Search

Background: The use of blood group genotyping for the prediction of antigen expression has been discussed in clinical transfusion settings, but much less for reagent red blood cells selection. In France, the Centre National de Référence pour les Groupes Sanguins (CNRGS) produces a reference panel of reagent red blood cells, mainly used for red cell antibody identification. The use of high-throughput DNA analysis has never been applied to blood donors whose red blood cells are used as reagents. The aim of this study was to compare the serological phenotype and that predicted from DNA analysis in such donors, and to determine the benefit of DNA analysis in reagent red blood cells selection strategy., Study Design and Method: Red blood cells of 346 blood donors were typed with two different reagents for each antigen. The genotyping was performed by using HEA v1.2 BeadChips, BioArray Solutions, Immucor. The comparison between the serologically determined 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. Four cases of discrepancy for RH, KEL, LU and DO blood group systems were analyzed. Genotyping precisions were of special interest for the Duffy blood group system., Conclusion: Systematic DNA analysis brings important information on reagent red blood cells selection. It can be used at a routine level. Especially, the notion of "antigen of double dose" which is specified in several countries by government bodies should evolve regarding data obtained from DNA analysis. This should improve the quality of reagent red blood cells as first step for antibody identification., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

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, if its prevalence in France is 4/1000 or less in the general population. An individual with a rare blood phenotype can develop a naturally-occurring or immune antibody corresponding to his rare specificity. In case an extremely low stock of compatible blood is available at the national level, a so-called "transfusion deadlock" is described. Most of the individuals with a rare blood group are coincidently identified when a routine pretransfusion testing or pregnancy follow-up is performed, if the antibody(ies) corresponding to the rare specificity is(are) present. Other individuals are discovered following a systematic red cell typing, or family investigations in siblings. One hundred and twenty-one rare blood specificities and 42 rare blood genotypes are currently defined at the French National Reference Laboratory for Blood Groups (CNRGS-Paris). The French national registry of individuals with a rare blood phenotype/genotype includes about 9600 people, who are urged to regularly donate blood for the National Rare Blood Bank. This bank, based on a homologous blood transfusion program, is in charge of the long-term storage of rare frozen blood units, that can only be delivered after receiving authorization from the CNRGS. The global and individual care management of the individuals with a rare blood group, concerning potentially several hundred thousand people in France, requires a close cooperation between all the protagonists within the transfusion chain.

For ten years, the working party of immunohaematology of the French Society of Blood Transfusion organizes a quality control. After the modification of the law about the realization of erythrocyte typing and detection of unexpected red cell allo-antibodies, the quality control was performed in order to determine the sensitivity of the indirect antiglobulin test by filtration with a standard anti-RH1(D) produces by the National Reference Center of Blood Groups.

In 2004, the French Reference Centre for Rare Blood Groups and Immunohaematology (CNRGS) developed 7 types of activities: 1) Studies of complex Immunohaematology issues (IH), 2) Studies of rare blood phenotypes, 3) the transfusion of patients showing complex issues, 4) IH reactive control in consistency with the 98/79/CE European Directive, 5) European studies and expertise on reactives and techniques, 6) Biotechnologies applied to blood groups, in particular RH, KEL, FY, JK, DO and CO, 7) Implementation of allo-immunization research programs (cellular immunology and grafting issues). The CNRGS efficiency is based on the 'reference-research' link thanks to the Inserm partnership and direct applications to patients allowing to a better risk management and control.

The French reference laboratory for rare blood groups (CNRGS) is working for all participants of the transfusion chain: from the donors to the recipients; from the French Establishment for Blood to medical laboratories; from hospital to the haemovigilance network; from governmental agencies to European structures. This laboratory is in charge of: (1) studies of complex problems of immunohaematology; (2) studies of rare blood group phenotypes; (3) reagents quality controls; (4) production of biological standards; (5) specific specimen banks; (6) molecular studies of blood group antigens and antibodies involved; (8) panels of reference cells or DNA; (9) international exchanges.

Until the 1980s blood group reagents had been produced from human or animal plasmas. Since then, the main change has been the increase of the use of monoclonal antibodies in laboratory reagents. Today, they are the basis of most reagents for blood group typing. They include murine (hybridomas) and human (Epstein-Barr virus immortalized lymphocytes and phage display) antibodies. The use of these antibodies leads to standardized methods of production and a better definition of the specificity through international works. The main drawback is the lack of antibodies for some blood group antigens. However, in the future these methods will be confronted with the development of DNA-based methods.

The reagents used for blood group analyses are subject in France to the same regulations as other reagents. A file must be submitted for each reagent to the Agence française de sécurité sanitaire des produits de santé. Furthermore, each production lot must be checked by the Centre national de référence pour les groupes sanguins (CNRGS) for control before marketing. These controls allow a comparative quality assessment of the reagents. The investigation by the CNRGS of the problems encountered in the every day use of these products makes this quality control even more accurate.

