[Red-blood-cell allo-immunization]. / Allo-immunisation anti-érythrocytaire.

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.

Blood group genotyping by high-throughput DNA analysis: application to the French panel of RBC reagents.

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.

Transfusion of rare cryopreserved red blood cell units stored at -80 degrees C: the French experience.

The technology allowing freezing of RBC units has been available for many decades. The high-glycerol method for RBC storage at -8 degrees C is predominantly used. Several studies have shown satisfactory results regarding the in vitro viability and function of cryopreserved RBCs. RBC freezing is nowadays mostly encountered in rare blood programs and military deployments. Preservation time of frozen RBCs appears to be virtually indefinite, but most countries apply a 10-year outdate. There is no mandatory time restriction in France. The National Rare Blood Bank currently includes 962 (17.5%) RBC units aged to years or more and 153 (2.8%) aged 20 years or more. Since 1994, 1957 RBC units have been thawed and transfused, among which 118 were aged 10 years or more and 8 were aged 20 years or more. Discarding RBC units older than to years may be highly sensitive for very rare blood groups, e.g., U-, of which approximately 30 percent of the cryopreserved units are aged to years or more. However, the lack of nucleic acid testing for HIV and HCV may be problematic for old RBC units drawn from donors who were not subsequently tested for these markers, which is now mandatory in most countries. Regarding the 118 transfused RBC units older than 10 years, no evidence of hemolysis of thawed RBCs and no transfusion reaction, clinical or biologic hemolysis, or transfusion ineffectiveness was reported, either by any of the parties involved in the transfusion supply of rare RBC units or through the French hemovigilance program, which requires a mandatory report of any transfusion reaction. It has recently been suggested to extend the 10-year restriction in some countries. Considering our experience and observational data, we may consider it safe and efficient to transfuse rare frozen RBC units older than 10 years. An international consensus for RBC cryopreservation time should ideally be established.

[Genotyping of blood group systems at the CNRGS. I: FY, JK, MNS systems]. / Génotypage des systèmes de groupe sanguin d'intérêt transfusionnel au CNRGS. I: les systèmes FY, JK, MNS.

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.

[The rare blood groups: a public health challenge]. / Les phénotypes érythrocytaires rares: un enjeu de santé publique.

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.

FY*X real-time polymerase chain reaction with melting curve analysis associated with a complete one-step real-time FY genotyping.

BACKGROUND AND OBJECTIVES: The Duffy (FY) blood group system is controlled by four major alleles: FY*A and FY*B, the Caucasian common alleles, encoding Fy(a) and Fy(b) antigens; FY*X allele responsible for a poorly expressed Fy(b) antigen, and FY*Fy a silent predominant allele among Black population. Despite the recent development of a real-time fluorescent polymerase chain reaction (PCR) method for FY genotyping FY*X genotyping has not been described by this method. This study focused on the real-time FY*X genotyping development associated with a complete, one-step real-time FY genotyping, based on fluorescence resonance energy transfer (FRET) technology. MATERIALS AND METHODS: Seventy-two blood samples from Fy(a+b-) Caucasian blood donors were studied by real-time PCR only. Forty-seven Caucasian and Black individual blood samples, referred to our laboratory, were studied by PCR-RFLP and real-time PCR. For each individual, the result of the genotype was compared to the known phenotype. RESULTS: The FY*X allele frequency calculated in an Fy(a+b-) Caucasian blood donors population was 0.014. With the Caucasian and Black patient samples we found a complete correlation between PCR-RFLP and the real-time PCR method whatever the alleles combination tested. When the known phenotype was not correlated to FY*X genotype, the presence of the Fy(b) antigen was always confirmed by adsorption-elution. CONCLUSION: The real-time technology method is rapid and accurate for FY genotyping. From now, we are able to detect the FY*X allele in all the alleles combinations studied. Regarding its significant frequency, the detection of the FY*X allele is useful for the correct typing of blood donors and recipients considering the therapeutic use of blood units and the preparation of test red blood cells for antibody screening.

