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Routine ABO blood group typing of apparently healthy individuals sporadically uncovers unexplained mixed-field reactions. Such blood group discrepancies can either result from a haematopoiesis-confined or body-wide dispersed chimerism or mosaicism. Taking the distinct clinical consequences of these four different possibilities into account, we explored the responsible cause in nine affected individuals. Genotype analyses revealed that more than three-quarters were chimaeras (two same-sex females, four same-sex males, one sex-mismatched male), while two were mosaics. Short tandem repeat analyses of buccal swab, hair root and nail DNA suggested a body-wide involvement in all instances. Moreover, genome-wide array analyses unveiled that in both mosaic cases the causative genetic defect was a unique copy-neutral loss of heterozygosity encompassing the entire long arm of chromosome 9. The practical transfusion- or transplantation-associated consequences of such incidental discoveries are well known and therefore easily manageable. Far less appreciated is the fact that such findings also call attention to potential problems that directly ensue from their specific genetic make-up. In case of chimerism, these are the appearance of seemingly implausible family relationships and pitfalls in forensic testing. In case of mosaicism, they concern with the necessity to delineate innocuous pre-existent or age-related from disease-predisposing and disease-indicating cell clones., (© 2024 The Author(s). British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Background: Gerbich-negative phenotypes of the Gerbich Blood Group System (ISBT 020) are very rare (with the exception of Papua New Guinea). The Gerbich-negative phenotypes Yus and Gerbich are negative for the antigens Ge2, and Ge2 and Ge3, respectively. In antigen-negative individuals, anti-Ge2 and anti-Ge3 antibodies can be naturally occurring, or are triggered during pregnancies and after transfusions. Previous studies suggested an elevated frequency of Gerbich-negative phenotypes for the Middle East. In the summer of 2015, a large-scale migration of people from the Middle East to Europe occurred raising the issue of question how to guarantee blood supply for patients and manage antenatal care for pregnant women from these countries., Materials and Methods: To investigate the frequency of rare Gerbich-negative phenotypes, 1,665 immigrants to Germany originating from the Middle East were genetically tested for the presence of rare Yus, i.e., GE * 01.-02 , and Gerbich, i.e., GE * 01-03 , alleles and compared to results obtained from 507 Germans., Results: Seven Yus GE * 01.-02.01 and one Gerbich GE * 01.-03.02 alleles were exclusively observed among people from the Middle East, with five of them clustering among 797 Syrians. No such alleles were observed in Germans. A cumulative Yus- and GE * 01.-03- type allele frequency of 0.00314 and resultant overall Gerbich-negative phenotype frequency of one among 101,633 Syrians were calculated., Conclusion: This manuscript describes for the first time an exclusively genetic screening for carriers of Gerbich-negative alleles. In conclusion, the Gerbich blood group system should be considered as one causative agent of unusual antibodies to red cell antigens, in routine patients and pregnant women, especially when originating from the Middle East., Competing Interests: C.G. acts as a consultant to inno-train Diagnostik GmbH, Kronberg i.T., Germany. Procedures for the molecular detection of GYPB deletions for S-s-U-phenotype diagnostics have been granted as a European patent (No. 3545102). Similar content patent US application is pending. All other authors declare no conflicts of interest., (Copyright © 2022 by The Author(s). Published by S. Karger AG, Basel.)

Background: All antigens described in the KN blood group system are located in the long homologous repeat D (LHR-D) of complement receptor 1 (CR1). While there have been reports that some sera react only with the long homologous repeat C (LHR-C), the antigens in LHR-C are unknown., Study Design and Methods: Recombinant LHR-C and LHR-D were used to identify antibodies directed against LHR-C of CR1, into which a point mutation was introduced to characterize the underlying blood group antigens. In addition, database studies to define haplotypes of CR1 were performed., Results: Several antisera were identified that were specific against CR1 p.1208His and against CR1 p.1208Arg, located in LHR-C. Fifteen KN haplotypes were found in the Ensembl genome browser. It was shown that due to a linkage disequilibrium anti-CR1 p.1208His may be mistaken for anti-KCAM., Conclusion: A novel antithetical KN blood group antigen pair was found at position p.1208 of CR1, for which the names DACY and YCAD are proposed. Antibodies against these two novel antigens seem to contribute to more than a quarter of all KN sera in Europe., (© 2020 AABB.)

