OBJECTIVE:By analyzing the correlation between genotypes and phenotypes, we explored the impact of the variant c.917T>C (p.L306P) in the ABO*B.01 allele on the expression and function of B-glycosyltransferase (GTB). This study aims to elucidate the molecular mechanisms underlying the occurrence of this subtype. METHODS:The study subjects included a blood donor specimen with incompatible forward and reverse ABO typing results. ABO phenotyping was determined using ABO blood group serology and GTB activity testing. Subsequently, Sanger sequencing and third-generation sequencing based on the PacBio platform were employed to sequence the ABO gene, resulting in the determination of haplotype sequences. Mutations were identified through sequence alignment. An in vitro cell expression system was established to assess the impact of the mutation site on antigen expression. RESULTS:The index case in this study was identified as B subtype with the allelic genotype c.917T>C in ABO*B.01/ABO*O.01.01 , which has not been previously reported. in vitro expression results revealed decreased levels of GTB expression and overall GTB activity in the mutant cells. Furthermore, the expression of the B antigen on the cell membrane was weaker in the mutant cells compared to the wild-type cells. CONCLUSION:The p.L306P variation caused by the c.917T>C mutation in the ABO*B.01 allele may be a genetic factor contributing to the reduced expression of B antigens on the surface of red blood cells.

BACKGROUND:Discrepancies in ABO typing can arise from genetic variations, posing risks in transfusions. Traditional sequencing handles point mutations well but struggles with recombination events, which long-read sequencing can accurately identify. Here, we report a case of Ael phenotype associated with a novel recombination allele in the ABO gene in a Chinese individual. STUDY DESIGN AND METHODS:We used serological methods to identify the phenotype, and applied sequencing-based typing (SBT) and third-generation long-read sequencing (TGS) to determine the ABO genotype. RESULTS:Serological testing revealed an Ael subtype. SBT sequencing identified seven heterozygous mutation sites, with one allele hypothesized as ABO*O.01.01 (c.261delG). The remaining six variants did not match any known allele combination. Two ABO haplotype sequences were obtained through TGS. Analysis confirmed a second haplotype as a hybrid of O.01.02-A02.05, with the breakpoint located between c.220C>T (g.chr9: 133258116C>T; p.Pro74Ser) and c.239+103delCCC (g.chr9: 133257993delCCC) . The novel allele sequence has been submitted to GenBank (Accession number: OR2834978). CONCLUSION:Our study demonstrates a case of a Chinese individual with an Ael phenotype caused by a novel allele resulting from the recombination of O.01.02 and A2.05. This finding underscores the significant advantage of long-read TGS technology in resolving structural variations.

By analyzing the correlation between genotypes and phenotypes, we explored the impact of the variant c.917T>C (p.L306P) in the ABO*B.01 allele on the expression and function of B-glycosyltransferase (GTB). This study aims to elucidate the molecular mechanisms underlying the occurrence of this subtype. The study subjects included a blood donor specimen with incompatible forward and reverse ABO typing results. ABO phenotyping was determined using ABO blood group serology and GTB activity testing. Subsequently, Sanger sequencing and third-generation sequencing based on the PacBio platform were employed to sequence the ABO gene, resulting in the determination of haplotype sequences. Mutations were identified through sequence alignment. An in vitro cell expression system was established to assess the impact of the mutation site on antigen expression. The index case in this study was identified as B subtype with the allelic genotype c.917T>C in ABO*B.01/ABO*O.01.01 , which has not been previously reported. in vitro expression results revealed decreased levels of GTB expression and overall GTB activity in the mutant cells. Furthermore, the expression of the B antigen on the cell membrane was weaker in the mutant cells compared to the wild-type cells. The p.L306P variation caused by the c.917T>C mutation in the ABO*B.01 allele may be a genetic factor contributing to the reduced expression of B antigens on the surface of red blood cells.

