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X-linked chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the CYBB gene (located on Xp21.1). Patients with large deletions on chromosome Xp21.1 can present with the McLeod phenotype and also Duchenne muscular dystrophy or retinitis pigmentosa. The objective of the present study was to describe a series of French patients with CGD and the McLeod phenotype. We retrospectively collected data from the medical records of 8 patients with CGD and the McLeod phenotype registered at the French National Reference Center for blood types. The median age at diagnosis of CGD was 1.2 years, the median age at diagnosis of the McLeod phenotype was 4.5 years, and the median length of follow-up was 15.2 years. Four patients displayed allo-immunization, with anti-KEL20 and anti-XK1 (formerly known as anti-KL) antibodies. Five of the 6 patients with available blood smears had acanthocytosis. Neuropsychiatric, muscle-related, and ocular manifestations were present in 4, 2, and 1 of the patients, respectively. Three of the 4 patients having undergone allogeneic hematopoietic stem cell transplantation (HSCT) are alive. Overall, 5 patients are alive, and 3 are alive and well. This is the largest yet descriptive study of a series of patients with X-linked CGD and the McLeod phenotype. Although this disease combination is rare, the timely, accurate diagnosis of the McLeod phenotype is critical because of the serious post-transfusion complications. However, HSCT can be considered in these patients.

The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3′,5′-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.

Background Mta (MNS14) is a low-prevalence antigen of the MNS system. A few cases of hemolytic disease of the fetus and newborn caused by anti-Mta have been reported in the literature, but up to now this antibody has never been associated with a hemolytic transfusion reaction (HTR). Case report A 38-year-old male with sickle cell disease undergoing exchange transfusion presented with shivering, nausea, dyspnea, and pain in the lower limbs. Biologic parameters showed increased hemolysis. The administered red blood cell (RBC) units had been issued by electronic crossmatch due to a negative antibody screening test. In the posttransfusion investigations, crossmatch of the transfused RBC units with the patient's serum showed incompatibility of one unit. The presence of an antibody against a low-prevalence antigen was suspected and further serologic testing was performed for identification. Results Anti-Mta was identified in the patient's serum. The RBCs of the incompatible unit implicated in the HTR were Mt(a+). An eluate of a posttransfusion blood sample of the patient was nonreactive with the incompatible RBCs, and the direct antiglobulin test was negative. Conclusion To our knowledge, this is the first case report of an HTR associated with anti-Mta .

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)

International audience; Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.

This is a related article to: Nucleoside ENTry modulates erythropoiesis (cf ci-dessous); International audience; Abstract The tight regulation of intracellular nucleotides is critical for the self-renewal and lineage specification of hematopoietic stem cells (HSCs). Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside transporters in the differentiation of HSCs to the erythroid lineage and in red cell biology remains to be fully defined. Here, we show that the absence of the equilibrative nucleoside transporter (ENT1) in human red blood cells with a rare Augustine-null blood type is associated with macrocytosis, anisopoikilocytosis, an abnormal nucleotide metabolome, and deregulated protein phosphorylation. A specific role for ENT1 in human erythropoiesis was demonstrated by a defective erythropoiesis of human CD34+ progenitors following short hairpin RNA-mediated knockdown of ENT1. Furthermore, genetic deletion of ENT1 in mice was associated with reduced erythroid progenitors in the bone marrow, anemia, and macrocytosis. Mechanistically, we found that ENT1-mediated adenosine transport is critical for cyclic adenosine monophosphate homeostasis and the regulation of erythroid transcription factors. Notably, genetic investigation of 2 ENT1null individuals demonstrated a compensation by a loss-of-function variant in the ABCC4 cyclic nucleotide exporter. Indeed, pharmacological inhibition of ABCC4 in Ent1−/− mice rescued erythropoiesis. Overall, our results highlight the importance of ENT1-mediated nucleotide metabolism in erythropoiesis.

Patients with sickle cell disease (SCD) show a high prevalence of red blood cell (RBC) alloimmunization, but few studies have focused on children. We aimed to study the prevalence and risk factors of RBC alloimmunization in SCD children. We retrospectively analysed the medical and transfusion files for 245 SCD children hospitalized in our centre in 2014 and included 175 patients who had received at least one RBC unit in their lifetime. The main clinical and immuno-haematological characteristics of alloimmunized and non-alloimmunized patients were compared. The prevalence of alloimmunization was 13·7% [95% confidence interval (CI) (8·6-18·6)], and 7·4% [95% CI (3·5-11·3)] after excluding the probable irregular natural antibodies (anti-M, anti-Lea , anti-Leb , anti-Lex ). Main risk factors for alloimmunization were increased number of RBC units received (median of 65 vs. 10 units per patient; P = 0·01) and the presence of one or more red cell autoantibodies (46·2% vs. 4·7%; P < 0·0001). The alloimmunization rate was higher for episodically transfused than chronically transfused patients (1·43 vs. 0·24/100 units received; P < 0·001). The presence of red cell autoantibodies appears to be a major risk factor for alloimmunization in SCD children and could justify specific transfusion guidelines.

