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Red blood cell allo-immunization is the immune response of an individual to foreign red blood cell antigens not present on the surface of their own cells. The aim of that paper is to clarify the different factors influencing the antibody response against red blood cell antigens., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

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.)

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 February 2006 and February 2009, 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.

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.

One hundred and forty five Mabs against RH antigens were tested. In this paper, we chose to detail reactivity of MoAbs directed against variant RBCs of the CNRGS collection for which we studied the molecular background. Because we developed procedures to identify variants of the RhD, RhC, RhE and Rhe antigens, we were especially interested in finding new monoclonal antibodies that could help us to characterize more accurately these variants. Therefore, we drew parallels between our procedures and results obtained with the 2001 workshop antibodies.

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.

Fifty-seven IgG monoclonal anti-D antibodies were evaluated in the Rh flow cytometry section, in which 12 laboratories participated. Staining protocols and a fluorescein (FITC)-conjugated Fab fragment goat anti-human IgG (H + L) as a secondary antibody were recommended but not mandatory. A CcDEe red blood cell (RBC) sample that was shown to be homozygous for RHD by molecular methods was supplied and used as internal 'standard RBC' throughout all experiments. An RBC panel comprising two partial D and four weak D types was supplied as well. The use of standard RBC reduced the variability of the data among the laboratories and allowed the conversion of fluorescence data into epitope densities, which were compounded in an antigen density (antigen D per RBC). The highest antigen density was determined for DVI type III, followed by DVII and weak D type 3; the lowest antigen density were determined for weak D type 1 and type 2. Nine of the 12 participating laboratories discriminated three groups of aberrant RhD that had similar Rhesus indices (RI): D category VI with RI = 0; weak D type 2 and type 3 with an high RI; and D category VII and weak D type 1 with an intermediate RI. The antigen densities and the Rhesus indices obtained correlated well among the laboratories of this Workshop section despite different staining protocols, secondary antibodies and instrumentation.

Immunoglobulin preparations of anti-D (RH1) are injected to prevent haemolytic disease of the newborn. Such preparations are obtained by the fractionation of plasma from immunized donors. Measurement of the concentration of IgG anti-D is required to estimate the potency of anti-D preparations and sera from immunized donors. We have developed an ELISA method for the quantification of IgG anti-D. This method included the following steps, sensitization of red cells by anti-D, solubilization of red cell membranes by Triton, and eventually, measurement of IgG anti-D concentration by ELISA. The international reference preparation of anti-D (68/419) was used as a reference. With this method, we measured IgG anti-D concentrations in 5 immunoglobulin preparations of anti-D and in the sera of 10 donors immunized by D antigen. The ELISA results were compared with those obtained by automated hemagglutination. A mean anti-D concentration of 56.2 micrograms/mL was found by ELISA in immunoglobulin preparations. Similar results were obtained by automated hemagglutination (mean 52 micrograms/mL). In the sera of 10 D-immunized donors, anti-D IgG concentration varied from 2.2 to 59.8 micrograms/mL. A good correlation between ELISA and automated hemagglutination was observed in these sera (r = 0.98, p < 10(-7)). In conclusion, the ELISA technique offers an alternative to automated hemagglutination. It requires only the standard equipment necessary for immuno-enzymatic methods.

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 immunologic risk associated to erythrocyte transfusions is bound to the polymorphism of blood group systems and to the respect of blood transfusion regulations. The results of three studies are presented, which were carried out respectively by the French Society of Blood Transfusion, the National Institute of Blood Transfusion and the National Haemovigilance Network. Two hundred and twenty-seven cases of immunologic accidents are analysed using the Kaplan's interpretation model and the traditional method of process analysis. The results show three critical factors in the occurrence of this type of incident: the relevance of the clinical examinations prescribed, the way in which the biological results are taken into account, and the relationship/exchange of information between private and public hospitals, and blood transfusion centers.

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 national reference Center for blood groups checked samples of reagents and devices used in France for a definitive verification of pretransfusion ABO tests performed at the patient's bedside, as defined by French health authority regulations. The results of an initial inquiry was published in 1991. The new study shows no significant improvement of the quality of reagents and devices. This is a major concern considering the importance of ABO incompatibility in severe hemolytic transfusion reactions.

Until the '80s blood group reagents were composed of human or animal polyclonal antibodies; nowadays they are mainly produced from monoclonal antibodies. In this paper two aspects will be considered; firstly the evolution in the use of monoclonal reagents in France and secondly the quality of these new reagents in comparison with polyclonal reagents. From 1981 to 1995, 17567 batches of blood group reagents were analyzed and controlled by the French Blood Group Reference Laboratory (CNRGS). All data are given in six tables.

The immunological risk of red blood cell transfusions now seems higher than the viral risk. According to studies, severe accidents due to blood incompatibility occur with a frequency estimated at 1/6000 to 1/29000; despite technical progress, the risk does not significantly diminish. The majority of accidents do not originate from laboratory or production stages but from defects in the application of clinical procedures. Preventive measures are based on (i) the elaboration of clinical guidelines, (ii) the compliance to strict rules in carrying out bedside ABO check, and (iii) the realization and interpretation of antibody screening tests. The implementation of quality assurance systems and of the epidemiological surveillance system, which define the basis of a prevention policy, leads to the expectation of an improvement of transfusion safety.