BACKGROUND:The drug antibodies can seriously affect the efficacy of the drug and/or destroy red blood cells (RBCs). In severe cases, the drug antibodies may lead to hemolytic anemia, ineffective transfusion, and even life-threatening conditions. However, current methods for detecting drug-induced antibodies present several limitations: cumbersome operational workflows, time-consuming processes, suboptimal sensitivity for low-titer antibodies, and ease of disturbance by autoantibodies and erythrocyte alloantibodies. Therefore, there is an urgent need for a drug antibody detection method with operational simplicity, ultrahigh sensitivity, and interference resistance. METHODS:Blood samples were collected from patients with drug-induced immune hemolytic anemia (DIIHA). Based on the established assay protocol, nine reaction systems were prepared and sequentially analyzed by flow cytometry. The results of flow cytometry were used to determine the presence or absence of drug antibodies, identify the specific type of drug antibody, and assess the antibody titer. RESULTS:Different drug antibodies, including ceftriaxone, cefoperazone, and amoxicillin, were identified in all six patients with DIIHA. Upon discontinuation of the implicated antibiotics, the efficacy of blood transfusions in those six patients showed significant improvement. CONCLUSIONS:This approach offers several advantages, including ease of operation, rapid detection time, high specificity and sensitivity, and excellent interference resistance. This new approach will be suitable for determining drug antibodies in clinical laboratories.

OBJECTIVE:To explore the serological characteristics and bioinformatics analysis results of 4 blood donors with RHCE*cE(281C, 282T) variant allele. METHODS:A total of 4 non-related blood donors with RHCE*cE (281C, 282T) variant allele (donors 1-4) were selected as the study objects. They donated blood at Shenzhen Blood Center from January 2022 to June 2023. The 4 blood donors were all Han. And 5 mL elbow venous blood was collected from these 4 blood donors. Regular serological assaying with 4 kinds of monoclonal antibody reagents was used for determination of the RhCcEe type. The nucleotide sequences of all 10 exons and adjacent flanking intron regions of RHCE gene in these 4 donors were analyzed by Sanger sequencing, and the full-length haplotype analysis of RHCE gene was performed by using the single-molecule real-time sequencing (SMRT) third-generation technology. DeepTMHMM software was used to analyze the structure of protein transmembrane region of wild type and variant RhCcEe protein and predict the location of amino acid substitution. The effects of mutations on RhCcEe protein function were analyzed using PolyPhen-2, SIFT and Mutation Taster bioinformatics software. Robetta and Swiss-PdbViewer v4.1.0 were used for modeling the tertiary structures of RhCcEe to analyze the difference between wild type and variant RhCcEe protein. The mutation was rated according to the standards and guidelines for the classification of genetic variants of the American College of Medical Genetics and Genomics (ACMG). This study has been approved by the Medical Ethics Committee of Shenzhen Blood Center (Approval No. SZBCMEC-2022-024). RESULTS:The RhCcEe phenotypes of the 4 blood donors were CCEweake by serological assaying. The RhE antigen were weakly expressed form 0 to 3+. The analysis of RHCE gene sequence indicated that all the 4 donors with RHCE*cE (281C, 282T) allele. The mutation caused the substitution of a single amino acid in the RhCcEe protein (p.Leu94 Pro) and the amino acid substitution was located in the transmembrane α3 chain resulted in significant changes in the 3D structure of the extracellular region of RhCcEe protein. The substitution was predicted to be "Probably damaging", "Damaging" and "Polymorphism" by PolyPhen-2, SIFT and Mutation Taster bioinformatics software. According to the guidelines of ACMG, the variant was rated to be likely pathogenic. CONCLUSION:The RHCE*cE (281C, 282T) variant allele was first found in the Han Chinese population. The serological data of this allele were enriched. It provides an important guarantee for the safety of blood transfusion. Bioinformatics analysis provided evidences for further study of the structure and functions of RhCcEe protein.

Background: RhD variants are categorized into partial D, weak D, and DEL. The detection of DEL can only be achieved through the adsorption and elution method or molecular techniques. Here, we report a case of DEL phenotypes associated with a novel allele in a Chinese individual. Study design and methods: We used serological methods such as saline, indirect anti-human globulin, and adsorption-elution. The RHD genotype was determined by the PCR-sequence specific primer (PCR-SSP) method as well as the Sanger dideoxy sequencing. Results: RBCs of the sample were found to be DEL phenotype by serological testing, with negative reactions in the saline and indirect anti-human globulin tests while positive reactions by the absorption-elution method. The genotyping results revealed a hemizygous ( RHD c .1127 T>G / RHD-). The novel allele sequence has been submitted to GenBank (Accession number: OR608456). Conclusion: Our study demonstrates a case of a Chinese individual with DEL phenotype caused by a novel allele RHD c .1127 T > G. It expands the database of the DEL variant.

