Search

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.

• A new approach to identifying complicated genome variations can be achieved through the long-read sequencing (LRS) technique represented by PacBio SMRT technology. • Based on LRS analysis of the core region of GYPA and GYPB , different genotypes of MNS can be determined accurately and reliably in one assay. • LRS can present a new approach for MNS blood group typing. The objective of this study was to devise a novel approach for determining MNS blood group utilizing long-read sequencing (LRS) and to identify intricate genome variations associated with this blood group system. In this study, a total of 60 blood samples were collected from randomly selected Chinese Han blood donors. The amplification of the full-length sequences of GYPA exon 2-7 (11 kb) and GYPB exon 2-6 (7 kb) was conducted on the blood samples obtained from these 60 donors. Subsequently, the sequencing of these amplified sequences was performed using the PacBio platform. The obtained sequencing data were then compared with the reference sequence of the human genome (GRCh38) utilizing the pbmm2 software, resulting in the acquisition of the haploid sequences of GYPA and GYPB. The serological typing prediction was conducted using the International Society of Blood Transfusion (ISBT) database, while the analysis of SNVs sites was performed using deepvariant v1.2.0 software and reference sequence alignment. A total of 60 samples yielded unambiguous high-quality haplotypes, which can serve as a standardized reference sequence for molecular biology typing of MNSs in the Chinese population. In a total of 60 serological samples, the LRS method successfully identified the M, N, S, and s blood group antigens by analyzing specific genetic variations (c.59, c.71, c.72 for GYPA , and c.143 for GYPB), which aligned with the results obtained through conventional serological techniques. 4 Mur samples that had been previously validated through serology and molecular biology were successfully confirmed, and complete haploid sequences were obtained. Notably, one of the Mur samples exhibited a novel breakpoint, GYP (B1-136-B ψ 137-212-A213-229-B230-366), thereby representing a newly identified subtype. Single molecule sequencing, which eliminates the necessity for PCR amplification, effectively encompasses GC and high repeat regions, enhancing accuracy in quantifying mutations with low abundance or frequency. By employing LRS analysis of the core region of GYPA and GYPB , diverse genotypes of MNS can be precisely and reliably identified in a single assay. This approach presents a comprehensive, expeditious, and precise novel method for the categorization and investigation of MNS blood group system. [ABSTRACT FROM AUTHOR]

Amr J Halawani,1 Mahmoud M Habibullah,2,3 Gasim Dobie,2 Alaa Alhazmi,2,3 Farkad Bantun,4 Mohammed H Nahari,5 Ibrahim Dawmary,6 Hisham I Abu-Tawil6,7 1Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi Arabia; 2Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Jazan University, Jazan, Saudi Arabia; 3SMIRES for Consultation in Specialized Medical Laboratories, Jazan University, Jazan, Saudi Arabia; 4Department of Microbiology, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia; 5Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Najran University, Najran, Saudi Arabia; 6Department of Laboratory and Blood Bank, Prince Mohammed bin Nasser Hospital, Ministry of Health, Jazan, Saudi Arabia; 7Department of Laboratory and Blood Bank, King Faisal Medical City for Southern Regions, Ministry of Health, Abha, Saudi ArabiaCorrespondence: Amr J Halawani Makkah, 24381 8156, Saudi ArabiaEmail ajjhalawani@uqu.edu.saPurpose: Knowledge of the prevalence of blood group antigens in a given population is important for the prevention of hemolytic reactions. The MNS blood group system (002) has four polymorphic antigens—M, N, S, and s. Anti-S and anti-s antibodies may result in immediate and delayed hemolytic transfusion reactions, and hemolytic disease of the fetus and newborn may occur. The present study investigated the frequencies of the main antigens and phenotypes of the MNS blood group system.Subjects and Methods: We randomly obtained 149 samples from anonymous Saudi blood donors living in Jazan Province. Serotyping was conducted using a gel card to investigate (M, N, S, and s) antigens and phenotypes.Results: The frequencies of MNS antigens were as follows: M = 89.26%, N = 51.67%, S = 61.07%, and s = 82.55%. Regarding the MNS phenotypes, nine phenotypes were observed in the study population. The most common phenotype was M+N–S+s+ (n = 36, 24.16%), in contrast to the least common phenotype M+N–S–s– (n = 1, 0.67%). The prevalence of the MNS phenotypes in the current study population was highly and significantly different from that in Europeans (P = 0.044) and African Americans (P = 0.000).Conclusion: In summary, this study reports the frequencies of the MNS antigens and phenotypes in Jazan Province, Saudi Arabia. The most common phenotype was M+N–S+s+, whereas the least observed phenotype was M+N–S–s–. The outcomes of this study may assist the blood banks in Jazan Province to establish an extended phenotyping protocol including the MNS antigens, in particular S and s antigens, to preclude any alloimmunization events.Keywords: MNS blood group, blood donors, transfusion, Saudi Arabia

