcOX40), i.e., fully human bivalent single-chain variable fragments (HuscFvs) linked to IgG Fc (bivalent HuscFv-Fcγ fusion antibodies) were generated by using phage-display technology and genetic engineering. The HuscFvs in the fusion antibodies bound to the cysteine-rich domain-2 of the EcOX40, which is known to be involved in OX40-OX40L signaling for NF-κB activation in T cells. The fusion antibodies caused proliferation, and increased the survival and cytokine production of CD3-CD28-activated human T cells. They showed enhancement trends for other effector T cell activities like granzyme B production and lysis of ovarian cancer cells when added to the activated T cells. (3) Conclusions: The novel OX40 agonistic fusion antibodies should be further tested step-by-step toward their safe use as an adjunctive non-immunogenic cancer immunotherapeutic agent.

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.

Application of a simplified PCR-SSP method to detect A4GALT*01 and A4GALT*02 typing among Thai blood donors.

OBJECTIVES: An intronic A4GALT single nucleotide variant, rs5751348:G>T, P2 or A4GALT*02 allele has a lower level of the enzyme-encoding A4GALT transcripts than the P1 individuals. Here, we first develop and validate a simple inhouse PCR-SSP method to detect A4GALT*01 and A4GALT*02 alleles, and second, apply this method to compare the allele frequencies between Thai and other populations. MATERIAL AND METHODS: The conventional test tube technique was used to detect the P1 antigen in 222 blood samples from Thai blood donors at Thammasat University Hospital. A PCR-SSP method was optimized and validated for reproducibility and specificity to identify these alleles and was subsequently tested on 1,840 DNA samples of unknown phenotypes obtained from central, northern and southern Thais. In addition, allele frequencies of central Thais were compared with those of other populations. RESULTS: In the tested cohort (n = 222), P1 and P2 phenotypes were typed in 26.13 and 73.87% of donors, respectively. The developed PCR-SSP was successfully optimized, and the outcomes were consistent with those of serological phenotyping and DNA sequencing results, demonstrating its validity for predicting P1/P2 phenotype. For central, northern and southern Thais, the A4GALT*01 frequency was 0.1579 (430/2,724), 0.1183 (71/600), and 0.2575 (206/800), whereas the A4GALT*02 frequency was 0.8421 (2,294/2,724), 0.8817 (529/600), and 0.7425 (594/800), respectively. Their observed frequencies among central Thais significantly differed from those in other populations (p < 0.05). CONCLUSION: Our study has successfully developed a simple, precise, and reliable method to genotype A4GALT*01 and A4GALT*02 using inhouse developed PCR-SSP for predicting P1/P2 status.

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.

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.

Genotyping Approach to Predict Coa and Cob Antigens in Thai Blood Donor Populations.

Purpose: Coa and Cob antigens of the Colton (CO) blood group system are implicated in acute and delayed hemolytic transfusion reactions (HTRs). Owing to the inadequate supply of specific antiserum, data on CO phenotypes remain limited. This study aimed to develop genotyping methods to predict Coa and Cob antigens and to estimate transfusion-induced alloimmunization risks in three Thai blood donor populations. Materials and Methods: The study included 2451 blood samples from unrelated healthy Thai blood donors obtained from central, northern, and southern Thailand. DNA sequencing was used to determine the CO*A and CO*B alleles. In-house PCR with sequence-specific primers (PCR-SSP) and high-resolution melting curve (HRM) assays were performed and genotyping results were compared using DNA sequencing. CO*A and CO*B allele frequencies among Thais were determined using PCR-SSP and their frequencies were compared with other populations. The risks of Coa and Cob transfusion-induced alloimmunization among Thai donor populations were calculated. Results: The validated genotyping results by PCR-SSP and HRM assays agreed with DNA sequencing. The CO*A/CO*A was the most common (100.0, 100.0, and 99.3%), followed by CO*A/CO*B (0.0, 0.0, and 0.7%) among central, northern and southern Thais. Homozygous CO*B/CO*B was not found. The CO*A and CO*B allele frequencies among central Thais significantly differed compared among southern Thais (p < 0.01) but not among northern Thais. Those allele frequencies among Thais were similar to those of Taiwanese, Chinese and Malay-Malaysian populations but not to South Asian, Southeast Asian, Korean, Japanese, Filipino, French Basque, and Maltese populations (p < 0.01). A higher risk of anti-Cob production rather than anti-Coa production was particularly noted in the southern Thai population. Conclusion: This study constitutes the first to determine CO*A and CO*B genotypes using PCR-SSP and HRM assays among Thais and this finding would be beneficial in predicting alloimmunization risk and providing safe transfusions among Thais.

