ALBERT

Beschreibung: Introduction: Storage of red blood cells (RBCs) for transfusion purposes is accompanied by a number of morphological and biochemical changes (storage lesions) that reduce post-transfusion survival/efficacy and increase risk for adverse reactions in the recipients. The clearance of altered and older RBCs from circulation is triggered by the clustering of Band 3, an aggregate state that is recognized by a low-affinity naturally occurring IgG antibody (Nab). Considering the key role of Band 3 in the maintenance of RBC structure and survival, elucidation of functional and structural modifications of Band 3 during storage should lead to new approaches aiming to improve RBC storage and post-transfusion viability. Results: Immunoblot analysis of RBC membrane proteins using an anti-phosphotyrosine antibody showed a progressive increase in the phosphorylation status of Band 3 during RBC storage (Figure 1). In addition, using the quenching fluorescence of eosin-5-maleimide (EMA), we showed an increase of the mobile fraction of Band 3. These findings are consistent with previous demonstration that tyrosine phosphorylation of Band 3 reduces its affinity for ankyrin, leading to the release of the immobile fraction of Band 3 from the skeleton complex, and enhancement of the lateral mobility of Band 3 into the lipid bilayer. Immunoblot experiments using an antibody that specifically recognizes the clustered form of Band 3 revealed an increase of Band 3 cluster formation from the 28th day of storage. We also showed that the release of microparticles (MPs) that occurs during RBC aging increases from the 28th day of storage (Figure 2). Finally, stopped-flow-based functional studies showed a decrease of the anion exchanger activity of Band 3 from the 28th day of storage. Conclusion: Altogether, our results suggest that the 28th of storage represents a key moment for the molecular processes leading to irreversible lesions of RBCs and allow us to propose a new Band 3 phosphorylation/clustering-based mechanism of RBC aging. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: INTRODUCTION Autoimmune haemolytic anaemia (AIHA) can complicate solid organ as well as bone marrow transplantation both in children and adults. We describe the first case report of a child with AIHA due to mixed type warm-acting IgM and warm IgG auto-antibodies, 8 months after liver transplantation. CASE REPORT A sixteen-month old boy (A Rh D positive blood group,), 8 months after an orthotopic liver transplant (full cadaveric liver, male A Rh D negative) presented with fever and moderate hepatitis. Two weeks after this episode he developed severe anemia with a Hb level of 39 g/L. His red cells were A1 Rh(D) positive. Plasma testing could not be interpreted due to positive reactions with all test cells. Screening for irregular antibodies was positive. The direct antiglobulin test was repeatedly negative. Further analysis revealed a cold agglutinin with low titre (8 at 15°C) but high thermal amplitude (22–37°C) although of IgM nature, and the absence of any underlying alloantibodies. Only Rh null red blood cells were not agglutinated by the autoantibody. Therapy included keeping body temperature over 37° C, transfusions of A Rh(D) positive warmed crossmatched blood units, intravenous immunoglobulins (1g/kg/d x 5 days), and methylprednisolone (20 mg/kg/d). Tacrolimus was replaced by cyclosporin A. Further investigations revealed panagglutinating IgG autoantibodies in the plasma and on the erythrocytes and the monospecific direct antiglobulin test became positive for IgM, IgG, C3c and C3d. French National Reference Laboratory for Immuno-Hematology and Rare Blood Groups confirmed these findings and showed both the IgM and IgG autoantibodies to be directed against the Rh proteins (anti-RH29 antibody) and to be strongly active at 37°C. Crossmatched group O Rh(D) positive red blood cell (RBC) units were transfused, however with limited effect and progressive haemolysis. Hb level dropped to 33 g/L. One cryopreserved O Rh null RBC unit was obtained from the French National Rare Blood Bank. Rituximab® (375 mg/m2 once weekly) (anti-CD 20 monoclonal antibody) was also introduced. Immediately following this transfusion and 24 hours after the first dose of rituximab, Hb levels increased and were stable at 80 g/L. No further transfusions were needed and the haemolytic parameters normalized slowly. Total 4 doses of rituximab were administered.Ten weeks after admission, the child could be discharged. At 18 months’ follow-up, there is no recurrence of AIHA. DISCUSSION Only few cases of AIHA due to warm acting “cold” agglutinins have been described, generally resulting in death. AIHA in patients with liver transplant has been previously reported, mainly due to warm autoantibodies or to classical cold agglutinin disease associated with viral infections, lymphoproliferative disease or autoimmune disorders. Our case is interesting in that this unusual AIHA occurred not only in a liver transplanted child, but also late after transplantation. Although extensive investigations revealed no aetiology, the hypothesis of a viral infection-triggered AIHA with first an IgM, then a mixture of IgM and IgG autoantibodies directed against the same epitope is plausible. This case illustrates the efficacy of rituximab in AIHA not responding to first-line therapy and the importance of international collaboration to provide extremely rare compatible blood units.

Beschreibung: 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.

Beschreibung: Abstract 3595 Poster Board III-532 Background Chronic Granulomatous Disease (CGD) is an inherited disease affecting innate immunity and leading to increased susceptibility to severe invasive fungal and bacterial infections. The X linked form of CGD is the most frequent and is caused by mutations in the gp91phox subunit of the NADPH oxidase complex encoding gene CYBB. McLeod syndrome patients present with late-onset neuromuscular troubles and a mild chronic haemolytic anaemia with acanthocytes. It is defined by the lack of expression of Kell antigens on erythrocytes and caused by mutations in the KX gene located close to the CYBB gene. The association of CGD and McLeod syndrome is a rare event. It can also be associated with Duchenne muscular dystrophy (DMD), retinitis pigmentosa or ornithine transcarbamylase deficiency. Patients : Four CGD patients patients were recently diagnosed with McLeod syndrome in Necker Hospital on a 2 year period of time. Their clinical and main biological characteristics are reviewed and a review of recent litterature Observations: In 1 patient, McLeod was evoked because of association of Duchenne myopathy and CGD revealed at 10 months with major hypotonia and recurrent infections. Initially, no acanthocytes were seen but Kell antigens testings led to confirmation of McLeod. Three other infants were diagnosed with CGD because of lung infections. One had lung biopsies revealing Nocardia sp. infection and received packed blood red cells. Four months after, an allo immunisation against red cells and a weakened Kell antigen expression led to McLeod diagnosis. For other patients, diagnosis was evoked because of anaemia and acanthocytes on blood smear analysis. All had confirmed by CYBB mutations with complete deletion. None had muscle weakness or retinal abnormality. For 2 infants, iron therapy and erythropoietin led to the correction of anemia. Conclusion Management and evaluation of X-linked CGD patients should require the search of McLeod syndrome. Biological diagnosis is sometimes uneasy, especially because acanthocytes are sometimes hard to identify on blood smears. Kell antigen reactivity screening could be easy to perform. When McLeod syndrome is diagnosed, patients are included in the French national registry for rare erythrocyte phenotype to receive appropriate transfusion management. Erythropoietin therapy could be useful in preventing chronic anemia. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Key Points SLC29A1 encoding the equilibrative nucleoside transporter 1 (ENT1) specifies a novel blood group system that includes the Ata antigen. Although At(a−) people of African ancestry have functional ENT1, 3 siblings of European ancestry were identified who do not express ENT1.

Beschreibung: 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.