Search

Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021.As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented.Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030).The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

Background and objectives There is concern regarding the lack of prevention of unnecessary transfusion of RhD negative red cells and unnecessary administration of Rh immunoglobulin (RhIG) to pregnant women. In this study, performance of ID RHD XT, a genotyping assay for identification of six RHD allelic variants and human platelet antigens HPA-1a/1b was assessed. Materials and methods Whole blood samples presenting weak, discrepant or inconclusive D phenotype results were genotyped with ID RHD XT and compared to reference molecular tests. Candidacy for RhIG prophylaxis was determined by analysing samples from pregnant women. Hands-on time to complete the procedures was measured. Results Overall, 167 samples were tested (55 donors, 56 patients, 52 pregnant women and four newborns). Agreement between ID RHD XT and the reference method was 100% (51% weak D type 1, 2 or 3; 35·5% weak D Types 1, 2 or 3 not detected; 4% RHD deletion; 1% RHD*Pseudogene; 1% RHD*DIIIa-CE(3-7)-D; and 4% no amplification variant detected for RHD genotype; and 64% HPA-1a/a; 30% HPA-1a/b; and 3% HPA-1b/b for HPA-1 genotype). Call rate was 98·2%. ID RHD XT identified 40% of the pregnant women that would not have required RhIG prophylaxis. Overall hands-on time was 25-45 min to process a batch of 24 samples, and four hours for total assay time. Conclusion ID RHD XT yielded reproducible results for RHD typing in serologically weak D phenotype individuals. ID RHD XT was proven useful for the correct management of patients with RhD serological discrepancies and the rational use of RhIG in pregnancy.

Hemolytic disease of the fetus and newborn (HDFN), as well as fetal and neonatal alloimmune thrombocytopenia (FNAIT), represent two important disease entities that are caused by maternal IgG antibodies directed against nonmaternally inherited antigens on the fetal blood cells. These antibodies are most frequently directed against the RhD antigen on red blood cells (RBCs) or the human platelet antigen 1a (HPA-1a) on platelets. For optimal management of pregnancies where HDFN or FNAIT is suspected, it is essential to determine the RhD or the HPA-1a type of the fetus. Noninvasive fetal RhD typing is also relevant for identifying which RhD-negative pregnant women should receive antenatal RhD prophylaxis. In this review, we will give an overview of the clinical indications and technical challenges related to the noninvasive analysis of fetal RBCs or platelet types. In addition, we will discuss the ethical implications associated with the routine administration of antenatal RhD to all pregnant RhD-negative women and likewise the ethical challenges related to making clinical decisions concerning the mother that have been based on samples collected from the (presumptive) father, which is a common practice when determining the risk of FNAIT.

The Sda histo-blood group antigen (GalNAcβ1-4(NeuAcα2-3)Galβ-R) is implicated in various infections and constitutes a potential biomarker for colon cancer. Sd(a−) individuals (2–4% of Europeans) may produce anti-Sda, which can lead to incompatible blood transfusions, especially if donors with the high-expressing Sd(a++)/Cad phenotype are involved. We previously reported the association of B4GALNT2 mutations with Sd(a−), which established the SID blood-group system. The present study provides causal proof underpinning this correlation. Sd(a−) HEK293 cells were transfected with different B4GALNT2 constructs and evaluated by immunostaining and glycoproteomics. The predominant SIDnull candidate allele with rs7224888:T>C (p.Cys406Arg) abolished Sda synthesis, while this antigen was detectable as N- or O-glycans on glycoproteins following transfection of wildtype B4GALNT2. Surprisingly, two rare missense variants, rs148441237:A>G and rs61743617:C>T, found in a Sd(a−) compound heterozygote, gave results similar to wildtype. To elucidate on whether Sd(a++)/Cad also depends on B4GALNT2 alterations, this gene was sequenced in five individuals. No Cad-specific changes were identified, but a detailed erythroid Cad glycoprotein profile was obtained, especially for glycophorin-A (GLPA) O-glycosylation, equilibrative nucleoside transporter 1 (S29A1) O-glycosylation, and band 3 anion transport protein (B3AT) N-glycosylation. In conclusion, the p.Cys406Arg β4GalNAc-T2 variant causes Sda-deficiency in humans, while the enigmatic Cad phenotype remains unresolved, albeit further characterized.

The Sd

Background and Objectives: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. Materials and Methods: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. Results: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. Conclusion: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays. (Less)

Objectives: To carry out genetic and serological analyses of a Swiss blood donor and a Danish patient carrying an aberrant ABO phenotype with weak A expression. Background: ABO is the most clinically important blood group system but also one of the most complex. The system antigens are determined by carbohydrate structures generated by A and B glycosyltransferases encoded by the ABO gene. Genetic variants of ABO may encode a glycosyltransferase with reduced activity, leading to weak expression of A antigen. Methods: Samples from two individuals were examined using genetic testing and extended immunohaematological evaluation, including standard serological methods, flow cytometry and analysis of plasma glycosyltransferase activity. Results: Both individuals were serologically determined to be AweakB. Genetic testing revealed that both were heterozygous for a novel ABO*A1.01-like allele with an in-frame duplication of 21 nucleotides in exon 7 (c.543_563dup), leading to the insertion of seven amino acids (QDVSMRR). Flow cytometric testing of native red blood cells (RBCs) showed very weak A antigen expression. This was in accordance with the enzyme activity test. Conclusion: In summary, we describe a novel A allele with a duplication of 21 nucleotides in exon 7 that significantly decreases the enzyme activity and leads to very weak expression of A antigen. (200 words).

