Comparison of the Detection Rate and Specificity of Irregular Red Blood Cell Antibodies Between First-Time Pregnant Women and Women With a History of Multiple Pregnancies Among 18,010 Chinese Women.

Background: There is insufficient evidence to assess the risk of the production of clinically important alloimmune irregular red blood cell (RBC) antibodies in first-time pregnant women. Methods: Using the microcolumn gel antiglobulin method, 18,010 Chinese women with a history of pregnancy and pregnant women were screened for irregular RBC antibodies, and for those with positive test results, antibody specificity was determined. The detection rate and specificity of irregular RBC antibodies in women with a history of multiple pregnancies (two or more) and first-time pregnant women were determined. Results: In addition to 25 patients who passively acquired anti-D antibodies via an intravenous anti-D immunoglobulin injection, irregular RBC antibodies were detected in 121 (0.67%) of the 18,010 women. Irregular RBC antibodies were detected in 93 (0.71%) of the 13,027 women with a history of multiple pregnancies, and antibody specificity was distributed mainly in the Rh, MNSs, Lewis, and Kidd blood group systems; irregular RBC antibodies were detected in 28 (0.56%) of the 4983 first-time pregnant women, and the antibody specificity was distributed mainly in the MNSs, Rh, and Lewis blood group systems. The difference in the percentage of patients with irregular RBC antibodies between the two groups was insignificant (χ 2 = 1.248, P > 0.05). Of the 121 women with irregular RBC antibodies, nine had anti-Mur antibodies, and one had anti-Dia antibodies; these antibodies are clinically important but easily missed because the antigenic profile of the reagent RBCs that are commonly used in antibody screens does not include the antigens that are recognized by these antibodies. Conclusion: Irregular RBC antibody detection is clinically important for both pregnant women with a history of multiple pregnancies and first-time pregnant women. Mur and Dia should be included in the antigenic profile of reagent RBCs that are used for performing antibody screens in the Chinese population.

Hemolytic disease of the fetus and newborn caused by anti-sD antibody in a GP.Mur/Mur Thai mother and review of the prevalence of sD in Thai blood donors.

BACKGROUND: Low-prevalence antigen sD (MNS23) is encoded by GYPB c.173C > G. Hemolytic disease of the fetus and newborn (HDFN) due to anti-sD is rare. A mother delivered a newborn whose red blood cells (RBCs) were DAT-positive and was later diagnosed with HDFN. Serum from the mother was incompatible with the father's RBCs and was used to screen 184 Thai blood donors. This study aimed to investigate the cause of HDFN in a Thai family and determine the prevalence of sD in Thai blood donors. MATERIALS AND METHODS: Three family members and four blood donors were investigated in the study. Massively Parallel Sequencing (MPS) was used for genotyping. Standard hemagglutination techniques were used in titration studies, phenotyping, and enzyme/chemical studies. Anti-s, anti-Mia , anti-JENU, and anti-sD reagents were used in serological investigations. RESULTS: The mother was GYP*Mur/Mur. The father and the four donors were GYPB*s/sD predicting S - s + sD +. The baby was GYP*Mur/sD and his RBCs were Mia +, s + w with anti-s (P3BER) and JENU+w . RBCs from two GYPB*sD -positive blood donors reacted with anti-sD (Dreyer). Proteolytic enzyme α-chymotrypsin-treated sD + cells did not react with anti-sD (Wat) produced by the GP.Mur/Mur mother but reacted with the original anti-sD (Dreyer). DISCUSSION: This is the first report of HDFN due to anti-sD in the Asian population. The genotype frequency for GYPB*sD in a selected Thai blood donor population is 2.2% (4/184). Anti-sD should be considered in mothers with Southeast Asian or East Asian background when antibody identification is unresolved in pregnancies affected by HDFN.

Fatal haemolytic transfusion reaction due to anti-Ena and identification of a novel GYPA c.295delG variant in a Thai family.