Conclusions: This update of the H blood group system (Scharberg EA, Olsen C, Bugert P. The H blood group system. Immunohematology 2016;32:112-8) reports 11 new FUT1 null alleles: 8 causative for the H- phenotype (Bombay phenotype, Oh), 3 in para-Bombay cases, and 5 new FUT1 alleles causative for a weak H phenotype (para-Bombay, H+w). The H blood group system (International Society of Blood Transfusion system 18) consists of a single antigen (H) defined by a terminal fucose residue found on red blood cells (RBCs) and in secretions. The H antigen is synthesized on the RBC surface by the FUT1 gene product fucosyltransferase 1. On epithelial cells and in body fluids, the H antigen is synthesized by the FUT2 gene product fucosyltransferase 2.

Background: The provision of compatible blood products to patients is the most essential task of transfusion medicine. Besides ABO and Rh, a number of additional blood group antigens often have to be considered for the blood supply of immunized or chronically transfused patients. It also applies for platelet antigens (HPA) and neutrophil antigens (HNA) for patients receiving platelet or granulocyte concentrates. Here, we describe the molecular screening for a number of blood group, HPA, and HNA alleles. Based on the screening results we are building up a regional blood donor registry to provide extended matched blood products on demand., Methods: We developed and validated TaqMan™ PCR and PCR-SSP methods for genetic markers defining 37 clinically relevant blood group antigens (beyond ABO and Rh), 10 HPA, and 11 HNA. Furthermore, we describe a feasible method for fast molecular screening of the HNA-2 null phenotype. All data were statistically evaluated regarding genotype distribution. Allele frequencies were compared to ExAC data from non-Finnish Europeans., Results: Up to now more than 2,000 non-selected regular blood donors in south-west Germany have been screened for blood group, HPA, and HNA alleles. The screening results were confirmed by serology and PCR-SSP methods for selected numbers of samples. The allele frequencies were similar to non-finnish Europeans in the ExAC database except for the alleles encoding the S, HPA-3b and HNA-4b antigens, with significantly lower prevalence in our cohort, as well as the LU14 and the HNA-3b antigens, with a higher frequency compared to the ExAC data. We identified 71 donors with rare blood groups such as Lu(a+b-), Kp(a+b-), Fy(a-b-) and Vel-, and 169 donors with less prevalent HPA or HNA types., Conclusion: Molecular screening for blood group alleles by using TaqMan™ PCR is an effective and reliable high-throughput method for establishing a rare donor registry.

The GYPC gene encodes the glycophorins C and D. The two moieties express 12 known antigens of the Gerbich blood group system and functionally stabilize red blood cell membranes through their intracellular interaction with protein 4.1 and p55. Three GYPC exon deletions are responsible for the lack of the high-frequency antigens Ge2 (Yus type, exon 2 deletion), Ge2 and Ge3 (Gerbich type, exon 3 deletion), and Ge2 to 4 (Leach type, exons 3 and 4 deletion), but lack exact molecular description. A total of 29 rare blood samples with Yus (GE:-2,3,4) and Gerbich (GE:-2,-3,4) phenotypes, including individuals of Middle-Eastern, North-African or Balkan ancestry were examined genetically. All phenotypes could be explained by 4 different Yus alleles, characterized by deletions of exon 2 and adjacent introns, and 3 different Gerbich alleles, with deletions of exon 3 and adjacent introns. A 3600 base pair GYPC region, encompassing exon 2 and flanking region, shares a high degree of sequence homology with a region flanking exon 3, probably representing an evolutionary duplication event. Defining the expression of Gerbich variants presently relies on rare serological reagents. Our approach substitutes the serological characterization with a precise genotype approach to identify the rare Yus and Gerbich alleles., (© 2017 John Wiley & Sons Ltd.)