This update on the P1PK blood group system (Hellberg Å, Westman JS, Thuresson B, Olsson ML. P1PK: the blood group system that changed its name and expanded. Immunohematology 2013;29:25-33) provides recent findings concerning the P1PK blood group system that have both challenged and confirmed old theories. The glycosphingolipids can no longer be considered the sole carriers of the antigens in this system because the P1 antigen has been detected on human red blood cell glycoproteins. New indications suggest that P1Pk synthase activity truly depends on the DXD motif, and the genetic background and molecular mechanism behind the common P1 and P2 phenotypes were found to depend on transcriptional regulation. Transcription factors bind the P1 allele selectively to a motif around rs5751348 in a regulatory region of A4GALT, which enhances transcription of the gene. Nonetheless, unexplained differences in antigen expression between individuals remain.

BACKGROUND: The P1 antigen was first described in 1927 and belongs to the P1PK histo-blood group system, together with Pk and NOR. The A4GALT-encoded 4-α-galactosyltransferase synthesizes these antigens and has been considered to extend glycolipids exclusively. However, contradicting studies have been published regarding the presence of P1 on human glycoproteins. In other species, P1 occurs on glycoproteins. Furthermore, human ABH antigens occur on both glycolipids and glycoproteins and are biochemically related to P1. Thus, we hypothesized that P1 is present on RBC glycoproteins in humans. STUDY DESIGN AND METHODS: RBCs of known P1/P2 status (phenotype and rs8138197 genotype) were used. The RBC surface glycans were modified with α-galactosidases, papain, and/or peptide-N-glycosidase F. RBC membrane proteins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/immunoblot. A new P1/P2-allelic discrimination assay based on rs5751348 was validated. RESULTS: P1 occurs on various glycoproteins, seen as smearlike patterns in anti-P1-stained immunoblots with RBC membranes of P1 but not P2 or p phenotype. There was a significant difference between the staining of P1-homozygous and P1-heterozygous RBCs (P1P1 > P1P2), as well as intragenotypic variation. Immunoblotting banding patterns show major carriers at approximately 50 and 100 kDa. P1 staining was lost after treatment of RBCs with α-galactosidase of broad Galα-1,3/4/6-specificity. Peptide-N-glycosidase F treatment reduced the P1 signal, while papain or α-1,3-specific galactosidase did not. P1/P2 status was confirmed by a new rs5751348 assay. CONCLUSION: Our data indicate that the P1 antigen can reside on human RBC glycoproteins. Glycosidase studies suggest that at least part of the epitopes occur on N-glycans. (Less)

P1 and Pk are glycosphingolipid antigens synthesized by the A4GALT-encoded α1,4-galactosyltransferase, using paragloboside and lactosylceramide as acceptor substrates, respectively. In addition to the compatibility aspects of these histo-blood group molecules, both constitute receptors for multiple microbes and toxins. Presence or absence of P1 antigen on erythrocytes determines the common P1 (P1+Pk+) and P2 (P1-Pk+weak) phenotypes. A4GALT transcript levels are higher in P1 individuals and single-nucleotide polymorphisms (SNPs) in noncoding regions of A4GALT, particularly rs5751348, correlate with P1/P2 status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by Kruppel-like factor 1 (KLF1) haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 regulates A4GALT expression. Intriguingly, P1 -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain P1 -specific shifts in electrophoretic mobility-shift assays and small interfering RNA silencing of KLF1 did not affect A4GALT transcript levels. Instead, protein pull-down experiments using P1 but not P2 oligonucleotide probes identified runt-related transcription factor 1 (RUNX1) by mass spectrometry. Furthermore, RUNX1 binds P1 alleles selectively, and knockdown of RUNX1 significantly decreased A4GALT transcription. These data indicate that RUNX1 regulates A4GALT and thereby the expression of clinically important glycosphingolipids implicated in blood group incompatibility and host-pathogen interactions.

TO THE EDITOR: Although frequently effective[1][1],[2][2] and usually benign, high-dose (2 g/kg) intravenous immunoglobulin (IVIG) therapy can result in marked red blood cell (RBC) hemolysis, which in some cases is life threatening in severity.[3][3][⇓][4]-[5][5] The mechanism by which this

Background and objectives: Fetal RHD genotyping of cell-free maternal plasma DNA from RhD negative pregnant women can be used to guide targeted antenatal and postnatal anti-D prophylaxis for the prevention of RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 31 laboratories. Materials and methods: Aliquots of pooled maternal plasma from gestational week 25 were sent to each laboratory. One sample was fetal RHD positive, and a second sample was fetal RHD negative. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. The samples were tested blindly. Results: Different methodological approaches were used; 29 laboratories used qPCR and two laboratories used ddPCR, employing a total of eight different combinations of RHD exon targets. Fetal RHD genotyping was performed with no false-negative and no false-positive results. One inconclusive result was reported for the RHD positive sample. All clinical conclusions were satisfactory. Conclusion: This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting. (Less)

Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic because of a disease state or recent transfusions. In addition, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A well-characterized renewable reference panel for standardization of blood group genotyping was developed. The panel consists of genomic DNA lyophilized and stored in glass vials. Genomic DNA was extracted in bulk from immortalized lymphoblastoid cell lines, generated by Epstein-Barr virus transformation of peripheral blood lymphocytes harvested from volunteer blood donors. The panel was validated by an international collaborative study involving 28 laboratories that tested each DNA panel member for 41 polymorphisms associated with 17 blood group systems. Overall, analysis of genotyping results showed >98% agreement with the expected outcomes, demonstrating suitability of the material for use as reference. Highest levels of discordance were observed for the genes CR1, CD55, BSG, and RHD. Although limited, observed inconsistencies and procedural limitations reinforce the importance of reference reagents to standardize and harmonize results. Results of stability and accelerated degradation studies support the suitability of this panel for use as reference reagent for blood group genotyping assay development and standardization.

This update on the P1PK blood group system (Hellberg Å, Westman JS, Thuresson B, Olsson ML. P1PK: the blood group system that changed its name and expanded. Immunohematology 2013;29:25–33) provides recent findings concerning the P1PK blood group system that have both challenged and confirmed old theories. The glycosphingolipids can no longer be considered the sole carriers of the antigens in this system because the P1 antigen has been detected on human red blood cell glycoproteins. New indications suggest that P1Pk synthase activity truly depends on the DXD motif, and the genetic background and molecular mechanism behind the common P1 and P2 phenotypes were found to depend on transcriptional regulation. Transcription factors bind the P1 allele selectively to a motif around rs5751348 in a regulatory region of A4GALT, which enhances transcription of the gene. Nonetheless, unexplained differences in antigen expression between individuals remain.

The main change that has occurred in the GLOB blood group system since the GLOB review published in this journal in 2013 is the addition of an antigen. The high-prevalence PX2 antigen, originally recognized as the x2 glycosphingolipid, is expressed on red blood cells of most individuals and is elevated in the rare PP1Pk-negative p blood group phenotype. P synthase, encoded by B3GALNT1, was found to elongate paragloboside to PX2 by adding the terminal β3GalNAc moiety. Hence, PX2 was moved from the GLOB collection to the GLOB system. The presence of naturally-occurring anti-PX2 was noted in P1k and P2k individuals exhibiting nonfunctional P synthase. Although the clinical significance of this specificity remains unclear, a recommendation to avoid transfusing Pk patients with p phenotype blood has been made. Currently, 13 mutations at the highly conserved B3GALNT1 locus have been found to abolish P synthase function and are recognized as null alleles by the International Society of Blood Transfusion. A new allele with a missense mutation but resulting in normal expression of P has been assigned GLOB*02. Finally, the GLOB collection was made obsolete after the move of LKE antigen to the 901 series.

BACKGROUND AND OBJECTIVES: ABO remains the clinically most important blood group system, but despite earlier extensive research, significant findings are still being made. The vast majority of catalogued ABO null alleles are based on the c.261delG polymorphism. Apart from c.802G>A, other mechanisms for O alleles are rare. While analysing the data set from the 1000 Genomes (1000G) project, we encountered two previously uncharacterized deletions, which needed further exploration.MATERIALS AND METHODS: The Erythrogene database, complemented with bioinformatics software, was used to analyse ABO in 2504 individuals from 1000G. DNA samples from selected 1000G donors and African blood donors were examined by allele-specific PCR and Sanger sequencing to characterize predicted deletions.RESULTS: A 5821-bp deletion encompassing exons 5-7 was called in twenty 1000G individuals, predominantly Africans. This allele was confirmed and its exact deletion point defined by bioinformatic analyses and in vitro experiments. A PCR assay was developed, and screening of African samples revealed three donors heterozygous for this deletion, which was thereby phenotypically established as an O allele. Analysis of upstream genetic markers indicated an ancestral origin from ABO*O.01.02. We estimate this deletion as the 3rd most common mechanism behind O alleles. A 24-bp deletion was called in nine individuals and showed greater diversity regarding ethnic distribution and allelic background. It could neither be confirmed by in silico nor in vitro experiments.CONCLUSION: A previously uncharacterized ABO deletion among Africans was comprehensively mapped and a genotyping strategy devised. The false prediction of another deletion emphasizes the need for cautious interpretation of NGS data and calls for strict validation routines. (Less)

P1 and P

Cells of the clinically important p histo-blood group phenotype lack P1, P(k) , and P glycosphingolipid antigens. All cases investigated so far are due to alterations in the 4-α-galactosyltransferase-encoding Exon 3 of A4GALT. Repetitive elements in the genome can mediate DNA rearrangements, the most abundant being the Alu family of repeats.