Mingchun Lin,1,2 Meixiu Liu,3 Shulian Zhang,1 Chao Chen,1 Jin Wang1 1Neonatal Department, Children’s Hospital of Fudan University, Shanghai, 201102, People’s Republic of China; 2Neonatal Fellowship Training at Children’s Hospital of Fudan University, Yueqing Maternal and Child Health Hospital, Wenzhou, Zhejiang Province, People’s Republic of China; 3Blood Bank, Children’s Hospital of Fudan University, Shanghai, 201102, People’s Republic of ChinaCorrespondence: Jin WangNeonatal Department, Children’s Hospital of Fudan University, No. 399, Wanyuan Road, Shanghai, 201102, People’s Republic of ChinaTel +86-21-64932960Email drwangjin@fudan.edu.cnPurpose: To review the neonatal cases with different types of minor blood group incompatible haemolytic diseases in China, and to improve the clinical understanding and management.Materials and Methods: Seven cases from January, 1st, 2013 to December 31st, 2019 were searched out and reviewed retrospectively. All clinical data and laboratory findings were collected.Results: There were totally seven cases enrolled including three cases of MNS, three of Diego, and one of Kidd combined with Rh, anti-RhE incompatibility. Among the seven cases, two had intrauterine transfusion, two underwent exchange transfusion, five received intravenous immune globulin, five cases developed anaemia, and three of them had transfusion. But among them, only four were found to have positive antibody screening and three were confirmed HDN with antibody types antenatally.Conclusion: The clinical presentation is diverse. Antibody screening followed by the technique of peak systolic velocity in the fetal middle cerebral artery (MCA-PSV) helps to filter out the severe cases.Keywords: neonatal haemolytic disease, HDN, alloimmunization, MNS blood group, Diego blood group, Kidd blood group

Background and Objectives: Antigens of the MNS blood group system are expressed on the red blood cell (RBC) membrane on glycophorin A (GPA) and glycophorin B (GPB) or on hybrid molecules of GPA and GPB. This study investigated the distribution of glycophorin variants and alloantibodies against Hil and MINY among Japanese individuals. Methods: Mi(a+) or Hil+ RBCs were screened using an automated blood grouping machine (PK7300) with monoclonal anti‐Mia or polyclonal anti‐Hil. Glycophorin variants were defined by serology with monoclonal antibodies against Mia, Vw, MUT and Mur, and polyclonal antibodies against Hil, MINY and Hop + Nob (KIPP). The glycophorin variants were further confirmed by immunoblotting and Sanger sequencing. Alloanti‐Hil and alloanti‐MINY in the plasma were screened using GP.Hil RBCs in an antiglobulin test. The specificity of anti‐Hil or anti‐MINY was assessed using GP.Hil (Hil+MINY+) and GP.JL (Hil‒MINY+) RBCs. Results: The GP.HF, GP.Mur, GP.Hut, GP.Vw, GP.Kip and GP.Bun frequencies in 1 005 594 individuals were 0·0357%, 0·0256%, 0·0181%, 0·0017%, 0·0009% and 0·0007%, respectively. GP.Hil was found in as four of the 13 546 individuals (0·0295%). Of 137 370 donors, 10 had anti‐Hil (0·0073%) and three had anti‐MINY (0·0022%). Conclusions: Glycophorin variants were relatively rare in Japanese individuals, with the major variants being GP.HF (0·0357%), GP.Hil (0·0295%) and GP.Mur (0·0256%). Only one example of anti‐MINY was previously reported, but we found three more in this study. [ABSTRACT FROM AUTHOR]

Background: The low‐incidence antigen Sta of the MNS system is usually associated with the GP(B‐A) hybrid molecule, which carries the 'N' antigen at the N terminus. The GP(A‐A) molecule with trypsin‐resistant M antigen has been found in a few St(a+) individuals. Materials and Methods: Among Japanese blood donors, we screened 24 292 individuals for the presence of St(a+) with trypsin‐resistant 'N' antigen and 193 009 individuals for the presence of St(a+) with trypsin‐resistant M antigen. The breakpoints responsible for the Sta antigen were analysed by sequencing the genomic DNAs. Results: A total of 1001 (4·1%) individuals were identified as St(a+) with trypsin‐resistant 'N' antigen. Out of 1001 individuals, 115 were selected randomly for sequencing. Two novel GYP*Sch (GYP*401) variants with new intron 3 breakpoints of GYPA were detected in three cases. Twenty‐five (0·013%) individuals were identified as St(a+) with trypsin‐resistant M antigen. Five individuals had the GYP(A‐ψB‐A) hybrid allele; two of these five individuals were GYP*Zan (GYP*101.01), and the remaining three had a novel GYP(A‐ψB‐A) allele with the first breakpoint in GYPA exon A3 between c.178 and c.203. Nine individuals had a novel GYP(A‐E‐A) allele with GYPE exon E2 and pseudoexon E3 instead of GYPA exon A2 and A3. The 11 remaining individuals had a novel GYP(A‐A) allele with a 9‐bp deletion that included the donor splice site of intron 3 of GYPA. Conclusion: Our finding on diversity of glycophorin genes responsible for Sta antigen provides evidence for further complexity in the MNS system. [ABSTRACT FROM AUTHOR]