The H blood group system, ISBT symbol H (018), consists of a single antigen (H) defined by a terminal fucose residue found on red blood cells and in secretions formed by the action of α-1,2-fucosyltransferases 1 (α2FucT1) and 2 (α2FucT2), respectively. Mutant alleles of the corresponding FUT1 and FUT2 genes result in either a H– phenotype (Bombay phenotype, Oh) or a weak H phenotype (para-Bombay, H+w). In addition, the FUT2 gene is the molecular basis of the secretor (Se) status, and homozygosity or compound heterozygosity for null alleles is associated with the nonsecretor (se) status. H– individuals have natural anti-H (mostly IgM), which can cause severe hemolytic transfusion reactions with intravascular hemolysis.

International rare blood donor panels or registries are important in the consistent availability of rare blood for patients who need this scarce resource. In countries where it has been possible to commit resources to this effort and often where the need is great, donors have been entered into a registry. The ISBT leadership recognized the importance of this very challenging inventory management activity and created a Working Party to support it. Individual countries support the WHO International Rare Donor Panel by submitting their donors' phenotype or genotype information to be catalogued into the database. It is extremely important that this database be cultivated and grown. The contributing countries keep their list updated and supply the blood product as they can when requested. It is known that some blood types are extremely scarce worldwide and requests for these are particularly difficult to fulfil. Thus, it is important to have a protocol to identify and recruit donors with rare blood types. It is equally or perhaps more important to ensure that the patients who need the rare blood are being managed appropriately in the presence and absence of rare blood products being available., (© 2015 International Society of Blood Transfusion.)

Background: The SMIM1 protein carries the Vel blood group antigen, and homozygosity for a 17 bp deletion in the coding region of the SMIM1 gene represents the molecular basis of the Vel- blood group phenotype. We developed PCR-based methods for typing the SMIM1 17 bp (64-80del) gene deletion and performed a molecular screening for the Vel- blood type in German blood donors., Methods: For SMIM1 genotyping, TaqMan-PCR and PCR-SSP methods were developed and validated using reference samples. Both methods were used for screening of donors with blood group O from southwestern Germany. Heterozygotes and homozygotes for the SMIM1 64-80del allele were serologically typed for the Vel blood group antigen. In addition, the rs1175550 SNP in SMIM1 was typed and correlated to the results of the phenotyping., Results: Both genotyping methods, TaqMan-PCR and PCR-SSP, represent reliable methods for the detection of the SMIM1 64-80del allele. Screening of 10,598 blood group O donors revealed 5 individuals homozygous for the deletional allele. They were confirmed Vel- by serological typing. Heterozygotes for the 64-80del allele showed different antigen expressions ranging from very weak to regular positive., Conclusion: Molecular screening of blood donors for the Vel- blood type is feasible and avoids the limitations of serological typing which might show false-negative results with heterozygous individuals. The identification of Vel- blood donors significantly contributes to the adequate blood supply of patients with anti-Vel.

PCR with sequence-specific primers (PCR-SSP) is a cost-effective and robust method for the analysis of single nucleotide polymorphisms (SNPs). Many blood group antigens and the antithetic antigens are based on a diallelic SNP in the coding region of the corresponding blood group gene. Here, we describe PCR-SSP protocols for genotyping 24 blood group antigens based on 12 diallelic SNPs. We also provide protocols for molecular determination of the rare blood group phenotypes Yk(a-) and Vel-.