International audience; The Cromer blood group system consists of 19 antigens (16 of high prevalence and 3 of low prevalence). This study describes the identification and characterization of a new Cromer high-prevalence antigen, named CORS. The CORS-negative proband carries a c.713G>A substitution in the CD55 gene, resulting in the substitution of glycine 238 into a glutamic acid (p.Gly238Glu).

A workshop on the clinical significance of red blood cell alloantibodies, organized by the Executive Committee of the Working Party on Immunohaematology of the International Society of Blood Transfusion (ISBT), took place in Dubai, United Arab Emirates, on 9 September 2016, in conjunction with the 34th International Congress of the ISBT. This event was funded by the ISBT Academy, founded in 2011, to support educational and knowledge activities. This report is a summary of that meeting.

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.

The blood group antigen Vel has been one of immunohematology’s greatest enigmas: the variation in antigen strength from one individual to another, the property of anti-Vel to readily hemolyze Vel+ red blood cells (RBCs), and the difficulty to screen for sufficient numbers of Vel– blood donors had made Vel a tough nut to crack. In 2013, a small, previously unknown protein called small integral membrane protein 1 (SMIM1) was identified on the RBC by three independent research groups using different approaches, and all three groups demonstrated that Vel– RBCs lacked SMIM1. This discovery correlated with homozygosity for deletion c.64_60del in SMIM1 and meant that for the first time there was a universal method to screen for Vel– blood donors. This finding was not the whole answer, however, and an explanation behind the variability in antigen strength was later shown to be due to polymorphism in SMIM1 intron 2, a region that is responsible for gene transcription. Clinically, anti-Vel is important and has caused severe transfusion reactions, although hemolytic disease of the fetus and newborn caused by anti-Vel is uncommon. However, while screening for Vel– blood donors has become easier, the function of SMIM1 is still unknown, and despite its well-conserved sequence across the animal kingdom, the enigma continues. Immunohematology 2017;33:56–59.

Background and objectives: The International Society of Blood Transfusion (ISBT) Working Party for Red Cell Immunogenetics and Blood Group Terminology meets in association with the ISBT congress and has met three times since the last report: at the international meetings held in Dubai, United Arab Emirates, September 2016 and Toronto, Canada, June 2018; and at a regional congress in Copenhagen, Denmark, June 2017 for an interim session. Methods: As in previous meetings, matters pertaining to blood group antigen nomenclature and classification were discussed. New blood group antigens were approved and named according to the serologic and molecular evidence presented. Results and conclusions: Fifteen new blood group antigens were added to eight blood group systems. One antigen was made obsolete based on additional data. Consequently, the current total of blood group antigens recognized by the ISBT is 360, of which 322 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known system. Clinically significant blood group antigens continue to be discovered, through serology/sequencing and/or recombinant or genomic technologies.

International audience; BACKGROUND : Sickle cell disease (SCD) patients undergo multiple red blood cell (RBC) transfusions and are regularly exposed to low‐prevalence (LP) antigens specific to individuals of African descent. This study evaluated the prevalence of antibodies against LP antigens in SCD patients and the need to identify these antibodies in everyday practice.STUDY DESIGN AND METHODS : Plasma from 211 SCD patients was tested with RBCs expressing the following LP antigens: RH10 (V), RH20 (VS), RH23 (DW), RH30 (Goa), KEL6 (Jsa), and MNS6 (He).RESULTS : Nine LP antibodies were found in eight patients (3.8%): five anti‐RH23, two anti‐RH30, and two anti‐MNS6. The exposure risk, calculated for each LP antigen, was below 3% per RBC unit, for all antigens tested. Thus, in this cohort of transfused SCD patients, the prevalence of LP antibodies was similar to that of antibodies against antigens of the FY, JK, and MNS blood group systems. These findings also reveal the occurrence of anti‐RH23 in SCD patients. No anti‐RH20 or anti‐KEL6 were found, despite the high frequency of mismatch situations.CONCLUSION : These results highlight the immunogenicity of these LP antigens, and the evanescence of antibodies against LP antigens. They also highlight the importance of appropriate pretransfusion testing for patients frequently transfused, who are likely to be exposed to multiple types of blood group antigens.

The Kidd blood group system currently comprises two polymorphic and antithetical antigens, Jka and Jkb, and one high-prevalence antigen, Jk3. Jknull individuals do not express any of the Kidd antigens, and are at risk of developing an anti-Jk3 which is known to be dangerous and responsible for acute or delayed hemolytic transfusion reaction. We report a case of an immunized Jknull patient, who was scheduled to undergo a heart transplant. In order to organize his blood provision management, two conference calls were held between the clinical team and the different staff involved in this challenging blood supply. In light of the blood needs, the available resources, and the constraints, a mix of fresh and frozen units were used. As the supply from France was not sufficient, Finland and New Zealand provided the majority of the fresh units. We report here how this international supply chain was organized, including the difficulties that we encountered. Anticipation, communication and flexibility were essential in making this heart transplant possible without needing to transfuse incompatible units.