Background and objectives: MNS is a highly polymorphic blood group comprising 49 antigens recognized by International Society of Blood Transfusion, some of which may have been generated by genomic recombination among the closely linked genes GYPA, GYPB and GYPE. The GYPE gene has an almost identical sequence to GYPA*01 allele in exon 2 (99% homology), which accounts for M antigen. We investigated an unusual glycophorin molecule with protease‐resistant M antigen. Methods: Blood samples were screened by an automated blood typing system (PK7300) using bromelain‐treated red blood cells (RBCs) and murine monoclonal anti‐M. The M‐positive RBC samples were analysed by immunoblotting using anti‐M as the primary antibody. GYPA, GYPB and GYPE genes were analysed by polymerase chain reaction (PCR), cloning and sequencing using reticulocyte mRNA and genomic DNA. Results: Serological tests and immunoblotting revealed that 103 of the 193 009 individuals (0·0534%) expressed protease‐resistant M‐active glycophorin having a molecular weight of 20 kDa. All the 103 individuals were S+ s− or S− s+. When reticulocyte mRNA from the individuals with M‐active glycophorin (20 kDa) was examined by PCR and cloning followed by sequencing, a novel GYPE‐B hybrid transcript was identified. Long‐range PCR and sequencing using genomic DNA revealed that the individuals had a GYPB‐E(2‐4)‐B hybrid gene. This hybrid gene was predicted to encode a 59‐amino‐acid mature glycoprotein that expresses no S or s antigens Conclusions: The prevalence of the M‐active glycophorin (20 kDa) in the Japanese population is 0·0534%. This glycophorin is predicted to be a 59 amino acids polypeptide encoded by the novel GYPB‐E(2‐4)‐B hybrid gene. [ABSTRACT FROM AUTHOR]

Background and objectives: The rare S–s– phenotype has two main molecular backgrounds. GYPB deletions give rise to the S–s–U– phenotype, which loses the expression of the U antigen, while variant GYPB alleles usually lead to the S–s–U+var phenotype, which express a variant U antigen. The S–s– phenotype is typically found in people of African origin and represents a challenge in transfusion sets, especially when S–s– patients develop anti‐U. Molecular analysis is the most reliable method for determining U antigen status. We studied the molecular basis of the S–s– phenotype in donors and patients at Regional Blood Center of Ribeirão Preto. Material and Methods: Five patients and 25 donors with the S–s– phenotype were investigated through real‐time PCR for the GYPB*S/s polymorphism, followed by an allele‐specific/RFLP‐PCR for GYPB deletion (GYPB*Null) and for its main variants: GYPB*P2 and GYPB*NY. DNA sequencing was conducted in one sample. Results: Two samples were heterozygous GYPB*P2/GYPB*NY, eight were homozygous/hemizygous for GYPB*P2 and 19 samples were homozygous for GYPB*Null. A hybrid gene (GYPB‐E‐B.Ros) was found in one sample after discrepant results in the initial tests. Conclusion: GYPB deletion is the main mechanism responsible for the S–s– phenotype in our donors and patients. It is essential to evaluate the main GYPB variant alleles when genotyping in order to obtain the correct prediction of the phenotype. Hybrid genes lead to discrepancies between genotype and phenotype and may not be detected by conventional molecular assays. [ABSTRACT FROM AUTHOR]

We found an individual with weakened S antigen expression on red blood cells (RBCs) during routine blood grouping. The proband was typed S+s+ by polyclonal antibodies, but the RBCs demonstrated different reactivity with three monoclonal anti‐S. The proband did not have alloanti‐S. Cloning and Sanger sequencing revealed that the proband had a c.166A>T (p.Thr56Ser) mutation in exon 4 of GYPB*S. When antibody screening of 60 455 blood donors was performed using the proband RBCs, no antibodies were detected. GYPB*S with c.166T should encode an unusual S antigen but the creation of a novel antigen remains to be investigated. [ABSTRACT FROM AUTHOR]

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.