BACKGROUND AND OBJECTIVES: High-frequency antigen Ena (MNS 28) is expressed on glycophorin A (GPA). En(a-) individuals can form anti-Ena when exposed to GPA. A Thai patient formed an antibody that reacted against all reagent red blood cells (RBCs). The patient received incompatible blood resulting in a fatal haemolytic transfusion reaction (HTR). This study aimed to characterize the antibody detected in the patient and investigate the cause of HTR. MATERIALS AND METHODS: Blood samples from the patient and three of his family members were investigated. Massively parallel sequencing (MPS) and DNA-microarray were used for genotyping. Standard haemagglutination techniques were used for phenotyping and antibody investigations. RESULTS: DNA sequencing showed the patient was homozygous for GYPA*M c.295delG (p.Val99Ter) predicting En(a-). Three family members were heterozygous for GYPA c.295delG. MPS and DNA-microarray predicted the patient was N- discordant with the N+ RBC phenotype. The patient's plasma was positive with enzyme/chemical-treated reagent RBCs but failed to react with En(a-) and Mk Mk RBCs. CONCLUSION: The GYPA c.295delG variant prevented GPA expression on RBCs resulting in En(a-) phenotype. The N+ phenotype result was probably due to the anti-N typing reagent detecting 'N' (MNS30) on GPB. The patient's alloantibody has anti-Ena specificity.

Characterization of alternatively spliced transcript variants of glycophorin A and glycophorin B genes in Chinese blood donors.

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.

Frequencies of glycophorin variants and alloantibodies against Hil and MINY antigens in Japanese.

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.

GP.MOT: A novel glycophorin variant identified in a Japanese blood donor.

BACKGROUND: In this study, we identified a novel glycophorin variant (GP.MOT) in a Mia -positive Japanese blood donor. The proband with this glycophorin variant was discovered by antigen screening of samples from 475,493 Japanese blood donors using monoclonal anti-Mia . STUDY DESIGN AND METHODS: Standard serological techniques and flow cytometry were performed. GP.MOT RBCs were examined by immunoblotting using anti-GPA, anti-MUT or anti-Mur. Genome DNA was extracted from whole blood, and the GYPA/GYPB was analyzed by polymerase chain reactions and Sanger sequencing. RESULTS: The MNS blood group of the proband was M + N + w S-s + with the presence of other low-frequency antigens including Mia , Mur, MUT, and KIPP. A 43-kDa molecule, which is almost equivalent in size to glycophorin A (GPA), was identified by immunoblotting using monoclonal anti-MUT and anti-Mur. Sanger sequencing clearly indicated that the proband had two different GYPA*M alleles at SNP rs62334651 (GYPA*M232 + 55A and GYPA*M232 + 55G), as well as a GYP(B-A) hybrid allele (GYP*MOT) with breakpoints located on pseudoexon 3 of GYPB from c.210 to c.219. DISCUSSION: We identified a hybrid glycophorin GP.MOT with the deduced unique amino acid sequence GPB (20-45)-GPΨB (46-70)-GPA (71-149), which has not been previously reported.

Allo-anti-M: Detection peaks around 2 years of age, but may be attenuated by red blood cell transfusion.

BACKGROUND: Anti-M is frequently observed as a naturally occurring antibody of little clinical significance. Naturally occurring anti-M is often found in children although the specific triggers of production, persistence, and evanescence of anti-M have yet to be elucidated. METHODS: In a retrospective, multicenter, nationwide cohort survey conducted from 2001 to 2015, alloantibody screening was performed before and after transfusion in 18,944 recipients younger than 20 years. Recipients were categorized into six cohorts based on their age at transfusion; within and among these cohorts, allo-anti-M was analyzed in regard to its production, persistence, and evanescence. RESULTS: In 44 patients, anti-M detected before and/or after transfusion was an age-related phenomenon, with a median age of 2 years and an interquartile range of 1-3 years; anti-M was most frequently detected in a cohort of children 1 to <5 years (0.77%, 31 of 4035). At least five patients were presumed to have concurrent infections. Among 1575 adolescents/young adults (15 to <20 years), no anti-M was detected. Of 29 patients with anti-M prior to transfusion, the antibody fell to undetectable levels in 17 recipients (89.5%, of whom at least 13 received only M-negative red cells) after anywhere from 5 days to 5.8 years; anti-M persisted in 2, and was not tested in 10. Only 15 recipients (0.08%) produced new anti-M after transfusion. CONCLUSION: Naturally occurring anti-M is a phenomenon of younger ages, predominantly between 1 and 3 years. After transfusion, it often falls to undetectable levels.