Antigens of the Gerbich blood group system are expressed on glycophorin C (GPC) and glycophorin D (GPD), minor sialoglycoproteins of human erythrocytes. GPC and GPD help maintain erythrocyte shape of and contributes to the stability of its membrane. There are six high-prevalence Gerbich antigens: Ge2, Ge3, Ge4, GEPL (GE10), GEAT (GE11), GETI (GE12) and five low-prevalence Gerbich antigens: Wb (GE5), Lsa (GE6), Ana (GE7), Dha (GE8), GEIS (GE9). Some Gerbich antigens (Ge4, Wb, Dha, GEAT) are expressed only on GPC, two (Ge2, Ana) are expressed only on GPD, while others (Ge3, Lsa, GEIS, GEPL, GETI) are expressed on both GPC and GPD. Antibodies recognizing GPC/GPD may arise naturally (so-called "naturally-occurring RBC antibodies") or as the result of alloimmunization, and some of them may be clinically relevant. Gerbich antibodies usually do not cause serious hemolytic transfusion reactions (HTR); autoantibodies of anti-Ge2- or anti-Ge3 specificity can cause autoimmune hemolytic anemia (AIHA).

Introduction Most blood antigen-carrying proteins have important functions not only in red blood cells (RBCs) but also in other tissues. Hence, because of their polymorphisms and the existence of natural null phenotypes, blood groups also represent powerful tools to investigate human diseases. The PEL-negative rare blood type is one of the last with an unknown genetic basis. Family studies showed that the PEL-negative phenotype was inherited as an autosomal recessive trait, but the PEL locus remained undetermined since the first description of the corresponding antibody in French-Canadian individuals in 1996. Results Combining whole-exome sequencing and comparative global proteomic approaches using genomic DNA and red cell membrane proteins from four unrelated French-Canadian PEL-negative individuals and from controls, we identified i) the Multidrug Resistance-Associated Protein 4 (MRP4), also known as ABCC4, as the only missing protein in all PEL-negative RBCs and ii) a large deletion removing the last 10 exons of the ABCC4 gene. Western blot analyses confirmed that ABCC4 was present in the membrane of PEL-positive control RBCs but totally absent in the PEL-negative RBCs (Fig 1A). Western blot and flow cytometry analyses of transfected HEK293 cells revealed that over-expression of ABCC4 results in a significant increase in PEL antigen cell-surface expression (Fig 1B). Conversely, similar analyses of ABCC4-knockout K562 cells generated by the CRISPR-Cas9 strategy showed that the complete loss of ABCC4 abolished the expression of the PEL antigen (Fig 1C). PEL-negative individuals are therefore the first ever reported population lacking the complete expression of the ABCC4 protein. Surprisingly, despite the expected biological importance of ABCC4 as a cyclic nucleotide transporter, no apparent mutual symptoms or diseases were identified after investigation of the clinical history of the 12 PEL-negative members within the 4 families analyzed in this study. However, platelet investigation from one PEL-negative individual supported the role of this protein in the modulation of platelet aggregation, as previously observed in ABCC4-knockout mice studies To better understand the function of ABCC4 in mature RBCs, we measured the intracellular cGMP level in RBCs from 5 PEL-negative individuals and 5 controls. We showed that the cGMP level was not significantly impaired in the absence of ABCC4 suggesting that another cGMP transporter could compensate for the absence of ABCC4 on the RBC membrane. Interestingly, proteomic and flow cytometry analyses indicated that among the five other ABC transporters (ABCC1, ABCA7, ABCB6, ABCC5 and ABCG2) expressed on RBCs, only ABCG2 was increased in PEL-negative/ABCC4null RBCs compared to controls (1.3-fold; p=0.0007). This suggested that the absence of ABCC4 in PEL-negative individuals could be compensated by the over-expression of ABCG2. Our data, together with previous studies indicating that both ABCC4 and ABCG2 export cGMP, strongly suggest that the over-expression of ABCG2 in PEL-negative RBCs represents a compensatory mechanism for the lack of ABCC4, which could explain the absence of hematological abnormalities in PEL-negative individuals. Discussion Using genetic, molecular and cellular approaches, we demonstrated that ABCC4 is the gene underlying the novel PEL blood group system and that homozygosity for a large deletion in this gene is responsible for the rare PEL-negative phenotype. ABCC4 represents the third ABC transporter carrying a blood group system after ABCG2 (JR) and ABCB6 (LAN). The expression level of ABCC4 is strongly involved in drug resistance and toxicity, and in the clinical course of leukemia. As ABCC4 carries the PEL antigen, PEL phenotyping could be useful in the adjustment of an optimal drug dose and prevention of chemotherapy side effects in leukemia. Our findings are of immediate and important relevance in transfusion medicine and in a personalized medicine approach for chemotherapy treatment follow-up in leukemia. Furthermore, the exceptional natural "knockouts" of ABC transporters are highly valuable in understanding their function, not only in erythroid cells, but also in other cells or tissues. Disclosures Hermine: Novartis: Research Funding; Celgene: Research Funding; AB Science: Consultancy, Equity Ownership, Honoraria, Research Funding.