Immunological transfusion reactions more than often lead to an activation of the complement proteins and mononuclear cells, inducing a haemolysis from which stem the observed clinical symptoms. In the case of incompatibility, the alloantibodies can lead to an immediate reaction, taking place in the first few minutes or, in the case of a delayed reaction, arising after 24 hours. A standardized clinical and biological evaluation is necessary in order to confirm the diagnosis and to assess the consequences of the antigen-antibody conflict.

IgG non anti-RH1(D) monoclonal Rh antibodies were evaluated by flow cytometry. The values obtained with these antibodies were less strong than those obtained with anti-RH1(D) antibodies. For a significant number of antibodies, the signal was not high enough to give reliable results for the antibody specificity. Despite these drawbacks, flow cytometry was an efficient tool to appreciate the variation of reactivity by different antibodies with normal or variant cells. These variations were not always obvious by serological means.

In this paper we chose to emphasize three aspects of our work. First we underlined that "low grade and high grade" D weak red blood cells studied at the DNA level could, when monoclonal antibodies were used, give patterns of positive and negative reactions like partial RH1(D) cells. Secondly, we showed the importance of the technical conditions of the study which are essential for establishing a pattern of reactivity defining an epitope. It appears that the use of papain treated cells at room temperature can be misleading for the definition of epitope especially with IgM antibodies. Lastly we pointed out the interest of Rh variant cells, defined at the gene level, to study the expression of RH1(D) epitopes on the external part of the membrane.

The Société Française de Transfusion Sanguine and the Centre National de Référence pour les Groupes Sanguins performed a quality control to evaluate the performances of two serological tests: the Direct Antiglobulin Test (DAT) and the Elution test. Among the 110 Blood Transfusion Centers participating in this control, 80 (73%) returned a result. Of these, 68 results were correct for the DAT (85%; positive for type IgG) and 31 results were correct for the elution (39%; anti-FY1). This control gave the opportunity to confirm the main procedures used in routine testing on a national scale. The analysis of the results underlines the importance of the choice of a standardized technique for these tests. Such controls are useful to appreciate the quality of the routine tests and to find the means to improve them.

Blood transfusion is mainly bound to immunological and infectious risks. The immunological risk originates from an incompatibility between the blood of the donor and that of the recipient; this risk remains insufficiently assessed. A multicentre study has been carried out by the French Blood Transfusion Society and the National Institute for Blood Transfusion. Sixty-one accidents due to an erythrocyte incompatibility were found: 26 cases with ABO incompatibility, and 35 cases with alloantibodies of other blood group systems. For the former category of accidents, the most frequent cause was due to a failure in the realization of the bedside ABO check. For the latter, the main problem was the achievement and the interpretation of antibody screening. The long term follow-up shows no chronic after-effects of immunological accidents. For each accident, errors have been identified and analysed. It was proven that they all originate from health care establishments.

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.

The serological investigation of auto-immune hemolytic anemias based on the direct antiglobulin test, the study of the serum and the eluate allows a classification of the auto-immune hemolytic anemia which is still valid and correlates well with the clinical features. The use of new techniques should increase the sensitivity of the tests but also stress the problem of the frontier between physiological and pathological states. The use of monoclonal antibodies of defined specificity would be an useful tool for the immunohematological classification of the auto-immune hemolytic anemias. The addition of new therapeutical means should improve the prognosis of these anemias. The increasing consumption of drugs and the development of the investigations of the adverse reactions including the hemolytic anemias have allowed the emergence of new pathogenic concepts. The gravity of the clinical evolution of some of these immune hemolytic anemias stress the necessity of an adequate therapeutic survey and the development of new means of diagnostic. The clinicians should be aware of the possibility of cross reactivity for drug dependent antibodies with other components of related chemical structure.

The general objective is the study, through scientific approaches, of the main components of immunological risks linked to red blood cell transfusions, as well as their consequences, in order to define precise rules for prevention, taking into account that 2,700,000 units were transfused in 1992. To reach this general aim, five intermediary objectives have to be achieved: 1) The sentinel study of the methodology used to collect information about transfusion accidents, as well as their identification and their early clinical expression; 2) The analysis of the occurrence mechanisms of incompatibilities and transfusion accidents; 3) The up-date definition of post-transfusion alloimmunization, in particular regarding 3 parameters: a) the immunogenicity of the different erythrocyte antigens, that will have to be reassessed; b) the modes of occurrence of post-transfusion anti-erythrocyte alloimmunization; c) the different types of chronology in the appearance and the persistence of anti-erythrocyte antibodies. 4) The search for significant criteria in order to assess the immunopathological correlations of the consequences of antigen-antibody conflicts; 5) The elaboration of the principles for tests evaluation and identification of the techniques linked to blood groups and the study of anti-erythrocyte antibodies.