RHD 1227 A and hybrid Rhesus box analysis in Thai RhD+ and RhD- blood donors: Prevalence, RHD zygosity, and molecular screening.

BACKGROUND AND OBJECTIVES: DEL (D-elute) red blood cells (RBCs) are typed as D- by routine serological methods, as they carry a very weak form of D variant. Asia type DEL (c.1227 G>A) is the most prevalent DEL allele in East Asian populations that can lead to alloimmunization in D negative transfusion recipients. This study aimed to screen D+ and D- Thai blood donors for the Asia type DEL and its zygosity. MATERIALS AND METHODS: Blood samples of 723 D+ , and 191 D- Thai blood donors were collected. D antigen was typed by the routine serological method and adsorption-elution test to screen DEL phenotype. The hybrid Rhesus box, RHD 1227 G, RHD 1227 A and RHD exon 4 were analyzed with polymerase chain reaction using sequence specific primers, PCR-SSP. RESULTS: Among 191 D- blood donors, 52 (27.2%) RHD 1227 A allele carriers were detected, and 39 out of these 52 (75.0%) were positive for the hybrid Rhesus box (i.e. hemizygous RHD 1227 A). The remaining carriers were RHD 1227 A homozygous. RHD zygosity analysis in Thai D+ blood donors showed that only 4% (29/723) had a Dd genotype (hemizygous RHD 1227 G). Five (0.69%) blood donors were detected with RHD 1227 A alleles among 723 D+ blood donors. They were all G/A heterozygous at the RHD 1227 site. CONCLUSION: This study indicates that the high prevalence of RHD 1227 A allele among serologically apparent D- blood donors in Thais. Furthermore, the screening of hybrid Rhesus box, RHD 1227 A combined with RHD exon 4 is useful for analyses of Asia type DEL and its zygosity.

Prevalence of leucocyte antibodies in non-transfused male and female platelet apheresis donors.

OBJECTIVES: In our study group of Thai PLT apheresis donors, we assessed the prevalence of anti-leucocyte antibodies. BACKGROUND: Antibodies against human leucocyte antigens (anti-HLA), neutrophil antigens (anti-HNA), and major histocompatibility complex class I related chain A (anti-MICA) in blood products can lead to transfusion-related acute lung injury (TRALI). To reduce the risk of TRALI, some blood centres are implementing strategies based on screening platelet (PLT) apheresis donors for the presence of anti-leucocyte antibodies. METHODS/MATERIALS: Blood samples were collected from non-transfused individuals, 340 males and 63 females (50 nulliparous and 13 parous). Anti-HLA class I and II and anti-MICA were analysed using the Luminex assay, and anti-HNA-3 was detected using the granulocyte agglutination test. RESULTS: Anti-HLA was found in 14 of 403 subjects (3.5%). Ten subjects (2.5%) tested positive for HLA class I, 2 (0.5%) for HLA class II, and 2 (0.5%) for both HLA class I and HLA class II. Anti-HLA class I or II were detected in 2 of 13 (15.4%) parous females and only anti-HLA class I was found in 4 (8.0%) nulliparous females. Six of 327 subjects tested (1.8%), all males, were positive for anti-MICA. Anti-HNA-3 was not found in any of the 403 individuals. CONCLUSIONS: Screening for anti-HLA class I and II should be implemented for Thai PLT apheresis donors. Although immunisation against HNA and MICA seems to be a rare event in Thais, further work is necessary to decide whether our PLT apheresis donors should be screened for HNA and MICA antibodies.

Prevalence and impact of HLA and MICA allele mismatching on donor-specific antibodies induction in kidney transplant rejection.