Here, we report the biochemical and genetic basis of the Vel blood group antigen, which has been a vexing mystery for decades, especially as anti-Vel regularly causes severe haemolytic transfusion reactions. The protein carrying the Vel blood group antigen was biochemically purified from red blood cell membranes. Mass spectrometry-based de novo peptide sequencing identified this protein to be small integral membrane protein 1 (SMIM1), a previously uncharacterized single-pass membrane protein. Expression of SMIM1 cDNA in Vel− cultured cells generated anti-Vel cell surface reactivity, confirming that SMIM1 encoded the Vel blood group antigen. A cohort of 70 Vel− individuals was found to be uniformly homozygous for a 17 nucleotide deletion in the coding sequence of SMIM1. The genetic homogeneity of the Vel− blood type, likely having a common origin, facilitated the development of two highly specific DNA-based tests for rapid Vel genotyping, which can be easily integrated into blood group genotyping platforms. These results answer a 60-year-old riddle and provide tools of immediate assistance to all clinicians involved in the care of Vel− patients.

Background and Objectives The Colton blood group antigens are carried by the AQP1 water channel. AQP1−/− individuals, also known as Colton-null since they express no Colton antigens, do not suffer any apparent clinical consequence but may develop a clinically significant alloantibody (anti-CO3) induced by transfusion or pregnancy. Identification and transfusion support of Colton-null patients are highly challenging, not only due to the extreme rarity of this phenotype, the lack of appropriate reagents in most laboratories, as well as the possibility of confusing its with the recently described CO:-1,-2,3,-4 phenotype where AQP1 is present. This study investigated a new Colton-null case and evaluated three commercially available anti-AQP1s to identify Colton-null red blood cell samples.

Lionel Arnaud and colleagues identify ABCB6 as the genetic basis of the novel Lan blood group system. The human ATP-binding cassette (ABC) transporter ABCB6 has been described as a mitochondrial porphyrin transporter essential for heme biosynthesis1, but it is also suspected to contribute to anticancer drug resistance2,3,4, as do other ABC transporters located at the plasma membrane. We identified ABCB6 as the genetic basis of the Lan blood group antigen expressed on red blood cells but also at the plasma membrane of hepatocellular carcinoma (HCC) cells, and we established that ABCB6 encodes a new blood group system (Langereis, Lan). Targeted sequencing of ABCB6 in 12 unrelated individuals of the Lan(−) blood type identified 10 different ABCB6 null mutations. This is the first report of deficient alleles of this human ABC transporter gene. Of note, Lan(−) (ABCB6−/−) individuals do not suffer any clinical consequences, although their deficiency in ABCB6 may place them at risk when determining drug dosage.

BACKGROUND: S, s, and U antigens belong to the MNS system. They are carried by glycophorin B (GPB), encoded by GYPB. Black people with the low-prevalence S−s− phenotype, either U− or U+var, can make a clinically significant anti-U. Anti-U-like, a cold immunoglobulin G autoantibody quite commonly observed in S−s+U+ black persons, was previously described to be nonreactive with ficin-, α-chymotrypsin-, and pronase-treated red blood cells (RBCs); nonreactive or weakly reactive with papain-treated RBCs; and reactive with trypsin-treated RBCs. Here we describe, in S−s− people from different molecular backgrounds, an alloantibody to a high-prevalence GPB antigen, which presents the same pattern of reactivity with proteases as autoanti-U-like. STUDY DESIGN AND METHODS: Four S−s− patients with an alloantibody to a high-prevalence GPB antigen were investigated by serologic and molecular methods. RESULTS: An alloantibody was observed in two S−s−U−/Del GYPB, one S−s−U+var/GYPB(P2), and one S−s−U+var/GYPB(NY) patients. As this alloantibody showed the same pattern of reactivity with proteases as autoanti-U-like, we decided to name it “anti-U-like.” Anti-U-like made by the two S−s−U− patients was reactive with the S−s−U+var RBCs of the two other patients. CONCLUSION: S−s−U−/Del GYPB, S−s−U+var/GYPB(P2), and S−s−U+var/GYPB(NY) patients can make an alloanti-U-like. Anti-U-like made by S−s−U− people appears reactive with GYPB(P2) and GYPB(NY) RBCs, which both express a weak and partial U-like reactivity. We recommend transfusing S−s−U− RBCs in S−s−U− patients showing alloanti-U-like. Our study contributes to a better understanding of alloimmunization to GPB in black people and confirms importance of genotyping in S−s− patients, especially those with sickle cell disease to be frequently transfused.

BACKGROUND: Whether anti-D produced by individuals with a weak D phenotype are allo- or autoantibodies remains a matter of debate even though blood transfusion practice is impacted. The aim of our study was to determine the serologic features of anti-D in individuals expressing the most frequent weak D type in Caucasians that are weak D Type 1 or weak D Type 2, to assess whether anti-D were allo- or autoantibodies. STUDY DESIGN AND METHODS: Serologic D typing and molecular analysis enabled the including of 121 weak D Type 1 individuals and 99 weak D Type 2 individuals in our study. Serologic features of anti-D included autologous controls, direct antiglobulin test, elution, and titration of anti-D before and after adsorption of serum on autologous red blood cells (RBCs). RESULTS: Serologic D typing showed a variable reactivity of RBCs expressing weak D Type 1 or weak D Type 2 (4+ to 0). Anti-D was identified in six weak D Type 1 and six weak D Type 2 individuals, respectively. The serologic data were in favor of autoantibodies. CONCLUSION: A complete anti-D investigation in individuals with a D variant (weak D or partial D identified by molecular analysis) should be systematically performed before any valid conclusion on the nature of the antibody. Transfusing weak D Type 1 or weak D Type 2 patients with D+ RBC units should be recommended. Weak D Type 1 or weak D Type 2 pregnant women do not need anti-D immunoprophylaxis.