Characteristics of Donors and Modelling of the Characteristics to Possible Forecast the Repeat Donors Profile at Thammasat University Hospital.

Blood donations are essential to the blood supply available for patients in life-saving treatments. We aimed to identify characteristics affecting repeat donations, and to model a tool to forecast repeat donation among Thammasat University Hospital (TUH) donors. A retrospective study for 4 years of donations at TUH was conducted to identify characteristics affecting continuous donations and model a scoring tool, as well as pilot test it, prospectively. Data concerning age, sex, ABO grouping, Rh(D) typing, and collection site were included. The outcome was dichotomized as controls and cases based on first time and repeat donations. Receiver operating characteristic curve was used to obtain the cut-off, while odds ratio was used to assign the score. During the study, 37,736 donations comprised 6,305 controls and 31,431 cases. Characteristics that positively predicted repeat donation included male, age ≥ 30 years, AB blood group and on-site donation, and they were chosen to model the score. The total score value of 3 was chosen as the rounded cut-off. A pilot study, the score was observed to have an accuracy of 67.5%. In conclusion, 4 significant characteristics appeared to positively influence repeat donation. The predictive scoring model is a simple reliable and valid tool exhibiting good accuracy.

Serum proteomic profiling reveals MTA2 and AGO2 as potential prognostic biomarkers associated with disease activity and adverse outcomes in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy accounting for approximately 10% of hematological malignancies. Identification of reliable biomarkers for better diagnosis and prognosis remains a major challenge. This study aimed to identify potential serum prognostic biomarkers corresponding to MM disease activity and evaluate their impact on patient outcomes. Serum proteomic profiles of patients with MM and age-matched controls were performed using LC-MS/MS. In the verification and validation phases, the concentration of the candidate biomarkers was measured using an ELISA technique. In addition, the association of the proposed biomarkers with clinical outcomes was assessed. We identified 23 upregulated and 15 downregulated proteins differentially expressed in newly diagnosed and relapsed/refractory MM patients compared with MM patients who achieved at least a very good partial response to treatment (≥VGPR). The top two candidate proteins, metastasis-associated protein-2 (MTA2) and argonaute-2 (AGO2), were selected for further verification and validation studies. Both MTA2 and AGO2 showed significantly higher levels in the disease-active states than in the remission states (p < 0.001). Regardless of the patient treatment profile, high MTA2 levels were associated with shorter progression-free survival (p = 0.044; HR = 2.48; 95% CI, 1.02 to 6.02). Conversely, high AGO2 levels were associated with IgG and kappa light-chains isotypes and an occurrence of bone involvement features (p < 0.05) and were associated with prolonged time to response (p = 0.045; HR = 3.00; 95% CI, 1.03 to 8.76). Moreover, the analytic results using a publicly available NCBI GEO dataset revealed that AGO2 overexpression was associated with shorter overall survival among patients with MM (p = 0.032, HR = 1.60, 95% CI, 1.04 to 2.46). In conclusion, MTA2 and AGO2 proteins were first identified as potential biomarkers that reflect disease activity, provide prognostic values and could serve as non-invasive indicators for disease monitoring and outcome predicting among patients with MM.

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.

RHCE*E and RHCE*e genotype incompatibility in a southern Thai Muslim population.