[Quality control: sensitivity of the indirect antiglobulin test by filtration]. / Evaluation externe de la qualité: étude de la sensibilité du procédé de filtration en test indirect à l'antiglobuline.

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.

[Annual Report 2004 - French National Reference Centre for Rare Blood Groups and Immunohaematology (CNRGS)]. / Rapport 2004 - Centre national de référence pour les groupes sanguins (CNRGS).

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.

Serological studies of monoclonal RH antibodies with RH1 (D), RH2 (C), RH3 (E) and RH5 (e) variant RBCs.

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: activities in 2001]. / Le Centre national de référence pour les groupes sanguins (CNRGS): activité 2001.

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.

Section 1B: Rh flow cytometry. Coordinator's report. Rhesus index and antigen density: an analysis of the reproducibility of flow cytometric determination.

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.

Two new alleles of the RHCE gene in Black individuals: the RHce allele ceMO and the RHcE allele cEMI.

Six unrelated individuals of Afro-Caribbean origin, whose red cells have a marked reduction of the Rhe antigen expression, have been identified. All exhibited the same serological profile with anti-e monoclonal antibodies and lacked expression of the high frequency e-related antigen hrS. Transcripts and genomic analysis showed that these phenotypes resulted from the presence of two new RHCE alleles, ceMO and cEMI. The ceMO allele corresponded to a RHce gene carrying a G667T mutation (exon 5) and was detected at the homozygous state in sample 1 and at the heterozygous state in samples 2-6. The G667T mutation resulted in a Val223Phe substitution on the Rhce polypeptide, in close proximity to Ala226 (e-antigen polymorphism), which might account for the altered expression of e. The ceMO allele is also associated with the lack of expression of the hrS antigen. The absence of the hrS antigen expression may have implications in transfusion as hrS-negative individuals may develop clinically significant antibodies. The cEMI allele corresponded to a silent RHE allele carrying a nine nucleotide deletion within exon 3 and was detected at the heterozygous state in sample 2. This deletion resulted in a shortened polypeptide of 414 residues (instead of 417) that was absent (or severely reduced) at the red cell surface, as the E antigen was undetectable using serology and Western blot analysis with anti-E reagents. In DNA-based polymerase chain reaction genotyping for RHE determination, the cEMI allele provided a false positive result as the cells carrying this allele are serologically phenotyped as E-negative. The incidence of this allele in the Black population is unknown but, as shown already for D genotyping, one must exercise caution when genotyping is performed to detect the e/E polymorphism.

[Quantitative estimation by ELISA of IgG anti-D (RH1) antibodies in immunoglobulin preparations and in the sera of immunized donors]. / Dosage par une technique ELISA des anticorps anti-D (RH1) dans les préparations d'immunoglobulines et dans le sérum de donneurs immunisés.

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.

[External quality evaluation]. / Evaluation externe de la qualité.

Since ten years, the immunohaematology working group of the French Society of Blood Transfusion has organized a quality control. Tests concern essentially the screening and identification of irregular antibodies, direct antiglobulin tests and elutions.

[Trends in erythrocyte immunohematology reagents]. / Evolution des réactifs en immunohématologie érythrocytaire.

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.

Haemovigilance and transfusion safety in France.

The risks associated to red cell and platelet transfusions are essentially bound to the polymorphism of blood group antigens and to transfusion transmitted agents including virus, bacterias.... In France, the haemovigilance system and several investigations allowed to measure these different kinds of risks. We also developed analysis of failures in order to prevent errors and accidents to increase blood safety.

[Immunologic risk analysis of blood transfusion: 1991-1998]. / Analyse des risques immunologiques en transfusion sanguine: période 1991-1998.

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.

[Quality control of reactant lots at the Nation Blood Group Reference Center (CNRGS). Data for the year 1998]. / Les contrôles de lots de réactifs au Centre National de Référence des Groupes Sanguins (CNRGS). Données de l'année 1998.

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.