BACKGROUND: DNA testing has enabled the documenting of numerous variants of RHCE alleles, especially in individuals of African origin. The risk for production of clinically significant alloantibodies to Rh antigens of patients carrying variant RHCE alleles has led us to analyze the different RhCE variants investigated by molecular biology. Alloimmunization was analyzed regarding the RHCE genetic profile. STUDY DESIGN AND METHODS: Samples from 806 individuals with altered expression of RhCE antigens and/or producing anti-RhCE in the presence of the corresponding antigen were analyzed. RESULTS: A total of 572 individuals were shown to express RhCE variants. Variant RHCE*ce alleles and RH haplotypes were identified in 83% of cases, the most frequent ones being the RN haplotype, the ceMO allele, the (C)ces haplotype/ces1006 allele, and the ceAR allele identified in 36, 23, 20, and 17% of the tested samples, respectively. The absence of a high-prevalence Rh antigen was documented in 93 individuals. Partial C and partial e were expressed by 53% of individuals with RhCE variants. Rh antibodies were identified in 127 (20%) of 623 patients. They were found to be alloantibodies in 48 (38%) of these 127 patients. Alloimmunization against a high-prevalence Rh antigen was detected in 25% of cases. CONCLUSION: The challenge in clinical red blood cell (RBC) transfusion of patients with sickle cell disease, notably, would be to provide not only phenotypically matched, but also genetically matched, RBC units regarding RhCE variants.

BACKGROUND: DNA analysis for the prediction of blood group antigen expression has broad implications in transfusion medicine. It may be of particular interest especially to detect variants, when antigen expression is weak or altered. The use of high-throughput DNA analysis has never been applied to donors whose red blood cells (RBCs) are selected for reagent RBCs. The aim of this study was to analyze the concordance between the serologic phenotype and that predicted from DNA analysis in panel donors, to determine the benefit of the use of DNA analysis in reagent RBC selection strategy. STUDY DESIGN AND METHODS: The "Panel National de Reference du Centre National de Reference sur les Groupes Sanguins" is a reference reagent mainly used for antibody identification. DNA genotyping of 356 panel donors was performed with BeadChips (human erythrocyte antigen v1.2 BeadChips, BioArray Solutions). The comparison between serologic phenotype and that predicted from DNA analysis held on 8876 paired results obtained from 10 blood group systems and 25 antigens. RESULTS: A 99.95% concordance was observed. Discrepancies in four cases (RH, KEL, LU, and DO systems) were analyzed. Genotyping precisions on the Duffy system were of particular interest. No new rare blood group was observed. CONCLUSION: Systematic DNA analysis of panel donors should unquestionably change the management of reagent RBC selection. The notion of "antigens in double dose" should evolve regarding data obtained from DNA analysis, allowing an improved quality of reagent RBCs for antibody screening and identification.

BACKGROUND: The KN blood group system, which consists of nine antigen specificities, is located on complement receptor Type 1 (CR1/CD35). CR1, a complement regulatory protein, acts as a vehicle for immune complex clearance. CR1 exhibits a red blood cell (RBC) density polymorphism. CR1 sites on RBCs in normal individuals range from 150 to 1200 molecules per cell. CR1 density polymorphism is regulated by HindIII restriction fragment length polymorphism and Q981H and P1786R polymorphisms in Caucasians. Yet, the role of the different polymorphisms in determining the CR1 density on RBCs remains unknown. The “null” serologic KN phenotype, known as Helgeson phenotype, was reported to be related with a very low CR1 density, less than 150 molecules per cell. STUDY DESIGN AND METHODS: The aim of this work was to investigate whether the KN-negative phenotype displayed by 60 individuals was related to the CR1 density by performing the phenotypic and genetic analysis of CR1 and to investigate the molecular background associated with the KN system. RESULTS: We showed that the Helgeson-like phenotype had a prevalence of 12% in this population. The overall genotype/phenotype concordance was 90%. Among individuals with a KN-negative phenotype, the prevalences of Kn(a‐), McC(a‐), Sl1-negative, Sl3negative, and KCAM-negative deduced phenotype were 37, 12, 29, 7, and 24%, respectively. CONCLUSION: From our data, we suggest that the definition of the Helgeson phenotype must be revised, since the latter may be due not only to a very low CR1 density on RBCs, but also to the absence of expression of a high-prevalence KN antigen.