CONTEXT: The formation of red cell alloantibodies resulting from both transfusion and pregnancy can cause adverse effects from allogeneic blood transfusions. Alloanti-E is commonly detected among Thai and Asian populations. AIMS: This study aimed to determine RHCE*E and RHCE*e genotype incompatibility in a southern Thai Muslim population and to compare it with those previously reported for other populations. SUBJECTS AND METHODS: Nine hundred and twenty-seven DNA samples obtained from 427 unrelated healthy blood donors from southern Thai Muslims and 500 samples from Central Thais were included. Samples were genotyped for RHCE*E and RHCE*e using an in-house polymerase chain reaction with the sequence-specific primer technique. RESULTS: Significant differences were found when we compared the allele frequencies of the RHCE*E and RHCE*e between southern Thai Muslims and Central Thais: RHCE*E 0.162 versus 0.197 and RHCE*e 0.838 versus 0.803 and also found in Chinese, American native, Japanese, Korean, Alaskan native, Hawaiian, South Asian, Brazilian Japanese-descendant, and Malay Malaysian populations (P < 0.05). In addition, the E/e incompatibilities among southern Thai Muslims and Central Thais were 24.23% and 26.71%, respectively. CONCLUSIONS: This study was the first to determine the RHCE*E and RHCE*e genotype incompatibility among southern Thai Muslims, enabling the estimation of their potential alloimmunization risk. These data could be useful to provide safe blood transfusions across ethnic populations.

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.

Monoclonal antibody specific to the Dia blood group antigen generated by phage display technology.

BACKGROUND: Alloanti-Dia can be implicated in mild to severe blood transfusion reactions. Given the concomitance of a high prevalence of the Dia antigen and antibody circulating in some populations, an anti-Dia typing reagent is required in order to enable safe blood transfusions. Limitations of hybridoma technology to produce such a reagent led to the use of phage display technology to generate an anti-Dia monoclonal antibody. MATERIALS AND METHODS: A library of phages displaying murine single-chain variable fragment antibody (scFv-phages) was consecutively adsorbed with different panels of Di(a-b+) red cells to eliminate scFc-phages that potentially bind irrelevant blood group antigens. Thereafter, the subtractive library was specifically selected for the scFv-phages that bound Dia antigen by sequentially biopanning the library with Di(a+b+) cell ghosts and Di(a+b-) intact red cells. A specific interaction between the selected scFv-phages and Dia epitope was validated with the Dia peptide by a competitive haemagglutination inhibition assay and confirmed with the red cells by flow cytometry. RESULTS: An scFv-phage clone specifically bound the Dia epitope, as shown by its binding competition with the human anti-Dia to the Dia peptide in a haemagglutination inhibition test. Moreover, it was highly reactive to Di(a+b+) red cells but not to Di(a-b+) red cells, as determined by flow cytometry. DISCUSSION: In this study, a Dia-specific scFv-phage antibody was successfully produced. The selection protocol might be a prototypic platform for producing monoclonal antibodies to relevant blood group antigens. The scFv-phage produced in this way warrants further development for use as a reagent for Dia red cell typing.

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.

Impact of using genotyping to predict SERF negative phenotype in Thai blood donor populations.

BACKGROUND: SERF(+) is a high prevalence antigen in the Cromer blood group system that is encoded by a CROM*01.12 allele. The SERF(-) on red cells is caused by a single nucleotide variation, c.647C＞T, in exon 5 of the Decay-accelerating factor, DAF gene. Alloanti-SERF was found in a pregnant Thai woman, and a SERF(-) individual was found among Thai blood donors. Since anti-SERF is commercially unavailable, this study aimed to develop appropriate genotyping methods for CROM*01.12 and CROM*01.-12 alleles and predict the SERF(-) phenotype in Thai blood donors. METHODS: DNA samples obtained from 1,580 central, 300 northern, and 427 southern Thai blood donors were genotyped for CROM*01.12 and CROM*01.-12 allele detection using in-house PCR with sequence-specific primer (PCR-SSP) confirmed by DNA sequencing. RESULTS: Validity of the PCR-SSP genotyping results agreed with DNA sequencing; CROM*01.12/ CROM*01.12 was the most common (98.42%, 98.00%, and 98.59%), followed by CROM*01.12/CROM*01.-12 (1.58%, 2.00%, and 1.41%) among central, northern, and southern Thais, respectively. CROM*01.-12/CROM*01.-12 was not detected in all three populations. The alleles found in central Thais did not significantly differ from those found in northern and southern Thais. CONCLUSION: This study is the first to distinguish the predicted SERF phenotypes from genotyping results obtained using in-house PCR-SSP, confirming that the CROM*01.-12 allele frequency ranged from 0.007 to 0.010 in three Thai populations. This helps identify the SERF(-) phenotype among donors and patients, ultimately preventing adverse transfusion reactions.