AIM: Donor-recipient antigen mismatching for anti-human leucocyte antigen (HLA) and MICA is one of the risk factors for antibody induction leading to graft rejection. Our aim was to analyze the incidence and specificity of the different DSAs developing and to investigate the impact of HLA and MICA allele mismatches on antibody production in kidney transplant patients experiencing antibody-mediated rejection (AMR). METHODS: We retrospectively reviewed 253 consecutive recipients of kidney transplant who were diagnosed as experiencing AMR. RESULTS: Our results showed that around 27% of our patients were positive for DSAs over a median follow-up period of 24 months. Antibody to HLA-DQ7 was the most prevalent DSA detected. The allele mismatch number was significantly lower for DQ loci than -A and -B loci (DQ vs. A, p < .001; DQ vs. B, p = .002). Considering each HLA antigen, the incidence rate of DQ-DSA [41.9 (32.92-51.46; 95%CI)] was much higher than the rate observed for DSA directed to -A, -DR and -B loci. Half of the recipients in the DQ-DSA-only group, and the DQ-DSA together with non-DQ group, had MFI > 5000. Only one case developed de novo MICA-DSA (MICA002). CONCLUSION: Our study indicates that mismatching for HLA and MICA alleles leads to the development of HLA and MICA antibodies in some kidney transplant recipients. We have also demonstrated that DSA to the DQ locus is the most prevalent in kidney transplant patients with AMR. Thus, matching the DQ locus in kidney allocation algorithms may reduce post-transplant development of DSA.

Frequency of CD36 deficiency in Thais analyzed by quantification of CD36 on cell surfaces and in plasma.

BACKGROUND: Anti-CD36s, developing after transfusion or during pregnancy, play an important role in immune-mediated bleeding disorders among Asian populations. Currently, little is known about the clinical relevance of anti-CD36. Here, we aimed to determine the frequency of CD36 deficiency in Thais by analyzing CD36 expression on cell surfaces and in plasma. STUDY DESIGN AND METHODS: The expression and deficiency of CD36 on platelets and monocytes were determined by flow cytometry. Mutations in the CD36 gene were analyzed by nucleotide sequencing. Soluble CD36 (sCD36) in plasma was quantified with enzyme-linked immunosorbent assay. RESULTS: Fifteen of 700 blood donors (2.14%) were identified as CD36 deficient. The frequencies of Type I and II CD36 deficiency were 0.43% and 1.71%, respectively. Type I individuals exhibited c.1163A > T, c.429 + 4insG, and c.1156C > T. Type II individuals exhibited c.879 T > C, c.329-330delAC, c.818 + 108delAACT, c.1125 + 13C > A, and c.1163A > T. CD36 on donor platelets (n = 685) showed a wide distribution of expression levels (mean fluorescence intensity, 16.71 ± 8.68). In the normal phenotype (n = 14), sCD36 concentration was 58.84 ± 11.68 ng/mL, which was significantly correlated with platelet CD36 expression (r2 = 0.8551). In Type II-deficient individuals (n = 6), a similar sCD36 concentration was detected (53.67 ± 8.17 ng/mL). However, sCD36 could not be detected in Type I individuals (n = 3). CONCLUSION: CD36 Type I deficiency was found, indicating the potential for immune-mediated platelet disorders in Thais. However, the underlying mutations differed from those reported in Japan and China. Interestingly, sCD36 could not be detected in plasma of Type I-deficient individuals. This finding may lead to the use of plasma to identify individuals at risk and to allow screening of large cohorts.

The prevalence, alloimmunization risk factors, antigenic exposure, and evaluation of antigen-matched red blood cells for thalassemia transfusions: a 10-year experience at a tertiary care hospital.