Background: Alloantibodies to high-prevalence red blood cell (RBC) antigens are not easily identified by routine serologic techniques. This multicenter study was conducted to test the effectiveness of recombinant blood group proteins (rBGPs) at regional and international RBC reference laboratories., Study Design and Methods: Single or mixed soluble rBGPs (Lu, Yt, Kn, JMH, Sc, Rg, Ch, Do, and Cr) were assessed for their ability to inhibit the reactivity of antibodies to specific antigens. Initially, the effect of rBGPs was validated by testing panels of well-characterized patient serum samples containing antibodies to high-prevalence antigens in the hemagglutination inhibition assay. Subsequently, the rBGPs were prospectively used for routine antibody identification and the results were compared to those obtained with RBC-based diagnostics., Results: Panels of predefined antibodies to high-prevalence antigens were completely and specifically neutralized by the corresponding rBGP specificities. For prospective identification, antibodies to high-prevalence antigens (n = 62) were specifically inhibited by the corresponding rBGP specificities except for some Complement Receptor 1-related antibodies, which may be directed to epitopes not expressed on the truncated recombinant Kn. In 14 cases, additional clinically relevant alloantibodies were identified. In cross-matching, the rBGPs were successfully used to inhibit the reactivity of clinically irrelevant antibodies to high-prevalence antigens to determine compatibility between donor and recipient., Conclusion: rBGPs enable the identification of antibodies to high-prevalence antigens without the need for rare RBC reagents, which are often unavailable. Underlying antibodies can be reliably detected and cross-matching results validated, resulting in a more efficient blood supply for immunized patients., (© 2014 AABB.)

The mechanical fragility index (MFI) is an in vitro measure of sublethal injury to RBCs. In our previous experiments, we demonstrated that an increase in sublethal injury (increasing MFI) was a component of the RBC storage lesion, and that the MFI was significantly higher amongst the RBC units from male donors compared to pre-menopausal female donors during storage. It was hypothesized that hormonal or menstrual factors contributed to this difference. In this study, we found that RBC units donated by post-menopausal women demonstrated an MFI that was significantly higher than those donated by pre-menopausal women throughout storage., (© 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.)

Background: The mechanical fragility index (MFI) is an in vitro measurement of the extent of RBC sublethal injury. Sublethal injury might constitute a component of the RBC storage lesion, thus the MFI was determined serially during routine RBC storage., Methods: Leucoreduced AS-5- and SAGM-preserved RBCs were stored under routine blood bank conditions. The mechanical fragility (MF) of each unit was serially measured during storage., Results: For both AS-5 and SAGM units, male and female RBCs demonstrated statistically significant increases in the MFI during storage. The MFI was significantly lower in AS-5 units compared to SAGM units throughout storage. Female RBCs had significantly lower MFI vs. male RBCs in both AS-5 and SAGM units at all times. No significant differences in MFI were observed between ABO groups for both genders for AS-5 RBCs., Conclusions: The MF of RBCs increases during storage. Both gender and preservation solution influenced the MFI; however, the male:female MFI ratios were similar at all time-points and remained stable, suggesting that gender-based biological differences exist independent of storage solution. The MF could be a useful test for evaluating the effect of novel interventions intended to mitigate the susceptibility of RBCs to sublethal injury during storage., (© 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.)

Background: Variant RHCE alleles with diminished expression of C, c, E, and e antigens have been described and indicate the genetic diversity of this gene locus in several populations. In this study the molecular background of variant RhCE antigens identified by standard serologic routine testing in German blood donors and patients was determined., Study Design and Methods: Samples from blood donors and patients were routinely analyzed for RhCE phenotype using the PK7200 analyzer with two sets of monoclonal anti-C, -c, -E, and -e reagents. Samples with confirmed variant RhCE antigens were analyzed by nucleotide sequencing of the 10 RHCE exons. A multiplex polymerase chain reaction with sequence-specific priming (PCR-SSP) method was established for rapid typing of the rare RHCE alleles., Results: We identified 43 samples with serologic RhCE variants. Molecular analysis revealed variant RHCE alleles in 34 samples. Altogether 22 RHCE alleles were detected; 10 have not been published before. Twenty alleles harbored distinct single-nucleotide substitutions, 18 of which encoded amino acid changes and 2 of which occurred in noncoding regions. Two samples represented RHCE-D-CE hybrid alleles involving different segments of the RHCE Exon 5. A multiplex PCR-SSP screening for 17 RHCE alleles was negative in 1344 samples of the DNA bank GerBS. The cumulative phenotype frequency was estimated between 1 in 488 (0.20%) and 1 in 8449 (0.012%)., Conclusion: Single-amino-acid substitutions were the molecular basis for variant RhCE antigen expression in most samples. Nucleotide substitutions in RHCE exons were excluded as possible mechanism of diminished RhCE antigen expression in one-fifth of the serologically identified samples.