BACKGROUND: In the Rh blood group system, published observations showed that the c antigen has the fewest variant forms of the principal antigens in this system. The partial nature of the c antigen was only reported in c+ Rh:−26 persons and to be associated with the ces(340) allele. This study reports the first case of alloanti-c related to a (C)ces haplotype. STUDY DESIGN AND METHODS: Serologic and genetic studies were performed on blood samples of a multitransfused 40-year-old African patient with sickle cell disease displaying a DCcee phenotype. RESULTS: Red blood cells (RBCs) of the patient displayed normal expression of C, c, e, ce antigens either with routine reagents or with monoclonal antibodies. Analyses of DNA and Rh transcripts showed that the patient carried a (C)ces/DCe genotype. The patient's serum contained anti-D, anti-c, anti-E, anti-e, anti-V, anti-Jsa, and anti-S. Anti-c was isolated from the mixture of antibodies by using absorption and adsorption-elution techniques. Anti-c provided consistent reactions with c+ RBCs. Reactions were stronger with c+ ce+ RBCs than with c+ ce− RBCs. No agglutination of RBCs from individuals carrying a homozygous (C)ces genotype was observed. CONCLUSION: These data provide the evidence that anti-c in our patient was an alloanti-c and, consequently, that (C)ces haplotype encodes a partial c antigen. The clinical significance of anti-c related to this haplotype should be evaluated in the future.

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.

The Augustine-negative alias At(a-) blood type, which seems to be restricted to people of African ancestry, was identified half a century ago but remains one of the last blood types with no known genetic basis. Here we report that a nonsynonymous single nucleotide polymorphism in SLC29A1 (rs45458701) is responsible for the At(a-) blood type. The resulting p.Glu391Lys variation in the last extracellular loop of the equilibrative nucleoside transporter 1 (ENT1; also called SLC29a1) is known not to alter its ability to transport nucleosides and nucleoside analog drugs. Furthermore, we identified 3 individuals of European ancestry who are homozygous for a null mutation in SLC29A1 (c.589+1GC) and thus have the Augustine-null blood type. These individuals lacking ENT1 exhibit periarticular and ectopic mineralization, which confirms an important role for ENT1/SLC29A1 in human bone homeostasis as recently suggested by the skeletal phenotype of aging Slc29a1(-/-) mice. Our results establish Augustine as a new blood group system and place SLC29A1 as a new candidate gene for idiopathic disorders characterized with ectopic calcification/mineralization.

Background: Holley (Hy) is a high-incidence antigen of the Dombrock blood group system (ISBT 014), present in almost 100% of most populations and more than 99% of Blacks. Since anti-Hy is an extremely rare antibody, data on its clinical relevance and in particular on a possible hemolytic disease of the fetus and newborn (HDFN) are scarce. Case Report: The pregnant patient underwent two autologous whole blood collections at weeks 17 and 19 of gestation with cryopreservation. In our case autologous whole blood collection was well tolerated. There were no signs of HDFN in the healthy newborn. Conclusion: Our case improves our understanding of anti-Hy alloantibodies during pregnancy. Additionally, autologous whole blood collection of RBC units with cryopreservation is a safe and feasible way to manage pregnancies in women with rare alloantibodies, when no compatible donor can be found.

KLF1 is an essential erythroid transcription factor (EKLF) involved both in the ß-globin switch from fetal to adult and in definitive erythropoiesis. KLF1 also activates genes encoding heme biosynthetic enzymes, as well as genes involved in the cell cycle and the synthesis of membrane proteins. Heterozygosity for one or several nucleotide changes in KLF1 may be responsible for the so-called dominant Lu(a-b-) phenotype, also known as In(Lu) for "Inhibitor Lutheran" and characterized by a dramatic reduction in the expression levels of the Lutheran blood group antigens and a slight elevation in hemoglobin F (HbF). Several other blood group antigens are also under the direct control of KLF1, but their number and expression level in In(Lu) red blood cells (RBCs) remain a matter of debate. In addition, mutations in KLF1 in humans lead to anemia, acantocytosis and some of these variants, such as CDA type IV, are characterized by ineffective terminal differentiation and defects in enucleation. Given the major role of KLF1 in erythropoiesis, it would not be surprising that In(Lu) RBCs show abnormal properties. In this work, we performed proteomic studies to quantify membrane proteins in In(Lu) vs control RBCs. Results: using label-free mass spectrometry, we analyzed the expression levels of membrane proteins in 5 In(Lu) and 4 control RBCs. Hierarchical clustering allowed to identify two modulation profiles of protein expression (Figure 1). The first profile (red color) is composed of 49 proteins over-expressed in In(Lu) RBCs, with the majority of them corresponding to the "26S proteasome complex" (PSMA, PSMB, PSMD, and PSME). In addition, we confirmed the overexpression of the 26S proteasome complex in In(Lu) RBCs by western blot analyses. The second profile (green color) includes 23 membrane proteins with a lower expression level in In(Lu) RBCs; these proteins correspond to blood group antigens (e.g. Lu/BCAM, CD44), and to cytoskeletal proteins (e.g. dematin, flotillin). Five proteins carrying blood group systems show a decrease of expression in In(Lu) RBCs over 40%: Chido/Rodgers, Xg, Indian, Duffy and Lutheran: antigens. While our results indicating of decreased expression of Indian and Chido/Rodgers systems are consistent with previous reports, the Duffy expression was unexpectedly found to be decreased by 86%. Since we evidenced an important increase in the proteasome complex, and taking into account that KLF1 is involved in the regulation or erythropoiesis, we, investigated the expression profile of the 26 S proteasome expression during erythropoiesis. We observed an important decrease of all proteins of the proteasome complex during erythropoiesis. Conclusion: from our preliminary results reported in this study, we hypothesized that the increase of the 26S proteasome in the In(Lu) individuals may result from defects in organelle loss and could explain the profound dyserytropoiesis observed in the "nan" mice null for KLF1. Figure 1: Comparative proteomics of red blood cell membrane proteins in In(Lu) blood group type. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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 The numerous antigens in the Kell blood group system result from missense nucleotide changes in KEL. Antibodies to antigens in this system can be clinically important. We describe six probands whose plasma contained antibodies to high-prevalence Kell antigens, and discuss their relationship.