The P blood group antigen of the GLOB system is a glycolipid structure, also known as globoside, on the red blood cells (RBCs) of almost all individuals worldwide. The P antigen is intimately related to the Pkand NOR antigens discussed in the review about the P1PK blood group system. Naturally occurring anti-P is present in the serum of individuals with the rare globoside-deficient phenotypes p, P1 k, and P2 kand has been implicated in hemolytic transfusion reactions as well as unfavorable outcomes of pregnancy. The molecular genetic basis of globoside deficiency is absence of functional P synthase as a result of mutations at the B3GALNT1locus. Other related glycolipid structures, the LKE and PX2 antigens, remain in the GLOB blood group collection pending further evidence about the genes and gene products responsible for their synthesis. Immunohematology 2013;29:19–24.

BACKGROUND: The 1061delC single-nucleotide polymorphism (SNP) has been reported mostly in the context of the common A(2)[A201] allele and typically produces an A(2) phenotype. This study evaluated new A(weak) alleles, each containing 1061delC. STUDY DESIGN AND METHODS: Twenty samples were referred to our laboratory for analysis due to suspected A(weak) phenotypes originally detected at the referring centers. ABO Exons 1 through 7 and flanking intronic regions were sequenced. A antigen expression on red blood cells was analyzed by flow cytometry. Plasma enzyme activity was studied in one case. Molecular three-dimensional modeling techniques studied the potential effects of amino acid changes on the resulting glycosyltransferases (GTs). RESULTS: Thirteen alleles were discovered, each featuring 1061delC with at least 1 of 12 additional SNPs in the coding region. One of these SNPs disrupts the translation initiation codon. Another constitutes the first reported change in the DVD motif. One SNP found in three alleles causes a substitution of one of the four amino acids that differentiates the wild-type A and B enzymes but plasma enzyme analysis by two methods showed only slightly decreased or normal A(2) activity. Flow cytometric analysis semiquantified the A antigen levels in 16 cases featuring 10 of the alleles and ranged from very weak to nearly A(2) levels. However, the majority of the samples displayed A(x)-like patterns. Molecular modeling of some of the GT variants indicated conformational changes that may explain the diminished A expression observed. CONCLUSION: Missense SNPs were identified in 13 novel A(2)-like alleles, which produced a variety of A subgroup phenotypes. (Less)

Un individuo con un fenotipo eritrocitario raro carece de uno o varios antígenos presentes en la mayor parte de la población de pertenencia. Cuando presenta el anticuerpo correspondiente, se pueden producir complicaciones perinatales, transfusionales y/o transplantológicas. Se presenta el caso de una embarazada aloinmunizada derivada a nuestro servicio en la semana 12 de su tercera gesta para su evaluación y seguimiento. El diagnóstico inmunohematológico le asignó el excepcional fenotipo "p" (aproximadamente 1/200 000 individuos), asociado con una mayor tasa de abortos espontáneos y a reacciones transfusionales graves cuando se transfunden unidades incompatibles. El estudio del gen A4GALT demostró la presencia de la mutación c.752C > T en doble dosis. Esta mutación lleva a un cambio de una prolina por una leucina en el residuo 251 de la 4-α-galactosiltransferasa. Por parto inducido por sufrimiento fetal, nace a las 36 semanas una bebé con prueba de antiglobulina (Coombs) directa negativa, eluido reactivo, con ictericia que requirió luminoterapia. Una semana después el neonato fue externado sin secuelas aparentes. Posteriormente, a raíz de una cirugía inminente y la improbabilidad de encontrar sangre compatible, se elaboró un plan para cubrir las posibles demandas. Este caso pone en evidencia la necesidad de contar a nivel nacional con un laboratorio de referencia de inmunohematología y un banco de sangre de grupos raros, que permita resolver con celeridad situaciones que requieran transfundir a estos individuos.A rare blood group is usually defined as the absence of a high prevalence antigen or the absence of several antigens within a single blood group system. These individuals may develop clinically significant red cell antibodies to the high incidence red cell antigens they lack. A 33-year-old alloimmunized woman was referred to our center at the 12th week of her third pregnancy for evaluation and follow up. The laboratory work-up grouped her as belonging to "p" phenotype, associated with difficulties to find compatible blood for transfusion and a high incidence of recurrent miscarriage. At 36 weeks, a baby girl was born by induced labor due to fetal suffering. With a negative direct antiglobulin test but a positive elution test, she was in the neonatology ward for one week receiving luminotherapy. Homozygosity for a missense mutation at position 752 (c.752C > T) in the A4GALT gene was found to be responsible for the p phenotype. This mutation changes a proline to a leucine at codon 251 of the 4-α-galactosyltransferase. Recently, due to an imminent chirurgical intervention and the impossibility to have compatible blood available for transfusion, an autologous donation plan was designed to satisfy probable demand. This case showed the need for blood bank facilities capable to respond satisfactorily to these situations in Argentina. This would facilitate the storage of cryopreserved blood from individuals with rare blood groups for homologous use or to develop rare blood donors programs.