OBJECTIVE:To develop a genotyping method for the Junior blood type and report on a rare blood type with Jr(a-).METHODS:Healthy O-type RhD+ volunteer donors of the Shenzhen Blood Center from January to May 2021 (n = 1 568) and a pedigree with difficult cross-matching (n = 3) were selected as the study subjects. Serological methods were used for proband's blood type identification, unexpected antibody identification, and antibody titer determination. Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used for typing the proband's RhD gene. ABCG2 gene coding region sequencing and a PCR-SSP genotyping method were established for determining the genotypes of the proband and his family members and screening of Jra antigen-negative rare blood type among the 1 568 blood donors.RESULTS:The proband's ABO and RhD blood types were respectively determined as B and partial D (RHDDVI.3/RHD01N.01), Junior blood type Jra antigen was negative, and plasma had contained anti-D and anti-Jra. Sequencing of the ABCG2 gene revealed that the proband's genotype was ABGG201N.01/ABGG201N.01 [homozygous c.376C>T (p.Gln126X) variants], which is the most common Jr(a-) blood type allele in the Asian population. Screening of the voluntary blood donors has detected no Jr(a-) rare blood type. Statistical analysis of the heterozygotes suggested that the allelic frequency for ABCG2*01N.01 (c.376T) was 0.45%, and the frequency of Jr(a-) rare blood type with this molecular background was about 0.2‰.CONCLUSION:A very rare case of partial DVI.3 type and Jr(a-) rare blood type has been identified. And a method for identifying the Junior blood type through sequencing the coding regions of the ABCG2 gene and PCR-SSP has been established.

Several cases of the hemolytic disease of the fetus and newborn (HDFN) caused by immunoglobulin G (IgG) anti-M antibodies have been reported, in which almost all the HDFN-associated anti-M were warmly reacting. Here we report two cases of severe HDFN associated with cold-reacting IgG anti-M. In both cases, pregnancy was terminated, in weeks 33 and 23 respectively, due to a diagnosis of fetal growth retardation (FGR). To our knowledge, these are the most severe HDFN cases caused by cold-reacting IgG anti-M.

OBJECTIVE:The characteristics of the full-length mRNA sequences of MNS blood group-related genes GYPA, GYPB and GYPE were analyzed to understand the polymorphism of MNS blood group genes.METHODS:Anticoagulated blood within 24 h from 500 unpaid blood donors (8 ml each) were randomly selected, and MN, Ss and Mia blood types were identified by serological methods. 5 samples with different combinations of MNS and Mia blood types were randomly selected from 500 samples, and peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, then total mRNA was extracted. cDNA was prepared by using the reverse transcription kit. The target fragments were amplified by nested PCR, and the full-length mRNA sequences of GYPA, GYPB and GYPE were sequenced after gel cutting and recycling, and the base sequences were analyzed by Oligo 6.0 software.RESULTS:The MN, Ss and Mia phenotypes were detected by serological methods, and there were differences in agglutination intensity of red blood cells (RBC) and anti-Mia serum between different individuals. The full-length mRNA sequences of GYPA, GYPB and GYPE genes in 5 samples of different phenotype combinations were detected. The exon-6 was completely deleted from the GYPA mRNA in 1 sample, and the full-length of GYPA mRNA in the other 4 samples were complete. The exon-2 was completely deleted from the GYPB mRNA in 2 samples, with Mia blood type negative. 2 samples showed complete full-length of GYPB mRNA, with Mia blood type positive. There was base substitution in exon-5 of GYPB mRNA in 1 sample. The full-length of GYPE mRNA was intact in 5 samples.CONCLUSION:MNS blood group related-genes have obvious polymorphism, and the detection of full-length mRNA sequence lays a foundation for the analysis of GYPA, GYPB and GYPE gene structure and in-depth study of MNS blood group antigen expression.