KLF1 encodes an erythroid transcription factor, whose essential function in erythropoiesis has been demonstrated by extensive studies in mouse models. The first reported mutations in human KLF1 were found in individuals with a rare and asymptomatic blood type called In(Lu). Here, we show that KLF1 haploinsufficiency is responsible for the In(Lu) blood type, after redefining this peculiar blood type using flow cytometry to quantify the levels of BCAM and CD44 on red blood cells. We found 10 (seven novel) heterozygous KLF1 mutations responsible for the In(Lu) blood type. Although most were obligate loss-of-function mutations due to the truncation of the DNA-binding domain of KLF1, three were missense mutations that were located in its DNA-binding domain and impaired the transactivation capacity of KLF1 in vitro. We further showed that the levels of the hemoglobin variants HbF and HbA(2) were increased in the In(Lu) blood type, albeit differently. The levels of the membrane glycoproteins BCAM and CD44 were also differently reduced on In(Lu) red blood cells. This biochemical and genetic analysis of the In(Lu) blood type tackles the phenotypic outcome of haploinsufficiency for a transcription factor.

International audience; The ABO blood group influences susceptibility to severe Plasmodium falciparum malaria. Recent evidence indicates that the protective effect of group O operates by virtue of reduced rosetting of infected red blood cells (iRBCs) with uninfected RBCs. Rosetting is mediated by a subgroup of PfEMP1 adhesins, with RBC binding being assigned to the N-terminal DBL1α1 domain. Here, we identify the ABO blood group as the main receptor for VarO rosetting, with a marked preference for group A over group B, which in turn is preferred to group O RBCs. We show that recombinant NTS-DBL1α1 and NTS-DBL1α1-CIDR1γ reproduce the VarO-iRBC blood group preference and document direct binding to blood group trisaccharides by surface plasmon resonance. More detailed RBC subgroup analysis showed preferred binding to group A1, weaker binding to groups A2 and B, and least binding to groups Ax and O. The 2.8 Å resolution crystal structure of the PfEMP1-VarO Head region, NTS-DBL1α1-CIDR1γ, reveals extensive contacts between the DBL1α1 and CIDR1γ and shows that the NTS-DBL1α1 hinge region is essential for RBC binding. Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBLα1. RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins, opening novel opportunities for intervention against severe malaria. By deciphering the structural basis of blood group preferences in rosetting, we provide a link between ABO blood grouppolymorphisms and rosette-forming adhesins, consistent with the selective role of falciparum malaria on human genetic makeup.

International audience; The breast cancer resistance protein, also known as ABCG2, is one of the most highly studied ATP-binding cassette (ABC) transporters because of its ability to confer multidrug resistance. The lack of information on the physiological role of ABCG2 in humans severely limits cancer chemotherapeutic approaches targeting this transporter. We report here that ABCG2 comprises the molecular basis of a new blood group system (Junior, Jr) and that individuals of the Jr(a-) blood type have inherited two null alleles of ABCG2. We identified five frameshift and three nonsense mutations in ABCG2. We also show that the prevalence of the Jr(a-) blood type in the Japanese and European Gypsy populations is related to the p.Gln126* and p.Arg236* protein alterations, respectively. The identification of ABCG2(-/-) (Jr(a-)) individuals who appear phenotypically normal is an essential step toward targeting ABCG2 in cancer and also in understanding the physiological and pharmacological roles of this promiscuous transporter in humans.

In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34+ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 1010 cRBCs generated under good manufacturing practice conditions and labeled with 51Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro–generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.

BACKGROUND:ceAR (RHCE*ceAR) is a rare RH allele encountered in people of African/Caribbean ancestry, known to encode a partial e antigen. The homozygous ceAR/ceAR genotype encodes the rare blood group Hr−. This study describes alloanti-c/ce in a ceAR/Ce patient, suggesting that ceAR also encodes a partial c antigen. CASE REPORT: A 21-year-old patient suffering from intermediate β-thalassemia, with transfusion history, was hospitalized for severe anemia. Blood samples were referred to the National Reference Laboratory for suspicion of a mixture of alloantibodies or an alloantibody to a high-prevalence antigen. MATERIALS AND METHODS: Standard hemagglutination methods were performed to investigate the patient's RBCs and serum. A molecular analysis of RHD and RHCE was carried out by allele-specific polymerase chain reaction and DNA sequencing. RESULTS: Blood type performed by the referring laboratory was B, D+C+E−c+e+, K−. Several antibodies were identified: anti-c/ce, anti-Fyb, anti-Jka, and anti-S. Full serologic investigations showed that anti-c/ce could be very likely considered as an alloantibody. The patient's genotype was ceAR/Ce. Anti-c/ce reacted with ceAR/ceEK, ceEK/ceEK, and ceAR/ceBI but not with ceAR/ceAR, ceMO/ceMO, and ces(340)/ces(340) RBCs. CONCLUSION: This is the first case of alloanti-c/ce related to ceAR, suggesting that this rare RHCE allele encodes a partial c antigen. The presence of the C antigen in the patient allowed for the partial expression of the c antigen encoded by ceAR. The c antigen encoded by ceAR appeared to be different than that encoded by ceEK and ceBI and may share common lacking epitopes with the c antigens encoded by ceMO and ces(340).