Molecular genetic analysis of Mia -positive hybrid glycophorins revealed two novel alleles of GP.Vw and multiple variant transcripts of GYPB existing in both the homozygous GP.Mur and wild-type GPB individuals.

BACKGROUND: The hybrid glycophorins of MNS blood group system express a series of low incidence antigens including Mia , which are commonly found in Southeast Asian populations. In this study, the molecular basis of Mia -positive hybrid glycophorins was firstly clarified in the Chinese Southern Han population. RNA transcripts of GYPB gene in the homozygous GP.Mur individuals were also analyzed. STUDY DESIGN AND METHODS: DNAs were extracted from the whole blood samples of 111 Mia -positive donors. Then, high-resolution melting (HRM) analysis for GYP(B-A-B) was used to analyze the genotypes. Sequencing of GYPB pseudoexon 3 was conducted in the samples with variant melting curves. TA-cloning and subsequent sequencing of GYPA exons 2-4 were performed in the Mia -positive samples with normal GYPB/GYPB genotype by HRM. The transcript analysis of GYPB was conducted in homozygous GP.Mur and wild-type glycophorin B (GPB) individuals using RNA extracted from the cultured erythroblast. RESULTS: The heterozygous GYP*Mur/GYPB (n = 101), homozygous GYP*Mur/GYP*Mur (n = 7) including one novel GYP*Mur allele with an extra GYPA/GYPE specific nucleotide substitution (c.229+110A>T), heterozygous GYP*Bun/GYPB (n = 1) and GYP*Vw/GYPA (n = 2) with two novel GYP*Vw alleles were identified. RNA transcript analysis revealed multiple transcripts of GYPB existing in both homozygous GP.Mur and normal GPB individuals. CONCLUSION: The results showed the genetic diversity of hybrid glycophorins in the Chinese population. Besides, the successful analysis of GYPB transcripts indicates that the cultured erythroblast is a good source for RNA transcript analysis for the protein only expressed on the red blood cells.

Successful prenatal management of two foetuses affected by antibodies against GP.Mur with prenatal genotyping analysis and a literature review.

BACKGROUND: GP.Mur belongs to the GP(B-A-B) hybrid glycophorin family, which is the most common hybrid glycophorin in Southeast Asia. Antibodies against GP.Mur may cause a clinically significant haemolytic disease of the foetus and newborn (HDFN) although, so far, not many cases have been reported in mainland China. MATERIALS AND METHODS: Two Chinese women with a history of severe hydrops foetalis were seen in our centre. Alloantibody identification and GYP.Mur genotyping analysis were used for prenatal evaluation. Intrauterine transfusion was performed in two pregnancies in case 1. The features of these two women are described and literature-reported cases of HDFN related to antibodies against GP.Mur are summarised. RESULTS: The phenotype of both mothers was Mia- Mur-, while the fathers' was Mia+ Mur+ with a heterozygous GYP.Mur hybrid gene as determined by a high-resolution melting method of genotyping. In case 1, the antibodies against GP.Mur were detected in the mother's serum and the cord blood of two foetuses. Fortunately, the latest foetus was successfully saved after intrauterine transfusion. In case 2, hydrops foetalis occurred in the first two pregnancies, but the risk of HDFN was excluded for the third foetus because of the GP.Mur negative phenotype. The literature review showed that 68.8% (11/16) of the reported cases of HDFN related to antibodies against GP.Mur occurred in the Chinese population, and that 37.5% (6/16) of them were cases of severe HDFN. DISCUSSION: More cases of severe HDFN caused by antibodies against GP.Mur are presumably undetected as GP.Mur cells are not included in the panel of obligatory screening tests in most Southeast Asian countries including mainland China. The high-resolution melting method for GYP.Mur genotyping and zygosity detection is helpful in prenatal management.