Background: KEL1 (K) is the most immunogenic red blood cell antigen of the Kell blood group system. The frequently occurring anti-KEL1 alloantibodies may cause hemolytic transfusion reactions as well as severe hemolytic disease of the fetus and newborn. So far, reports on weak KEL phenotypes are scarce., Study Design and Methods: Blood samples of two unrelated Central European propositi with faint reactions in routine KEL1 typing were analyzed by extended serologic phenotyping, flow cytometry, genotyping by polymerase chain reaction with sequence-specific priming, and genomic DNA sequencing of separated parental KEL alleles., Results: Both propositi exhibited an unusual KEL:1,2,3,4 phenotype: markedly weakened and negative reactions were observed in serologic KEL1 typing in gel and tube technique, respectively. No KEL1 epitope loss was detected using five different monoclonal anti-KEL1 reagents. KEL genotyping confirmed KEL*1/2 and KEL*3/4 (Kpa/Kpb) heterozygosity of both individuals. Importantly, DNA sequencing of the two separated parental alleles of both propositi revealed a KEL*1-specific coding nucleotide T578 and a KEL*3-specific T841 on the same allele. This novel KEL*1,3 (KKpa)allele was associated with an approximately 80 percent reduction in KEL1 expression, compared to KEL:1,2,-3,4 controls. The low KEL1 density was attributed to acis-modifier effect of KEL3, so far only reported in association with weakened expression of KEL2 (k)., Conclusion: This is the first description of the KEL*1,3 allele encoding KEL1 and KEL3 on the same molecule. In individuals with weakened KEL1 because of KEL3 in cis, very sensitive serologic or molecular genetic techniques may be required for reliable Kell typing.

Background and Objectives: RH48 (JAL) is a low-incidence Rh antigen of unknown molecular background associated with weakened expression of RhCE antigens. The objective of this study was to establish the molecular basis of JAL., Materials and Methods: Seventeen JAL+ samples, from seven black (one of them a Brazilian of mixed race: black/Caucasian), nine European Caucasians and one Asian individuals, were typed with anti-D, -C, -c, -E and -e. Some samples were also tested for V/VS and ce (f). Titration studies and flow cytometry were used to analyse the expression of the JAL antigen and genomic DNA sequencing of all RHCE exons was conducted on all samples. Routine genotyping for RHCE was carried out on all samples. Screening of RHD exons 1-10, which included detection of the DAU allele, was carried out on all except one of the black samples. The Caucasian samples and remaining black sample were screened for the DAU mutation 1136C>T (T379M)., Results: Six black individuals had the Dce haplotype with RHCE mutations 340C>T (R114W) and 733C>G (L245V) [V/VS] and the RHD mutation T379M [DAU]. One mixed race individual had the Dce haplotype with the RHCE mutation 340C>T (R114W) but without the V/VS or DAU mutation. Eight Caucasians had the DCe haplotype with the 340C>T mutation. One Caucasian and one Asian had the Dce haplotype with a different mutation in an adjacent nucleotide, 341G>A (R114Q). All Caucasian individuals were negative for the DAU mutation 1136C>T (T379M). Previously described weakness of CE-related Rh antigens when present in single dose on JAL+ samples of DCe and Dce haplotypes was observed. Weak expression of V/VS was observed in the three black samples tested and weakness of JAL was observed in the black samples compared to the Caucasian samples., Conclusion: The same mutation (340C>T, R114W) in two different haplotypes (DCe and Dce) and another mutation (341G>A, R114Q) in one of these haplotypes (Dce) are associated with expression of the JAL antigen. One of the RHCE mutations detected in our samples (340C>T) has been previously described but not in association with the JAL antigen. Our results indicate that the previously described RhCeMA and ce(s)(340) alleles encode the JAL antigen. Expression of V/VS antigen is weakened in the presence of JAL and expression of JAL is usually weaker when associated with the Dce haplotype compared to DCe.