Background and Objectives Gene conversion events between GYPA and GYPB or GYPA and GYPE are facilitated by the close chromosomal proximity and high degree of sequence homology and can lead to the formation of GP hybrid genes. Discrepant results between blood group genotyping and haemagglutination in 22 random blood donors induced molecular characterization. Materials and Methods Sequence analysis of GYPA exons 1-7 and GYPB exons 1-5 was performed for g DNA and c DNA. The linkage of the nucleotide alterations was defined by haplotype separation. Results DNA analysis demonstrated a normal GYPA haplotype ( GYPA* N n = 20, GYPA* M n = 2) with an altered GP hybrid nucleotide sequence in trans. A GYPB homologue sequence of minimal 10-bp encompassing intron 1 and exon 2 was translated into GYPA, accounting for an amino acid substitution from arginine to glutamic acid at position 13 (38 C>A). Genomic DNA analysis demonstrated the cis-linkage of the hybrid nucleotide sequence with each GYPA( Ser20, Gly24) ( n = 20) associated with the expression of M and GYPA( Leu20, Glu24) ( n = 2) encoding the N phenotype. The serologic data indicate that the changes do not affect the expression of a normal M and N antigen. c DNA sequences confirmed the g DNA results and furthermore identified a heterozygous deletion of GYPB exon 2 in all probands. Conclusion The results document a GYPA- B- A hybrid gene, probably produced via a single unequal homologous recombination event. A segmental transfer of GYPB seems most likely accounting for the allelic dropout. [ABSTRACT FROM AUTHOR]

Serological methods are unreliable for accurate determination of blood group antigens in multi-transfused thalassemia patients. The MNS blood group system has five high-frequency antigens. Many studies demonstrated that some antibodies including anti-S, anti-s, and anti-U may cause acute and delayed transfusion reactions and hemolytic disease of the fetus and newborn. This study aimed to determine the genotype of the MNS blood group in thalassemia patients with alloantibodies by molecular methods. In this study, 104 blood samples from thalassemia patients were collected. The blood group phenotype for M, N, S and s antigens was determined by the tube hemagglutination method. MNS blood group genotyping was performed using PCR-SSP and DNA Sequencing methods. All patients were genotyped with a total of 6 pairs of primers. Discrepancies between genotype and phenotype were observed in 22 patients with S/s alleles and 2 patients with M/N alleles, however, there was full accordance between the results of SSP-PCR and DNA sequencing. The frequency of MNS blood group alleles was determined as follows: 25 % MNSs, 23 % MNss, 21 % MMSs, 9% MMSS, 9% MMss, 8% NNss, 2%MNSS, and NNSS, NNSs, MM genotypes at 1% each. In conclusion, molecular genotyping is more reliable than serological methods in multiple transfusion patients and can lead to a more compatible blood unit for transfusion in these patients. [ABSTRACT FROM AUTHOR]

Background: The low-incidence antigen St a of the MNS system is usually associated with the GP(B-A) hybrid molecule, which carries the 'N' antigen at the N terminus. The GP(A-A) molecule with trypsin-resistant M antigen has been found in a few St(a+) individuals., Materials and Methods: Among Japanese blood donors, we screened 24 292 individuals for the presence of St(a+) with trypsin-resistant 'N' antigen and 193 009 individuals for the presence of St(a+) with trypsin-resistant M antigen. The breakpoints responsible for the St a antigen were analysed by sequencing the genomic DNAs., Results: A total of 1001 (4·1%) individuals were identified as St(a+) with trypsin-resistant 'N' antigen. Out of 1001 individuals, 115 were selected randomly for sequencing. Two novel GYP*Sch (GYP*401) variants with new intron 3 breakpoints of GYPA were detected in three cases. Twenty-five (0·013%) individuals were identified as St(a+) with trypsin-resistant M antigen. Five individuals had the GYP(A-ψB-A) hybrid allele; two of these five individuals were GYP*Zan (GYP*101.01), and the remaining three had a novel GYP(A-ψB-A) allele with the first breakpoint in GYPA exon A3 between c.178 and c.203. Nine individuals had a novel GYP(A-E-A) allele with GYPE exon E2 and pseudoexon E3 instead of GYPA exon A2 and A3. The 11 remaining individuals had a novel GYP(A-A) allele with a 9-bp deletion that included the donor splice site of intron 3 of GYPA., Conclusion: Our finding on diversity of glycophorin genes responsible for St a antigen provides evidence for further complexity in the MNS system., (© 2020 International Society of Blood Transfusion.)

Background: Various techniques of genotyping the MNSs blood group have been described, but none of them enables the complete detection of all MNS antigens., Materials and Methods: Blood samples were obtained from blood donors. Primers were created using the published DNA sequences for glycophorins A and B. Genotyping was performed using polymerase chain reaction sequence-specific primers (PCR-SSP)., Results: A total of seven primers were found to specifically amplify the most common MNS antigens. The use of these primers has enabled us to correctly genotype all blood samples tested so far (n=116)., Discussion: Specifically created primers enable genotyping of the MNS antigens in a single PCR-SSP run. The method is reliable, easy to perform, and can be used in routine practice.