Several human histo-blood groups are glycosphingolipids, including P/P1/P(k). Glycosphingolipids are implicated in HIV-host-cell-fusion and some bind to HIV-gp120 in vitro. Based on our previous studies on Fabry disease, where P(k) accumulates and reduces infection, and a soluble P(k) analog that inhibits infection, we investigated cell surface-expressed P(k) in HIV infection. HIV-1 infection of peripheral blood-derived mononuclear cells (PBMCs) from otherwise healthy persons, with blood group P(1)(k), where P(k) is overexpressed, or blood group p, that completely lacks P(k), were compared with draw date-matched controls. Fluorescence-activated cell sorter analysis and/or thin layer chromatography were used to verify P(k) levels. P(1)(k) PBMCs were highly resistant to R5 and X4 HIV-1 infection. In contrast, p PBMCs showed 10- to 1000-fold increased susceptibility to HIV-1 infection. Surface and total cell expression of P(k), but not CD4 or chemokine coreceptor expression, correlated with infection. P(k) liposome-fused cells and CD4(+) HeLa cells manipulated to express high or low P(k) levels confirmed a protective effect of P(k). We conclude that P(k) expression strongly influences susceptibility to HIV-1 infection, which implicates P(k) as a new endogenous cell-surface factor that may provide protection against HIV-1 infection.

The Jk(a–b–) phenotype results from alterations in the JK gene and is characterized by absence of the RBC urea transporter in the cell membrane. The frequency of Jk(a–b–) varies among populations, but this phenotype is most commonly found in people of Polynesian and Finnish descent. Although rare, Jk(a–b–) individuals present a clinical challenge because anti-Jk3 is produced readily in response to transfusion and pregnancy, and Jk(a–b–) blood is not routinely available. Identification of Jk(a–b–) patients and donors is most often performed serologically. However, ten JK*0 alleles have been identified, and this information can be used in DNA-based typing. We selected five JK*0 alleles that had been encountered by our reference laboratory in two or more samples from unrelated individuals and designed an allele-specific primer PCR assay for use as an initial screening tool. After in-house validation, we tested genomic DNA from a family: a mother and her two sons referred to us for genetic investigation of their Jk(a–b–) phenotypes. Two different nucleotide substitutions, –1g>a in intron 5 (IVS5) and 956C>T in exon 10, originally associated with Polynesian and Indian/African populations respectively, were identified in the family. The mother and one son were compound heterozygotes, and the second son was homozygous for IVS5– 1g>a. We conclude that the effort to design and validate such a screening assay was cost-efficient when compared with DNA sequencing costs. Furthermore, selection of the more common JK*0 mutations was a practical approach that resulted in rapid identification of the genetic bases behind the Jk(a–b–) phenotypes in this unusual family. Although an obvious target for eventual inclusion into high-throughput genotyping platforms for clinical diagnostic services, current systems are very limited. Our approach provides a simple and inexpensive method for the identification of these rare alleles. Immunohematology 2009;25:165–169.

BACKGROUND: The rare p phenotype is found at a higher frequency in Amish people than in other populations. Different mutations in the 4-alpha-galactosyltransferase gene (A4GALT), responsible for synthesis of P(k) (Gb(3)) antigen, have been found to cause the P(k)-deficient p phenotype. The aim of this study was to explore the molecular background of the p phenotype in people of Amish origin. STUDY DESIGN AND METHODS: Twenty blood samples with the p phenotype, 19 of them from Amish individuals and 1 Pakistani, were investigated. Amplification of genomic DNA by polymerase chain reaction (PCR) and sequencing by capillary electrophoresis were performed. Blood donors of different geographic origin were screened with PCR-allele-specific primer to investigate whether the novel mutation occurs among individuals with common phenotypes. The mutation was also cloned into an expression vector and transfected to Namalwa cells, which do not normally express P(k). P(k) expression on the transfected cells and P/P(k) on red blood cells (RBCs), both with p and with common phenotypes, were analyzed by flow cytometry. RESULTS: All 20 samples were homozygous for 299C>T changing serine to leucine in a region that is highly conserved in homologous genes across species borders. The mutation was not found in any of the 500 alleles of blood donors investigated. P(k) expression was neither observed by serology and flow cytometry on p RBCs from Amish individuals nor following transfection of cells with constructs containing the novel missense mutation. CONCLUSION: A novel A4GALT missense mutation causes the p phenotype in Amish individuals.

The x2 glycosphingolipid is expressed on erythrocytes from individuals of all common blood group phenotypes and elevated on cells of the rare P/P1/P(k)-negative p blood group phenotype. Globoside or P antigen is synthesized by UDP-N-acetylgalactosamine:globotriaosyl-ceramide 3-β-N-acetylgalactosaminyltransferase encoded by B3GALNT1. It is the most abundant non-acid glycosphingolipid on erythrocytes and displays the same terminal disaccharide, GalNAcβ3Gal, as x2. We encountered a patient with mutations in B3GALNT1 causing the rare P-deficient P1 (k) phenotype and whose pretransfusion plasma was unexpectedly incompatible with p erythrocytes. The same phenomenon was also noted in seven other unrelated P-deficient individuals. Thin-layer chromatography, mass spectrometry, and flow cytometry were used to show that the naturally occurring antibodies made by p individuals recognize x2 and sialylated forms of x2, whereas x2 is lacking on P-deficient erythrocytes. Overexpression of B3GALNT1 resulted in synthesis of both P and x2. Knockdown experiments with siRNA against B3GALNT1 diminished x2 levels. We conclude that x2 fulfills blood group criteria and is synthesized by UDP-N-acetylgalactosamine: globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase. Based on this linkage, we proposed that x2 joins P in the GLOB blood group system (ISBT 028) and is renamed PX2 (GLOB2). Thus, in the absence of a functional P synthase, neither P nor PX2 are formed. As a consequence, naturally occurring anti-P and anti-PX2 can be made. Until the clinical significance of anti-PX2 is known, we also recommend that rare P1 (k) or P2 (k) erythrocyte units are preferentially selected for transfusion to P(k) patients because p erythrocytes may pose a risk for hemolytic transfusion reactions due to their elevated PX2 levels.