SUMMARY: BACKGROUND: The gene locus for the ABO blood group system encodes a glycosyltransferase. Alterations in the DNA sequence are associated with the blood groups and the expression levels of antigens on red blood cells. A number of ABO alleles have been described as the molecular basis of weak A or B antigens. PATIENTS AND METHODS: Here, we describe a novel variant B allele in a blood donor with discrepant results in routine forward (group A) and reverse (very weak anti-B isoagglutinins) ABO blood grouping. RESULTS: Determination of the ABO genotype using polymerase chain reaction-sequence-specific primers (PCR-SSP) indicated blood group A(2)B. Sequencing of the ABO gene exons 6 and 7 showed for 1 allele a G insertion into the GGGGGG sequence at position 811-816 of exon 7. The 816insG mutation (designated ABO*Bw20) led to a frame shift of the coding sequence and subsequent alteration of the protein sequence. The location of the mutation on a B allele was proven by PCR-SSP. Screening for the novel mutation in 211 blood donors with regular ABO phenotypes indicated that *Bw20 is a rare variant. CONCLUSIONS: The low levels of anti-B isoagglutinins associated with this novel variant indicate that residual undetectable amounts of B antigen may be present on red blood cells. The serological and molecular analysis of members of the blood donor's family further proved the phenotype-genotype correlation of the *Bw20 allele with antigen O and individually variable levels of anti-B isoagglutinins. The characterization of novel alleles associated with ABO subgroups may ensure the correct determination of blood groups in which serological methods are combined with molecular genetic approaches.

Background: The JAHK antigen was first described in 1995 as a low-frequency red blood cell antigen. Family studies confirmed the association of the antigen with the rare r(G) phenotype of the Rh blood group system, which is associated with weak expression of C and e, but normal G expression. JAHK was allocated the Rh number RH53. The serologic findings indicated the location of the antigen on the RhCE protein, although the molecular basis for JAHK has not been known., Study Design and Methods: The RHCE gene of eight persons from three unrelated families was analyzed by exon amplification and direct sequencing. Four of the samples were JAHK+ the remaining four were JAHK-. In one JAHK+ sample, the entire RHCE gene was sequenced. The remaining samples were sequenced for exons 1 to 3. A polymerase chain reaction procedure with sequence specific primers was developed for the specific detection of the JAHK allele., Results: Analysis of the entire RHCE gene of one JAHK+ sample showed the expected CcEe-specific nucleotide sequences and revealed an additional nucleotide change (365C>T) in exon 3. This change represented a missense mutation, which led to an amino acid substitution from serine to leucine at position 122 of the RhCE protein. Three JAHK+ samples from two other unrelated families showed the 365C>T mutation and confirmed the association of the Ser122Leu substitution with the JAHK+ phenotype., Conclusion: The molecular basis of the JAHK antigen (RH53) is defined by a 365C>T mutation in exon 3 of the RHCE gene leading to the amino acid substitution Ser122Leu. Because the position 122 is predicted to be located in the transmembrane region adjacent to the second loop, the substitution of the neutral serine by the hydrophobic leucine seems to be the cause of the JAHK antigen by a conformational change of the RhCE protein. This structural change may also cause the weakened expression of the C and e antigens observed in JAHK+ individuals. Based on our results it is concluded that the JAHK-specific mutation is associated with a dCe haplotype.