A new antigen SUMI carried on glycophorin A encoded by the GYPA*M with c.91A>C (p.Thr31Pro) belongs to the MNS blood group system.

BACKGROUND: MNS is one of the highly polymorphic blood groups comprising many antigens generated by genomic recombination among the GYPA, GYPB, and GYPE genes as well as by single-nucleotide changes. We report a patient with red blood cell (RBC) antibody against an unknown low-frequency antigen, tentatively named SUMI, and investigated its carrier molecule and causal gene. STUDY DESIGN AND METHODS: Standard serologic tests, including enzyme tests, were performed. Monoclonal anti-SUMI-producing cells (HIRO-305) were established by transformation and hybridization methods using lymphocytes from a donor having anti-SUMI. SUMI+ RBCs were examined by immunocomplex capture fluorescence analysis (ICFA) using HIRO-305 and murine monoclonal antibodies against RBC membrane proteins carrying blood group antigens. Genomic DNA was extracted from whole blood, and the GYPA gene was analyzed by polymerase chain reactions and Sanger sequencing. RESULTS: Serologic screening revealed that 23 of the 541,522 individuals (0.0042%) were SUMI+, whereas 1351 of the 10,392 individuals (13.0%) had alloanti-SUMI. SUMI antigen was sensitive to ficin, trypsin, pronase, and neuraminidase, but resistant to α-chymotrypsin and sulfydryl-reducing agents. ICFA revealed that the SUMI antigen was carried on glycophorin A (GPA). According to Sanger sequencing and cloning, the SUMI+ individuals had a GYPA*M allele with c.91A>C (p.Thr31Pro), which may abolish the O-glycan attachment site. CONCLUSIONS: The new low-frequency antigen SUMI is carried on GPA encoded by the GYPA*M allele with c.91A>C (p.Thr31Pro). Neuraminidase sensitivity suggests that glycophorin around Pro31 are involved in the SUMI determinant.

Novel hybrid genes and a splice site mutation encoding the Sta antigen among Japanese blood donors.

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.

Multiple GYPB gene deletions associated with the U- phenotype in those of African ancestry.

BACKGROUND: The MNS blood group system is defined by three homologous genes: GYPA, GYPB, and GYPE. GYPB encodes for glycophorin B (GPB) carrying S/s and the "universal" antigen U. RBCs of approximately 1% of individuals of African ancestry are U- due to absence of GPB. The U- phenotype has long been attributed to a deletion encompassing GYPB exons 2 to 5 and GYPE exon 1 (GYPB*01N). STUDY DESIGN AND METHODS: Samples from two U-individuals underwent Illumina short read whole genome sequencing (WGS) and Nanopore long read WGS. In addition, two existing WGS datasets, MedSeq (n = 110) and 1000 Genomes (1000G, n = 2535), were analyzed for GYPB deletions. Deletions were confirmed by Sanger sequencing. Twenty known U- donor samples were tested by a PCR assay to determine the specific deletion alleles present in African Americans. RESULTS: Two large GYPB deletions in U- samples of African ancestry were identified: a 110 kb deletion extending left of GYPB (DEL_B_LEFT) and a 103 kb deletion extending right (DEL_B_RIGHT). DEL_B_LEFT and DEL_B_RIGHT were the most common GYPB deletions in the 1000 Genomes Project 669 African genomes (allele frequencies 0.04 and 0.02). Seven additional deletions involving GYPB were seen in African, Admixed American, and South Asian samples. No samples analyzed had GYPB*01N. CONCLUSIONS: The U- phenotype in those of African ancestry is primarily associated with two different complete deletions of GYPB (with intact GYPE). Seven additional less common GYPB deletion backgrounds were found. GYPB*01N, long assumed to be the allele commonly encoding U- phenotypes, appears to be rare.

Management of transfusion needs in a case of immunizing anti-M antibody in a patient with acute myeloid leukemia.

Anti-M is a relatively common "naturally occurring" antibody. Unexpected alloantibodies in patient's serum other than ABO isoagglutinins (e.g., anti-M) may cause a discrepancy in the reverse grouping. As long as anti-M does not react at 37°C, it is clinically insignificant for transfusion. However, we found this antibody to be of "immunizing" type which was reactive at 37°C and AHG phase and showing problems in blood grouping and crossmatch. This antibody had both IgM and IgG components. When "M" antibodies active at 37°C are encountered, antigen-negative or red cells that are compatible with an indirect antiglobulin test should be provided.