Background and Objectives The chemical basis of the subgroups of A is largely unknown. We used thin-layer chromatography immunochemical staining techniques together with a range of characterized monoclonal reagents to analyse glycolipids isolated from a variety of weak subgroups. Materials and Methods Glycolipids isolated from red cells collected from nine genetically defined individuals of the rare subgroups of A, including a novel A(3) allele (A(2) 539G > A) not described previously, were subjected to a highly sensitive thin-layer chromatographic immunochemical analysis. Results Semicharacterized monoclonal antibodies revealed that, in addition to the expected quantitative differences between common phenotypes and the weak subgroups, qualitative glycolipid differences (or at least an apparent qualitative basis), caused by major changes in the ratios of different structures exist. Specifically it was found that the weakest A-expressing samples (A(el) phenotype) appeared to express an unusual A structure in the 8-12 sugar region. Variable expression of several structures in one of the A weak samples were suggestive of novel blood group A structures. Conclusions Although no structural characterization could be undertaken, the results are clearly indicative that the variant glycosyltransferases of the rare ABO subgroups are not only inefficient, but they may potentially synthesize novel ABO structures. (Less)

SummaryThe aim of this study was to further explore the molecular genetic bases of theclinically important but rare blood group phenotypes p, P 1k and P 2k by analysisof the 4-a-galactosyltransferase (P k ) and 3-b-N-acetylgalactosaminyltrans-ferase (P) genes responsible for synthesis of the related P k (Gb 3 ) and P (Gb 4 )antigens respectively. Lack of these glycolipid moieties is associated with severetransfusion reactions and recurrent spontaneous abortions but also offersimmunity against certaininfectious agents.Blood samples from 20pand 11P 1k or P 2k individuals of different geographic and ethnic origin were investigated.DNA sequencing by capillary electrophoresis was performed followingamplification of the coding regions in the P k or P genes. In the P k gene, ninenovel and five previously described mutations were detected. One of the newlyfound mutations introduced an immediate stop, five shifted the reading frameintroducingpremature stop codonsandthreeweremissense mutationscausingamino acid substitutions in conserved regions of the transferase. Four new andtwopreviouslydescribedmutationsinthePgenewerefound.Threeofthenovelalleles reported here carried nonsense mutations whilst the fourth allele had amissense mutation. The finding of 13 novel mutations in 14 alleles emphasizesfurther the genetic heterogeneity at the glycosyltransferase loci underlying theGLOB blood group system and collection.Keywords: blood group, allele, erythrocyte, glycosyltransferase, transfusion.

We report a novel RHCE*02 allele in a Swedish blood donor that is characterized by the change c.460A>G (Arg154Gly). The blood donor's red blood cells showed variable reactivity with different monoclonal anti-C and anti-e and antigen strength was markedly weakened. We believe that these changes represent both a quantitative and qualitative alteration of the antigens encoded by this allele.

BACKGROUND: The purpose of this study was to explore the molecular basis of the p phenotype by analysis of the recently cloned 4-α-galactosyltransferase gene responsible for synthesis of Pk (Gb3) antigen. STUDY DESIGN AND METHODS: Forty samples from individuals of eight different nationalities were investigated by serologic methods and DNA sequencing of the Pk gene. RESULTS: Ten different Pk-null alleles, of which 6 are novel, were encountered. The 29 Swedes were homozygous for M183K or G187D, with the former as the predominant allele. Three Israelis were homozygous for a single-nucleotide deletion at codon 219 that shifts and truncates the reading frame by 5 amino acids. Two Italians were homozygous for a triplet deletion causing F81del, while an English donor was heterozygous for F81del but also carried another allele with a combined deletion and insertion. A Pole was heterozygous for alleles with either a single-base deletion at codon 257 or a mutation causing S97L. A Norwegian person and a Japanese person were homozygous for single-base insertions causing a premature stop at codon 282 or extension of the protein by 92 residues, respectively. In 2 samples no mutations were detected. CONCLUSION: The genetic heterogeneity underlying the p phenotype is further emphasized by this study. To date, 11 p-specific mutations have been found in 14 distinct alleles.