BACKGROUND: Hemoglobin E-ß0 thalassemia and homozygous ß0 -thalassemia are the most common chronic transfusion-dependent thalassemias in Thailand. Patients with these conditions can experience clinical complications such as RBC alloimmunization. In this study we aimed to determine the prevalence, alloimmunization risk factors, antigenic exposure, and evaluation of antigen- (C, c, E, e, Mia ) matched RBC transfusion. STUDY DESIGN AND METHODS: Thalassemia patients were recruited from a tertiary care hospital for 10 years from 2008 to 2017. The medical records of transfusion history were reviewed for red cell phenotype both of patients and donors, number of units transfused, and type of alloantibodies. RESULTS: A total of 383 thalassemia patients were identified (178 males and 205 females). The frequency of RBC alloantibodies was 19.3%. Some patients tested positive for more than one antibody type. Autoantibodies were detected in nine individuals. Anti-E (49 [39.5%]), anti-Mia (24 [19.4%]), and anti-c (19 [15.3%]) were the most common antibodies detected. A high rate of alloimmunization was found in splenectomized patients. Risk of alloimmunization increased when more total units of blood had been transfused. A trend toward low alloimmunization rates was noted in the antigen-matched RBC group, where 3.5% (5/143) of patients were alloimmunized. Anti-E and anti-Mia , which may be naturally occurring, were identified in this group. CONCLUSION: Thai patients are more prone to develop antibodies against the Rh and Mia than to the Kell blood group antigens. Provision of at least antigen-matched (C, c, E, e, Mia ) RBCs appears to improve the efficacy of transfusion in thalassemia patients.

Phenotype frequencies of Rh (C, c, E, e), M, Mia and Kidd blood group systems among ethnic Thai blood donors from the north-east of Thailand.

We here report the first study of antigen and phenotype frequencies of Rh (C, c, E, e), M, Mia and Kidd antigens in north-east Thai blood donors. Blood transfusion services aim to ensure availability of adequate and safe blood to minimize the development of transfusion reactions. For pre-transfusion testing, the most important blood group systems are ABO and RhD. The transfusion of ABO-compatible otherwise unknown phenotype blood may result in alloimmunization, especially in multi-transfused patients. Extended red blood cell (RBC) phenotyping and selection of blood negative for specific antigens reduce post-transfusion complications and allow for effective blood transfusion regimens to be achieved. A total of 13,567 regular repeated, voluntary Thai blood donors were included for red-cell antigen typing of Rh (D, C, E, c, e). Samples from 12,768, 9,389 and 13,059 donors were typed for Kidd, M and Mia antigens, respectively. Amongst Rh antigens, e was the most common (96.80%) followed by C (95.50%), c (34.40%) and E (32.20%) with CCDee (60.00%) being the most common phenotype. For Kidd phenotypes, Jk(a+b+) was the most common (46.73%) and Jk(a-b-) was rare (0.07%). For the M and Mia antigen, M(+) was most frequently found (94.96%) and Mia (+) was found in 17.97% of individuals. Knowledge of red-cell antigen phenotype frequencies in a population is helpful for creating a phenotype database of blood donors which can provide antigen-negative compatible blood to patients with multiple alloantibodies. Moreover, provision of antigen-matched blood can prevent alloimmunization in multi-transfused patients.

Molecular and Functional Characterization of Fcγ Receptor IIIb-Ligand Interaction: Implications for Neutrophil-Mediated Immune Mechanisms in Malaria.

The Fcγ receptor IIIb (FcγRIIIb) is a low-affinity receptor of IgG and is essential in neutrophil-mediated effector functions. Different allelic forms of FcγRIIIb carrying human neutrophil antigen (HNA-1a, -1b, -1c, and -1d) have been identified. Here, we have generated stable transfected HEK293 cell lines expressing HNA-1aa, -1bb, and -1bc. Of these, cells expressing HNA-1bc interacted significantly stronger (binding affinities, 2.277 versus 0.743) with human IgG than cells expressing the HNA-1aa or -1bb alloforms. The higher affinity of IgG toward the HNA-1c alloform was confirmed using neutrophils derived from German blood donors. Neutrophils from HNA-1abc-phenotyped individuals bound IgG significantly stronger (1.825 versus 0.903) than did neutrophils from HNA-1ab-typed individuals. These findings were confirmed by surface plasmon resonance (SPR) analysis demonstrating that recombinant HNA-1bc had a higher affinity (dissociation constant [Kd ], 7.24 × 10-6 M) than recombinant HNA-1bb (Kd , 1.15 × 10-5 M) against normal IgG. Finally, we demonstrated that Plasmodium falciparum merozoites opsonized with human IgG affinity purified against P. falciparum glutamate-rich protein (GLURP) enhanced stronger reactive oxygen species (ROS) emission in neutrophils obtained from HNA-1abc donors than in neutrophils from HNA-1ab donors. Collectively, these results indicate that the amino acid substitution Ala78Asp resulting in the HNA-1c allotype leads to higher affinity toward human IgG, enhancement of neutrophil activation, and possibly effective clearance of malaria by intracellular ROS.