目的 分析MNS血型相关基因GYPA、GYPBmRNA剪接体多态性,探讨各种剪接体编码的GPA和GPB蛋白异构体的亚细胞定位与MNS血型抗原表达的相关机理.方法 随机选取无偿献血者10份血液,提取外周血总mRNA,反转录为cDNA后,以巢式PCR方法扩增GYPA、GYPB基因的开放阅读框并进行Sanger测序,碱基序列与GYPA(NCBI:NM_002099)、GYPB(NCBI:NM_002100.5)比对.得到的GYPA、GYPB开放阅读框的野生型及各种剪接异构体编码序列后,通过融合PCR技术分别与绿色荧光蛋白(GFP)编码基因融合并克隆后转染到HEK293细胞进行过表达,通过聚焦激光扫描显微镜监测GPA-GFP和GPB-GFP融合荧光蛋白的亚细胞定位.结果 2例标本的GYPA mRNA中缺失外显子1与外显子2,预测的GPA蛋白异构体缺失2～26位氨基酸,GYPB mRNA全长序列完整.6例标本的GYPA mRNA完整,GYPB mRNA中缺失外显子2,预测的GPB蛋白异构体缺失13～45位氨基酸,其他外显子序列完整.1例标本的GYPA mRNA完整,GYPB mRNA的外显子5中364～385位碱基被AG替换,显示氨基酸信号肽截短.其他标本的GYPmRNA全长序列完整.GP-GFP融合蛋白的荧光信号的观察结果表明,根据各RNA剪接体克隆表达的GPA、GPB糖蛋白异构体均可表现出细胞膜表面定位分布,其中的一些可变剪接导致蛋白异构体发生不同程度的细胞内弥散,影响蛋白在细胞表面分布的速度和比例,可能构成MNS抗原强弱的原因之一.结论 GYP mRNA剪接体有明显多态性特征,但GYP mRNA的部分片段缺失不影响其编码的蛋白异构体在细胞表面的定位分布,能够确保其展示MNS抗原特性.

Objective To investigate the family inheritance of α-Thalassemla gene and the risk of severe anemia in neonates caused by cold IgG anti-M. Methods ABO, Rh, MN blood groups and the specificity of unexpected antibody were identified by blood group serology. The IgG subtype and antibody titer of anti-M antibody were detected. The etiology of neonatal hemolytic disease was identified by three tests and α-Thalassemla gene diagnosis. Results Family investigation showed that father was B, CCDee, MN with no α-Thalassemla gene detected; Mother B, CcDee, NN, carrying α-Thalassemla gene; both the proband and his brother were B, CCDee, MN, carrying α-Thalassemla gene. Cold IgG anti-M was present in plasma of both the mother and the proband. The titer of the mother was 128 and that of the proband was 64. The subtype of IgG anti-M was IgG1 and IgG3. The direct anti-globulin test, release test and free test of the proband and his brother were negative, and the diagnosis was severe anemia and hemolysis caused by α-Thalassemla combined with cold IgG anti-M. Conclusion The direct antiglobulin test of neonatal hemolytic disease caused by IgG anti-M can be negative or weakly positive, and α-Thalassemla gene could be hereditary in families. The presence of α-Thalassemla gene can cause anemia, hemolysis and splenomegalysis in neonates, which could be aggravated when accompanied by cold-type IgG anti-M. In the presence of high-valency IgG antibody in plasma, blood exchange combined with transfusion can improve the curative effect.

Objective To study the molecular mechanism of 9 samples with rare RhD variants and their RhD epitopes and protein structure. Methods The 9 blood samples with rare RhD variants were collected from 210 644 blood donors of Shenzhen Blood Center. Regular serological assaying was used for determination of Rh type for the 9 samples. Indirect anti-human globulin test (IAT) was used to confirm the RhD antigen and to screen the antibodies. D-screen reagent was sued to analyze the RhD epitopes of the samples. RHD zygosity testing of the samples was detected by PCR-SSP. The nucleotide sequences of all 9 exons and adjacent flanking intron regions of RHD gene were sequenced. The prediction of the effects of mutations on RhD protein function were analyzed using PROVEAN, SIFT, PolyPhen-2 and MutationTaster software. Robetta and Swiss-PdbViewer 4.1.0 were used for modeling the tertiary structures of RhD. Results A total of 9 individuals with rare RhD variants were identified as follows: RHD*weak D type 25, RHD*weak D type 50, RHD*weak D type 95, RHD*weak D type 12, RHD*weak D type 128 and four novel RHD alleles. The prediction of the tertiary structures showed that the RhD protein conformation was disrupted in the 9 rare RhD variants samples. Conclusion Five rare and four novel RHD alleles have been identified. Their phenotypic and genotypic descriptions enrich the database of reported RHD alleles. Bioinformatics analysis provided evidences for further study of the structure and functions of RhD protein.