Fetal inheritance of GP*Mur causing severe HDFN in an unrecognized case of maternal alloimmunization.

BACKGROUND: The clinical and laboratory features of hemolytic disease of the newborn can be challenging to diagnose during pregnancy in the apparent absence of a blood group antibody. Low-frequency antibodies go undetected due to the lack of appropriate antigen-positive reagent red blood cells (RBCs). CASE REPORT: A pregnant woman of Southeast Asian descent was referred to a maternal-fetal medicine outpatient clinic due to a complicated obstetric history and a negative antibody screen. This initial visit at 29 weeks and 0 days' gestational age (GA) was unremarkable. A hydropic infant, born at 29 weeks and 5 days' GA, succumbed on the seventh day of life. Comprehensive laboratory testing was performed after birth. The hospital blood bank performed a maternal antibody identification. Direct antiglobulin test was performed on the cord blood. A reference laboratory confirmed an anti-Mia , performed paternal Mia phenotyping, and identified a hybrid glycophorin B-A-B GP*Mur allele. DISCUSSION: Maternal alloimmunization to low-frequency antigens remains a challenge. Southeast Asians make up a significant percentage in some US locations. Worldwide reports on the frequency of maternal alloimmunization of the MNS system can be used to guide the use of specific reagent RBCs for testing. Such strategies rely on the identification of blood donor units for reagent manufacture and use in perinatal antibody screens. CONCLUSION: The incidence of Mia and related antibodies is significant among Southeast Asians. In North America, prenatal antibody screening cells are not routinely chosen to match this population. The clinical and societal implications are discussed.

Management of patients with rare blood groups in maternity.

We report on our experiences since 2010 with pregnant women with rare blood types. The lack of compatible blood is a challenge for the anaesthetist whose priority is to prevent and treat anaemia in late pregnancy in order to avoid immunisation after transfusion of incompatible blood. In our hospital, the blood type is checked during the first obstetric consult, which is variable, starting from the fourth month of pregnancy. Rare blood types are most often diagnosed in an advanced stage of pregnancy (30 weeks of gestation: WG) due to the late inscription for obstetrics consult, resulting in even later anaesthetic visit. In our 13 patients, the most common blood systems are Duffy, MNS, and RH. 61.5% of the patients have associated antibodies (anti-MNS5). The majority of patients received iron with significant increase of ferritin (17.24 ± 12.95 µg/L versus 262.2 ± 404.4 µg/L, p = .033). Six of the patients had 2-3 injections of EPO between 29 - 36 + 1 WG. There were no transfers for paediatric management of haemolytic disease in the newborn following the birth. Overall, this treatment of patients with a rare blood group has also changed our practices for the follow-up of other pregnant women, and ferritin is more regularly prescribed.Impact statementWhat is already known on this subject? For rare blood groups, the frequency in the general population is less than 1/4000. The most common antibodies at risk of haemolytic disease and 'hydrops fetalis' are anti-D, anti-E, anti-C, and anti-K. The survey of pregnant women with a rare blood type takes into account the maternal risk of 'transfusion deadlock' and haemolytic disease of the newborn.What do the results of this study add? Rare blood types are most often diagnosed in an advanced stage of pregnancy (30 WG) due to the late inscription for obstetrics consults at Maternity. The most common blood systems are Duffy, MNS, RH, and 61.5% of the patients have associated antibodies (anti-MNS5). The most efficient treatment of prenatal anaemia was iron perfusions who allowed significant increase of ferritin and a maternal haemoglobin concentration of 12.1±1.46 g/dL in the ninth month of pregnancy.What are the implications of these findings for clinical practice and/or further research? A pregnant woman with a rare blood group is a situation that requires a technical platform specialised in haemorrhagic risk and a multidisciplinary team, including a blood bank as well as anaesthetic and obstetrical teams, with excellent interdisciplinary coordination.