Improvement of monoclonal antibody-immobilized granulocyte antigen assay for the detection of anti-HNA-1 alloantibodies.

BACKGROUND: Currently, the gold standard for the identification of antibodies against human neutrophil antigens (HNAs) is the monoclonal antibody-immobilized granulocyte antigen (MAIGA) assay. However, the handling of this assay is laborious and therefore cumbersome for the rapid screening of neutrophil antibodies. STUDY DESIGN AND METHODS: In this study, we simplified the performance of the conventional MAIGA procedure and approved it for the identification of anti-HNA-1 with HNA-1-typed neutrophils and stable transfected (HEK293) cell line expressing HNA-1a, -1b, and -1c. RESULTS: In contrast to the conventional MAIGA, all working steps including the incubation of antibodies with cells, washings, cell lysis, and subsequent antibody detection could be performed on microtiter plates. This modification accelerates the work schedule of MAIGA and reduces pipetting errors. Comparison between both assay performances using neutrophils as target showed concordant results. Subsequently, stable mammalian cell lines were tested. In comparison to neutrophils, cell lines were stable for a longer period of time (>4 weeks) and results obtained with these cell lines showed better interassay precision. Analysis of different FcγRIIIb capture monoclonal antibodies (MoAbs) for the MAIGA assay showed that MoAb LNK16 is superior for the detection of anti-HNA-1a, -1b, and -1c, whereas MoAb 3G8 showed false-negative results, caused by competitive inhibition of anti-HNA-1c alloantibodies. CONCLUSION: The modification of MAIGA and the use of transfected HEK293 cells improve the detection of anti-HNA-1 alloantibodies that may allow screening analysis in large cohort of samples.

Polymorphisms of killer immunoglobulin-like receptors (KIRs) and HLA ligands in northeastern Thais.

Killer cell immunoglobulin-like receptors (KIRs) are cell surface receptors on natural killer (NK) cells and subsets of T cells. The functions of NK cells are partly regulated by interactions between KIRs and HLA ligands on target cells. In this study, the presence or absence of 17 KIR genes and their known HLA ligands have been investigated in 235 unrelated individuals living in northeastern Thailand (NET). Subtypes of KIR2DS4 including full length (KIR2DS4F) and deleted forms (KIR2DS4D) have also been determined. Framework genes (KIR2DL4, 3DL2, 3DL3, and 3DP1) were found in all individuals and KIR genes belonging to the A haplotype (KIR2DL1, 2DL3, 3DL1, and 2DS4) were present in more than 90% of NET. KIR2DS4D (61.7%) was more common than KIR2DS4F (52.8%). A total of 33 different KIR genotypes were observed. Of these, three new genotypes were identified. The most common genotype (AA) was observed in 35.7% of NET, and HLA-C alleles bearing the C1 epitope (HLA-C1) had the highest frequency (97%). All individuals had at least one inhibitory KIR and its corresponding HLA ligand; 40.9% of NET had three pairs of receptor-ligand combinations, and 18.3% had all three receptor-ligand combinations of KIR2DL3+C1, 3DL1+Bw4, and 3DL2+A11. Surprisingly, the patterns of KIR gene frequencies in NET are more similar to those of Caucasians than Japanese, Korean, and Chinese. This is the first report on complete analysis of KIR and known HLA ligands in Thais. These data provide basic knowledge on KIR for further studies on disease associations and transplantation in northeastern Thais.

Preparation of single donor platelet with low antibody titers for all patients.