BACKGROUND:The null phenotype in P1PK blood group, known as "p," is extremely rare in the whole world. Individuals of p phenotype spontaneously form anti-PP1PK isoantibody. Here, we report a case of p phenotype with naturally occurring anti-PP1PK isoantibodies in a Chinese individual.STUDY DESIGN AND METHODS:Serology tests, containing alloantibodies screening and identification, were conducted to demonstrate the phenotype in P1PK blood group. The genotype of A4GALT gene was identified by haplotypes separation and sequencing.RESULTS:The serological assay demonstrated the p phenotype of the proband, presenting with 1:64 titer of anti-PP1PK . The sequencing data revealed a compound heterozygote consisting of A4GALT*P1.01 with c.343A>T and a novel allele based on A4GALT*01N.05 with an addition polymorphism c.100G>A. The sequence of the novel allele has been submitted to GenBank and the accession number OM912503 was assigned.CONCLUSION:Our study demonstrates a case of naturally occurring anti-PP1Pk in a Chinese individual with p phenotype, which is based on compound heterozygosity including one novel allele. As the proband is a young lady, monitoring the titer of anti-PP1PK and early initiation of medical intervention are essential after her pregnancy.

目的 探讨α-Thalassemla基因的家系遗传及合并冷型IgG抗-M导致新生儿重度贫血的危害.方法 以血型血清学方法鉴定家系ABO、Rh、MN血型与意外抗体特异性,检测抗-M IgG亚型及抗体效价,结合新生儿溶血病诊断的三项试验与α-Thalassemla基因的诊断,明确病因后换血与输血治疗.结果 家系调查显示父亲:B,CCDee,MN,未检到 a-Thalassemla 基因;母亲:B,CcDee,NN,携带 α-Thalassemla 基因;先证者与弟弟均为:B,CCDee,MN,携带 α-Thalas-semla基因.母亲与先证者的血浆均存在冷型IgG抗-M,母亲抗体效价:128,先证者抗体效价:64.IgG抗-M亚型为IgG1与IgG3型,先证者与弟弟的直接抗球蛋白试验阴性,放散试验阴性,游离试验阳性,诊断为α-Thalassemla合并冷型IgG抗-M引起重度贫血与溶血.结论 由IgG抗-M引起新生儿溶血病的直接抗球蛋白试验可以表现为阴性或者弱阳性,α-Thalassemla基因存在家系遗传.携带α-Thalassemla基因可以引起新生儿贫血、溶血和脾肿大,合并冷型IgG抗-M可加重胎儿期及新生儿期的贫血与溶血程度,血浆中存在高效价IgG抗体时,换血合并输血可以提高疗效.

目的 研究1例RhD血型鉴定部分凝集结果个体及其家系血清学表现和RHD基因.方法 通过血型微柱凝胶卡检测先证者ABO及RhD血型;盐水试管法检测先证者及其父母RhCcEe抗原;间接抗人球蛋白试验(indirect anti-human globulin test,IAT)及流式细胞术检测先证者RhD抗原.PCR序列特异性引物(PCR sequence specific primer,PCR-SSP)检测RHD基因以及RhD杂合型分析,基因测序方法分析RHD基因编码区序列.结果 血清学检测发现先证者血型为A型RhCcee,血型微柱凝胶卡、盐水试管法以及IAT法检测RhD抗原,结果呈部分凝集现象.流式细胞术结果显示先证者RhD抗原性减弱.经RHD基因编码序列分析发现,RHD基因第9外显子上的第1212位碱基发生C>A纯合突变,为RHD*weak D type 72的特征性突变点.家系调查显示,先证者父亲为O型RhCCDee,母亲为A型RhCcDee.父亲携带RHD*weak D type 72等位基因,基因型为RHD*weak D type 72/RHD+;母亲一条染色体缺失了全部的RHD基因,基因型为RHD+/RHD-.证明先证者分别从父亲和母亲遗传RHD*weak D type 72和RHD-等位基因,基因型为RHD*weak D type 72/RHD-.结论 发现了1例RHD*weak D type 72/RHD-基因型个体,丰富了RHD*weak D type 72变异型的研究数据.根据家系调查证明,RHD*weak D type 72等位基因由遗传获得,而非由个体基因变异形成.