The LW blood group is carried by a 42-kD glycoprotein that belongs to the family of intercellular adhesion molecules. The LW gene is organized into three exons spanning an HindIII fragment of approximately 2.65 kb. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members except that the signal peptide and the first Ig-like domain are encoded by the first exon. The 5'UT region (nucleotides -289 to +9) includes potential binding sites for various transcription factors (Ets, CACC, SP1, GATA-1, AP2) and exhibited a significant transcriptional activity after transfection in the erythroleukemic K562 cells. No obvious abnormality of the LW gene, including the 5'UT region, has been detected by sequencing polymerase chain reaction-amplified genomic DNA from RhD+ or RhD- donors and from an Rhnull variant that lacks the Rh and LW proteins on red blood cells. However, a deletion of 10 bp in exon 1 of the LW gene was identified in the genome of an LW (a- b-) individual (Big) deficient for LW antigens but carrying a normal Rh phenotype. The 10-bp deletion generates a premature stop codon and encodes a truncated protein without transmembrane and cytoplasmic domain. No detectable abnormality of the LW gene or transcript could be detected in another LW(a- b-) individual (Nic), suggesting the heterogeneity of these phenotypes.

Erythrocyte blood group-reference laboratory activities have to develop in accordance with new scientific knowledge brought by molecular biology. Reference laboratories will have to use immunology as well as molecular biology for a long time yet; both approaches are complementary. Recombinant cells expressing specific antigens will thus be used in parallel to red blood cells with selected phenotype combinations. To be efficient, Reference laboratory activities must be performed together with research and education activities with a view to spreading knowledge within a national network.

A patient suffering from chronic hepatitis exhibited severe transfusion reactions after administration of fresh frozen plasma and a plasma fraction: PPSB (prothrombin complex concentrate). 1 month before these reactions, she received fresh frozen plasma during plasma exchange therapy. The patient's serum obtained 1 week and 6 months after the second reaction gave a precipitation arc against PPSB preparations when examined by double-diffusion technique in agarose gel. An antibody of IgG class present in these sera reacted with a purified preparation of the fourth complement component (C4). This was demonstrated by various experiments (protein A radioimmunoassay and passive hemagglutination) using purified C4 as antigen. The antibody had a limited specificity and reacted only with C4 of Rodgers specificity. Phenotype determination of the patient's C4 group showed that she was Chido positive and Rodgers negative. Her HLA group was A1, Aw30; B8,-; DR3,-. The patient had neither detectable anti-IgA nor other anti-immunoglobulin antibodies. She had not received blood or plasma transfusion before her hepatitis. The coexistence of a precipitating anti-C4 antibody and adverse transfusion reactions to plasma fractions containing large amounts of C4 indicates that in the absence of antibodies of other specificities, this antibody can be considered as the cause of the transfusion reaction.

Fya antibodies were detected in 5 Fy(a-b-) black patients during a 5-year period. The Duffy phenotype was confirmed using anti-Fya, anti-Fyb, and anti-Fy3 by an adsorption-elution technique. These 5 patients had sickle cell disease and had received multiple blood transfusions. The antibodies were all found during the investigation of a hemolytic transfusion reaction or failure to achieve the desired red cell increments after transfusion.

Abstract: Aim of the study: Determination of blood group antigens from data obtained by using molecular methods (genotyping) has become an indispensable tool in the specialized immunohematology laboratories. The French National Reference Centre for Blood group typing (CNRGS) routinely performs genotyping of the FY, JK and MNS system (common genotyping), providing a phenotype deduced from genotyping data for FY1, FY2, JK1, JK2, MNS3 and MNS4 antigens. Patients and methods: We performed a study to evaluate the common genotyping prescriptions referred to the CNRGS over the last three years. Results: Between February2006 and February2009, the CNRGS performed 2392 genotyping, including 981 common genotyping. Analysis of 172 common genotyping performed in 2008 showed that 63.8% of the prescriptions expressed a genotyping demand. Of the latter, 42.7% were genotyping prescriptions only, whereas 57.2% were prescriptions of genotyping associated with alloantibody identification. All prescriptions refer to blood group genotyping indications issued from guidelines, with no incorrect prescription, that are patients transfused within four months before blood sampling in 63.6% of cases or a positive direct antiglobulin test in 24.5% of cases. Lastly, 36% of the blood samples referred to the CNRGS had no genotyping prescription. Yet, common genotyping was performed by the CNRGS to get complete immunohematology data for antibody identification. Conclusion: Usefulness of blood group genotyping in specialized immunohematology laboratories is obvious. However, the strategy for implementation of molecular methods remains to be defined. Use of high-throughput DNA analysis should change our way of working. [Copyright &y& Elsevier]