The biochemistry and molecular genetics underlying the related carbohydrate blood group antigens P, Pk, and LKE in the GLOB collection and P1 in the P blood group system are complex and not fully understood. Individuals with the rare but clinically important erythrocyte phenotypes P1k and P2klack the capability to synthesize P antigen identified as globoside, the cellular receptor for Parvo-B19 virus and some P-fimbriatedEscherichia coli. As in the ABO system, naturally occurring antibodies, anti-P of the IgM and IgG class with hemolytic and cytotoxic capacity, are formed. To define the molecular basis of the Pk phenotype we analyzed the full coding region of a candidate gene reported in 1998 as a member of the 3-β-galactosyltransferase family but later shown to possess UDP-N-acetylgalactosamine:globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase or globoside synthase activity. Homozygosity for different nonsense mutations (C202 → T and 538insA) resulting in premature stop codons was found in blood samples from two individuals of the P2k phenotype. Two individuals with P1k and P2k phenotypes were homozygous for missense mutations causing amino acid substitutions (E266A or G271R) in a highly conserved region of the enzymatically active carboxyl-terminal domain in the transferase. We conclude that crucial mutations in the globoside synthase gene cause the Pk phenotype.

BACKGROUND AND OBJECTIVES: During the past 10 years several DNA-typing methods have been developed to complement routine serological typing for determination of polymorphisms in the ABO, RH, KEL, JK and FY blood group genes. However, the molecular basis of blood groups can differ widely between ethnic groups. The purpose of this study was to evaluate selected DNA-based methods for phenotype prediction in a population not previously investigated. MATERIALS AND METHODS: Blood samples from a random sample of Jordanian blood donors were collected and red cells isolated from these blood samples were phenotyped for common ABO (n = 150) and KEL/FY/JK (n = 90) antigens. RHD-negative and -positive donors were selected for RH typing (n = 120 and 30, respectively). DNA was prepared and blood group genotyping performed according to selected methods in current use. Discordant samples required further investigation by extended serology and DNA sequencing. RESULTS: The degree of concordance between phenotype and genotype was high, but some exceptions were noted. Two of 14 A2/A2B samples lacked all mutations associated with known A2 alleles of the ABO system. RH typing revealed four samples with the c(cyt48) marker, causing false-positive RHC typing. A single D-negative sample was positive for D-specific exon 10 markers. The RHD pseudogene was not found in the 150 donors tested. Nine samples revealed discrepancies that were associated with unknown silent or weakly expressing Fyb-like alleles. CONCLUSIONS: With the exception of the FY system, we conclude that the molecular background of the clinically important blood group antigens studied here is similar to that reported for Caucasoids.

The rare but clinically important null phenotypes of the P1PK and GLOB blood group systems are due to alterations in A4GALT and B3GALNT1, respectively. A recently identified single-nucleotide polymorphism in Exon 2a of A4GALT predicts the common P1 and P2 phenotypes but rare variants have not been tested.

BACKGROUND: There are two principal types of group O alleles; deletional alleles feature 261delG leading to nonfunctional truncated protein. Nondeletional alleles have the consensus guanosine at residue 261. The major nondeletional allele, O-2, encodes full-length protein with Gly268Arg. While reports vary, O-2 has been proposed to encode weakly functional A-glycosyltransferase (GTA). The main objective of this study was to evaluate if GTA activity is detectable in O-2 donors. STUDY DESIGN AND METHODS: Donor samples from Pittsburgh and Lund were ABO typed by automated methods. DNA was extracted from 779 group O donors whose red blood cells (RBCs) were available for transfusion. ABO genotyping identified those with O-2 alleles. The following tests were performed on randomly selected O-2 samples (number): adsorption-elution with anti-A (3), flow cytometry (15), plasma enzyme activity (4), and attempts to convert group O RBCs to A (2) with O-2 plasma and titration of plasma anti-A/-A(1) (3). RESULTS: Forty O-2-heterozygous donors were identified (5.1%). Adsorption-elution and sensitive flow cytometry did not reveal A antigens on O-2 RBCs. Plasma enzyme analysis failed to show GTA activity above baseline; O-2 plasma was unable to add measurable A antigens to O RBCs. Titers of anti-A/-A(1) appeared reduced in O-2 plasma but did not cause ABO typing discrepancies. No immediate hemolysis or adverse reactions were reported following transfusion of O-2 RBCs to six evaluable group O recipients. CONCLUSIONS: Other than lower plasma anti-A titers, GTA activity was not found in these O-2 samples. Neither automated blood grouping discrepancies nor clinical problems related to transfusing these O-2 units were observed. (Less)

summary. Apparent deviation from Mendelian rules of blood group inheritance is rarely observed. Blood group O parents with children expressing weak A subgroups have occasionally been described but not explained. A detailed serological investigation of such a family is described here. The ABO locus was analysed by PCR-ASP/restriction fragment length polymorphism genotyping and DNA sequencing. The propositus' RBCs were very weakly agglutinated with monoclonal anti-A but distinctly with polyclonal anti-A,B, i.e. typical for Ax. Serum anti-A1 (titre 4) and -B were present. Her parents' blood groups were both clearly O, with titres of serum anti-A1, and -A at 16 and 4, respectively. Adsorption/ elution studies demonstrated A antigen on the daughter's cells only. The ABO genotypes were: mother, AxO1; father, O1vO2; and propositus, AxO2. The Ax allele was an A1-O1v hybrid allele with a crossing-over breakpoint between positions 235 and 446 in intron 6 (Ax-4). Compared to the A1 glycosyltransferase, this allele predicts a protein with two amino acid substitutions (Phe216Ile and Met277Val) known to yield either weakly expressed or no A antigen on RBCs. This study suggests that the nature of the ABO allele in trans can influence A antigen expression, a phenomenon previously described as allelic enhancement (or reinforcement). Potential mechanisms for this are discussed.