Objective To analyze the polymorphisms of GYPA and GYPB mRNA spliceosomes associated with MNS blood group, and to explore the mechanism of subcellular localization of GPA and GPB protein isomerism encoded by various spliceosomes as well as the expression of MNS blood group antigen. Methods Ten blood samples of voluntary blood donors were randomly selected. The total mRNA of peripheral blood was extracted and reversed into cDNA. Nested PCR was used to amplify reading open frame of GYPA and GYPB gene, and sequencing was performed by Sanger. The base sequence obtained was compared with GYPA(NCBI: NM_002099) and GYPB(NCBI: Nm_002100.5). After the wild type and various splicing isomer of the open reading frame of GYPA and GYPB had been obtained, they were fused with the encoding gene of green fluorescent protein (GFP) by fusion PCR technology, then cloned and transfected into HEK293 cells for over expression. The subcellular localization of GPA-GFP and GPB-GFP fused fluorescent proteins was monitored by focusing laser scanning microscope. Results Exon-1 and Exon-2 were missing in GYPA mRNA of the 2 samples, and 2~26 amino acids were missing in the predicted GPA isomer, and the full length sequence of GYPB mRNA was complete. GYPA mRNA was intact in 6 samples, exon-2 was missing in GYPB mRNA, 13~45 amino acids were missing in the predicted GPB protein isomer, and other exon sequences were intact. One sample had intact GYPA mRNA, and 364~385 bases in exon-5 of GYPB mRNA were replaced by AG, indicating truncation of amino acid signal peptide. The GYP mRNA sequences of other samples were complete. The fluorescence signal of GP-GFP fusion protein showed that all GPA and GPB glycoprotein isomers, cloned according to various RNA splicing, could demonstrate the orientation distribution on the cell membrane surface, while some alternative splicing leaded to different degrees of protein dispersion in the cell, and affected the distribution speed and proportion of protein on the cell surface, which might be one of the reasons for the strength variation of MNS antigen. Conclusion The GYP mRNA spliceosome is obviously polymorphic, but the partial deletion of GYP mRNA fragment does not affect the localization and distribution of the protein isomers encoded by GYP mRNA on the cell surface, which can ensure the expression of MNS antigen characteristics.

The authors have disclosed no conflicts of interest. Appendix S1. Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND AND OBJECTIVES:The molecular basis of MNS blood group variants is not fully clear yet. In this study, we have characterized mRNA variants of GYPA and GYPB genes to reveal whether alternative RNA splicing may cause antigenic diversity of the MNS system.MATERIALS AND METHODS:Total RNA was extracted from peripheral blood of Chinese blood donors and full-length cDNA products were generated. A nested polymerase chain reaction (PCR)-based method was established for fragment amplification and Sanger sequencing. Resulted full-length mRNA sequences were aligned with GYPA or GYPB genomic sequences respectively for exon identification. Amino acid (AA) sequences of GPA and GPB proteins were extrapolated and GYPA-EGFP, GYPB-EGFP fusion genes were generated to monitor subcellular distribution of the encoded glycophorin (GP) proteins.RESULTS:Totally 10 blood samples were analysed. GYPB mRNAs of all the subjects demonstrated frequent exon insertion or deletion whereas this kind of variation was only observed in 3 of 10 GYPA mRNA samples. None of the reported Miltenberger hybrids was detected in any of the mRNA samples. The alternative splicing resulted in changes of AA sequences in N-terminal domains where the MNS antigenic motifs resided; however, subcellular localizations of GP-EGFP fusion proteins showed that the above-mentioned AA changes did not affect cell surface distribution of the encoded GP proteins.CONCLUSIONS:Alternative RNA splicing may influence the antigenic features of GP proteins but not their cell surface distribution. Therefore, GYPA and GYPB mRNA characterization might be an invaluable supplement to serological phenotyping and DNA-based genotyping in MNS blood grouping.