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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.)

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.

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.

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.

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.

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.

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.

Alloimmunization following multiple transfusions, a frequently observed complication in sickle cell anemia patients, may make subsequent transfusion attempts extremely hazardous. The case of a young woman with sickle-cell anemia is reported; she was hospitalized for the treatment of extensive invalidating leg ulcers. The transfusion program that was initiated as a prerequisite to skin allografts had to be stopped, due to rapid occurrence of multiple alloimmunization. Broad spectrum alloantibodies precluded further selection of compatible blood units. In an attempt to get round this impossibility, a treatment with Hydroxyurea was initiated in order to boost synthesis of F-hemoglobin. After one year under this treatment, the patient's clinical status has clearly improved without requiring any new blood transfusion.

The serological investigation of immunohaemolytic anaemias is based on the direct antiglobulin test, on the study of the serum and on the eluate, thus allowing a classification, which is still valid and correlates well with the clinical features. The use of more sensitive techniques and of reagents made from monoclonal antibodies should improve the study of these anaemias, but emphasizes the issue of the limits between physiological and pathological states. The addition of new therapeutical approaches should improve their prognosis. The increasing consumption of drugs and the development of the investigations of the adverse reactions including the haemolytic anaemias have allowed the emergence of new pathogenic concepts. The severity of the course of some of these immune haemolytic anaemia stresses the importance 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 chimical structure.

The use of red blood cells coated with complement fractions using low ionic strength saline conditions is part of the evaluation of anti-complement antibodies, specially monoclonal antibodies used for antiglobulin reagents. This work shows that such cells stored in liquid nitrogen allow satisfactory long-term studies except for the anti-C3g antibodies.

In 1987-1988 the National Reference Center for Blood Groups checked samples of reagents and cards utilized in ultimate pretransfusion tests at the patient's bedside as described in the circular published on the 17th of May (1985) by the "Direction Générale de la Santé". This inquiry shaved some deficiencies concerning the storage conditions, the patency of the reagents and the absence of anti-A + B reagent. Taking these results into account, some modifications are considered in order to improve the realization of these tests which are regarded in France as the last major step for the security of the red blood cells transfusion.

The red blood cells and sera from 21 RN/RN individuals were studied. The study confirmed the Rh type D+C+E-c-e+, CW-, characterized by an increased expression of the D antigen, a markedly decreased expression of the C and e antigens, the presence of a low incidence antigen (Rh32) and the absence of a high incidence antigen (Rh46) associated with an epitope recognized by a murine monoclonal antibody (MR 432). Four individuals exposed to Rh46 cells by pregnancy and/or transfusion had an anti-Rh46 antibody. This antibody gave positive reactions with all red blood cells of common and rare Rh phenotype except Rh null, D--, D.., DC(W-) and RN/RN cells. This antibody is considered to be of clinical significance in case of transfusion or pregnancy.