A rare erythrocyte phenotype is characterized by the absence of expression of a high-frequency antigen on red blood cells or the absence of several antigens within the same blood group. In France, a blood group is considered rare when the frequency in the general population is less than 1/4000. Allo-immunization against lacking antigens through transfusion or pregnancy requires providing adapted transfusion-support in order to secure further transfusions. A national file of individuals including both patients and donors has been implemented for more than 20 years, along with a national rare blood bank (BNSPR) designed to provide rare blood 24 hours a day. The organization is based on cooperation between the National Blood Service (EFS) and the National Reference Center for Blood Groups (CNRGS). Rare blood donation is anonymous, homologous and non paid. Special dispensations are applied compared to the standard blood donation rules: age of donor, absence of leucodepletion, specific agreement for markers except HIV. The BNSPR is in charge of the rare blood unit management (reception, freezing, storage, thawing, distribution, delivering, and shipping) and of the sample storage. RBCs are freezed under −80°C with glycerol according to the Valeri’s method. The post wash storage period is limited to 24h in the open system of cryopreservation because the deglycerolization step is performed in an open system. In the automated closed system, RBCs can be both glycerolized and deglycerolized, and then the post wash storage in SAGM at +4°C is extended to 7 days. In both systems, the mean value of freeze-thaw wash process recovery is 79.5%. The mean percentage of haemolysis and residual extracellular glycerol, measured on the day of deglycerolization, are 0.2% and 0.15g respectively. From 2002 to 2007, 4064 units were frozen, 1076 were thawed for 211 patients and 2426 were destroyed for storage regulation. The aim of regulation is to reach for almost all units the standard blood donation rules, but also to improve the phenotypic and genotypic characteristics of the units. As an example, in the FY blood group, only the RH:−20, KEL: −6 units will be maintained in the frozen stock in order to avoid allo-immunization against low frequency antigens, difficult to detect in a routine laboratory. At the end of 2007, 9600 individuals are listed in the national file, 5470 blood units corresponding to 1603 donors are frozen. 97% of the donors are typed at least in the RH, KEL, FY, JK and MNS. More than 125 rare specificities are represented with the following repartition of the units: FY: −1, −2 (25%), KEL: −2 (18%), YT: −1 (15%), rare RH (8%), Vel-negative (6.5%), MNS: −3, −4, −5 (6%) LU: −2 (5%). Units from Afro-Caribbean donors represent 35% of the stock. 83% of the units are stored for less than 10 years and 84% are tested for HIV and HCV NAT. 2% of the units present biological abnormality markers, 2% have medical counter-indications in the donors and 0.9 % are not leuco-depleted. The mean number of transfused units per patient is 5.8 (min=1, max=36 for a KEL: −2 individual). The mean number of transfusion episodes per patient is 2.5 (min=1, max=16 for a FY: −1, −2 individual). Since 2002, the BNSPR has been helping other countries and 16 units have been shipped to different regions e.g. Canada, The Netherlands, Belgium, Germany, Switzerland. France should now define its policy regarding the shipping of rare blood units to foreign countries in order to extend this service to longer populations. This may be done in the cooperation frame of the European Council.

The human red blood cell (hRBC) has an exceptionally high permeability for water (Pf) and urea (Purea). However, the contribution of several blood group proteins to permeability of both molecules is still a matter of debate. Here, we have taken advantage of 3 extremely rare blood group phenotypes Colton-null, Kidd-null and Gil-null characterized by total deficiency of aquaporin-1 (AQP1), urea transporter (UT-B) and aquaporin-3 (AQP3), respectively. To evaluate the possible contribution of each protein to water and urea permeation, these hRBC were analyzed using a stopped-flow spectrometer. Functional studies showed that AQP1 and UT-B proteins substantially contribute (96%) to the high water permeability of the hRBC membrane. Furthermore, molecular dynamics simulations (MDS) of water diffusion through urea transporter reveal a permeation mechanism different from that observed in AQP1. Interestingly, uptake studies using 14C-urea indicated that the rate of urea transport through UT-B pore was reduced in the absence of AQP1 (Colton-null) and can be modulated by changing the osmolarity conditions. Overall, these data provide evidence that the urea transport through UT-B was controlled by the movement of water molecules into this channel.Taken together, this result allowed to propose molecular mechanisms of permeation and selectivity of UT-B for urea permeability.