Platelet concentrates from ABO-identical donors are the components of choice for patients. However, since inventories are generally insufficient and because there is usually a relative abundance of group O donors, perfect matches are not always possible. It is therefore the accepted practice for platelets to be transfused out of the ABO group when ABO-identical platelets are unavailable. Notwithstanding, the transfusion of platelets containing high titers of antibodies to the antigens on the red blood cells of the patient can cause clinically significant hemolysis. The way to improve the safety of group O platelets has focused on defining a safe level of antibodies or reducing the volume of incompatible plasma. In the current study, 107 group O single donor platelets (SDP) were modified after collecting the platelet pellet in a bag. The AB plasma was added instead of the donor's own plasma. The direct agglutination titers of anti-A/anti-B in the original group O SDPs' plasma were performed by doing a gel test, resulting in from 1:4 to 1:1024. The prevalence of high titers (i.e., at least 1:64 in our study) was relatively high, ∼63% for anti-A and 78% for anti-B. The titer of residual anti-A/anti-B in the modified SDPs ranged from negative to 1:8. In most of the modified SDPs anti-A/anti-B could not be detected in the plasma (58.9% and 52.3%, respectively). The results indicate that our modified SDPs have very low titers; that is, acting as a universal SDP which is safe for all ABO patients. This modified SDP form is a more convenient way to overcome the risk from incompatible plasma or loss of platelets during the process of volume reduction and can help effectively manage our inventory.

Polymorphisms of NKG2D ligands: diverse RAET1/ULBP genes in northeastern Thais.

Unique long 16 (UL-16)-binding proteins (ULBP) or retinoic acid early transcripts-1 (RAET1) are ligands to the activating receptor, NKG2D. The human RAET1/ULBP gene family is identified as ten members (RAET1E to N) with six loci encoding for potentially functional proteins. These are ULBP1 or RAET1I, ULBP2 or RAET1H, ULBP3 or RAET1N, and RAET1L, which are glycosylinositol phospholipid (GPI)-linked glycoproteins and ULBP4 or RAET1E and ULBP5 or RAET1G, which are transmembrane glycoproteins. The RAET1 products contain the alpha1 and alpha2 domains but lack the alpha3 domain and do not associate with beta2-microglobulin. RAET1/ULBPs have tissue-specific expressions, and some of them are also polymorphic. In the present study, polymorphic exons 2 and 3 of the RAET1E, G, H, I, L, and N were analyzed using sequence-based typing. One hundred and seventy-six unrelated healthy Northeastern Thais were included in this study. For RAET1E, RAET1G, RAET1H, and RAET1L, there were seven, two, five, and four single nucleotide polymorphisms (SNPs), respectively. Six of these are new SNPs, which are rare in this population. Of these, six new SNPs, two of two in RAET1E, two of three in RAET1H, and none of one in RAET1L are nonsynonymous substitutions. Interestingly, although the RAET1N is polymorphic in Caucasians, RAET1N and RAET1I had no variation in Thais indicating diverse RAET1 genes in different ethnic groups. These data provide the important basis for future analysis on the role of RAET1 genes in immune responses especially in cancer and infectious diseases.

Alu polymorphism within the MICB gene and association with HLA-B alleles.

We describe the finding of an Alu repeat dimorphism within the first intron of the MICB gene. The frequencies of the two AluyMICB alleles, AluyMICB*0(absence of insertion) and AluyMICB*1(presence of insertion), and their associations with the highly polymorphic HLA-B locus were determined for 51 human cell lines and for 109 and 200 Caucasians and northeastern Thais, respectively. Analysis of the AluyMICB and HLA-B allelic relationships revealed that AluyMICB*1 occurred at relatively low gene frequency (0.118-0.157) [corrected] but was strongly associated with HLA-B17 (HLA-B57,HLA-B58) and HLA-B13. The AluyMICB locus provides a useful dimorphic marker for investigations on the level of linkage disequilibrium between MICB, MICA, and HLA-B loci.