Background The molecular genetics of the P blood group system and the absence of P1 antigen in the p phenotype are still enigmatic. One theory proposes that the same gene encodes for both the P1 and Pk glycosyltransferases, but no polymorphisms in the coding region of the P k gene explain the P1/P2 phenotypes. We investigated the potential regulatory regions up- and downstream of the A4GALT (P k ) gene exons. Results P1 (n = 18) and P2 (n = 9) samples from donors of mainly Swedish descent were analysed by direct sequencing of PCR-amplified 5'- and 3'-fragments surrounding the P k coding region. Seventy-eight P1 and P2 samples were investigated with PCR using allele-specific primers (ASP) for two polymorphisms previously proposed as P2-related genetic markers (-551_-550insC, -160A>G). Haplotype analysis of single nucleotide polymorphisms was also performed with PCR-ASP. In ~1.5 kbp of the 3'-untranslated region one new insertion and four new substitutions compared to a GenBank sequence (AL049757) were found. In addition to the polymorphisms at positions -550 and -160, one insertion, two deletions and one substitution were found in ~1.0 kbp of the 5'-upstream region. All 20 P2 samples investigated with PCR-ASP were homozygous for -550insC. However, so were 18 of the 58 P1 samples investigated. Both the 20 P2 and the 18 P1 samples were also homozygous for -160G. Conclusion The proposed P2-specific polymorphisms, -551_-550insC and -160G, found in P2 samples in a Japanese study were found here in homozygous form in both P1 and P2 donors. Since P 2 is the null allele in the P blood group system it is difficult to envision how these mutations would cause the P2 phenotype. None of the novel polymorphisms reported in this study correlated with P1/P2 status and the P1/p mystery remains unsolved.

Background and Objectives Reagent red blood cells (RBCs) for antibody detection should express certain important antigens as a double dose, that is, the donors must be homozygous for the corresponding alleles. Traditionally, dose is determined by serological typing and known allele frequencies. However, RHD zygosity cannot be predicted serologically owing to the absence of an antithetical antigen, and FY zygosity is confounded by two variant haplotypes, FY*0 and FY*X. Furthermore, lack of reagents hampers our ability to type for some clinically important antigen pairs such as Do(a)/Do(b). Materials and Methods Genomic DNA was isolated from reagent RBC samples. Established, validated methods were used to determine the RHD, FY, and DO genotypes. Results Three of 52 D+ samples gave results that differed from the predicted genotype: two presumed (RR1)-R-1 samples and an (RR2)-R-2 sample were shown to be R(1)r" and R(2)r', respectively. Five of 59 samples that were from presumed homozygotes for either FY*A or FY*B were heterozygous, together with either FY*X (three samples) or FY*0 (two samples). Seventy-five samples tested for DO were DO*A/A (n = 14), DO*A/B (n = 39), or DO*B/B (n = 22). Conclusions The results show that serologically determined RhD and Duffy phenotypes of reagent RBCs are unreliable and that antigens we thought were represented as a double dose were single dose. The addition of Dombrock genotyping provides information which is useful in antibody identification. We conclude that selected genotype analyses are a valuable quality assurance measure to ensure that reagent RBCs comply with national and international recommendations for test sensitivity. (Less)

BACKGROUND: In the ABO blood group system mutations in the A gene may lead to weak A subgroups owing to a dysfunctional 3-α-N-acetylgalactosaminyltransferase. STUDY DESIGN AND METHODS: Blood and DNA were investigated to correlate weak A phenotypes with genotype, and an overrepresentation of the infrequent O2 allele was observed. Consequently, 57 available O2 alleles were examined in detail. RESULTS: Two new O2 alleles were identified having mutations resulting in Gly229Asp with or without Arg217Cys. A recently described O2 variant (488C>T; Thr163Met) was also found. Surprisingly, both the original and the variant O2 alleles were associated with either O or Aweak phenotypes. Three novel O alleles surfaced in six other samples with suspected A subgroups. These were A1-like alleles having nonsense mutations causing premature truncation at codons 56, 107, or 181. A second example of the rare O3 allele was also identified. A newly described O1 allele having 768C>A was found to be the third most frequent O allele among Swedish donors. Of the five novel O alleles, three were incorrectly interpreted as A1 following routine ABO genotyping. CONCLUSION: Apparent O alleles lacking 261delG may cause weak A expression on red blood cells and/or inhibit anti-A production. A hypothesis that exchange of genetic material between principally dissimilar O alleles during mitosis (“autologous chimerism”) restores glycosyltransferase activity in some cells would explain this interesting phenomenon.