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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.

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.

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.

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.

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.

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.

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 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.

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.

Background: Anti-D immunoglobulin preparations are injected to prevent hemolytic disease of the newborn. The concentration of IgG anti-D in these preparations is usually determined by an automated hemagglutination technique using as a reference a calibrated preparation of anti-D, but the method requires special equipment and cannot be routinely applied to measure the IgG subclasses of anti-D in these preparations., Study Design and Methods: Taking advantage of a recently described enzyme-linked immunosorbent assay (ELISA) for the determination of the anti-D concentration in sera of alloimmunized pregnant women, IgG anti-D and IgG subclass concentrations were measured in the international reference preparation (IRP) coded 68/419, 10 anti-D immunoglobulin preparations, and sera of 15 D-immunized volunteers., Results: An IgG anti-D concentration of 61.5 +/- 4.8 microg per ampoule (mean +/- SD) was found by ELISA in IRP 68/419. This result was in agreement with previous determinations obtained by radioimmunoassay (60 microg/ampoule). The IgG subclass concentration of anti-D in this preparation was 48.4 microg of IgG1 (78.6%), 3.0 microg of IgG2 (4.8%), 9.7 microg of IgG3 (15.8%), and 0.4 microg of IgG4 (0.7%). The mean proportion of IgG subclasses of anti-D in 10 immunoglobulin preparations was similar (81.7% for IgG1, 5.0% for IgG2, 12.7% for IgG3, and 0.6% for IgG4). In the sera of 15 immunized volunteers, the IgG anti-D concentration varied from 3.1 to 68.4 microg per mL. The mean IgG subclass composition of anti-D was 79.3 percent for IgG1, 2.2 percent for IgG2, 18.1 percent for IgG3, and 0.4 percent for IgG4. The proportions of IgG3 anti-D in these sera were found to range between 1 percent and 87 percent, as in the sera of D-alloimmunized pregnant women., Conclusion: ELISA provides an alternative to the radioimmunoassay and the automated hemagglutination technique. In addition, it allows the evaluation of the absolute concentration of each IgG subclass of anti-D in immunoglobulin preparations and necessitates only the conventional equipment required for an immunoenzymatic assay.

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.

After testing red cells from 12 RhE variants with a panel of anti-E monoclonal antibodies (MoAbs), four patterns of reactivity were detected indicating that the MoAbs may recognize four distinct E epitopes designated epE1, epE2, epE3 and epE4. The variants were classified into four categories (cat EI to EIV) which carried epE1 and epE2, epE1 and epE4, epE1, epE3 and epE4, and all four epitopes, respectively. Molecular analysis of the transcripts and genomic DNA of the variants from cat EI, EII and EIII displayed three distinct genetic alterations. Cat EI variants exhibited a point mutation (T500A) in exon 4 of the RHCE gene that resulted in a Met167Lys substitution in the third extracellular loop of the RhcE protein. Cat EII variant carried a hybrid gene structure characterized by replacement of exons 1-3 (or 2-3) of the RHCE gene by their specific counterparts in the RHD gene. This latter variant was also associated with a weak expression of the RhC antigen. In cat EIII variants there was a partial DNA exchange of exon 5 sequences (nt 697 and 712) between the RHCE and the RHD genes, generating a hybrid Rh cE-D-cE protein carrying the Glu233 and Val238 substitutions. The serological and molecular studies of the RhE variants indicated that: (i) the RhE antigen is a mosaic composed of at least four epitopes and proline at position 226 is necessary but not sufficient for the full expression of the E antigen, (ii) the lack of RhE epitope(s) is associated with heterogenous molecular alterations of the RHCE gene, and (iii) amino-acids located on the third and fourth extracellular loops of the RhCE polypeptide are critical for some RhE epitopes expression.

The Duffy (FY) blood group antigens are carried by the DARC glycoprotein, a widely expressed chemokine receptor. The molecular basis of the Fya/Fyb and Fy(a-b-) polymorphisms has been clarified, but little is known about the Fyx antigen and the FY*X allele associated with weak expression of Fyb, Fy3, Fy5, and Fy6 antigens. We analyzed here the structure and expression of the FY gene in 4 Fy(a-bweak) individuals. As compared with Fy(a-b+) controls, the Fy(a-bweak) red blood cell membranes contained residual amount of DARC polypeptide and these cells were poorly bound by anti-Fy antibodies and chemokines. The FY gene from Fy(a-b+) and Fy(a-bweak) individuals differed by one substitution, C286T. The resulting Arg89Cys amino acid change reduced the binding of anti-Fy antibodies and chemokines to DARC transfectants. We concluded that the Fybweak donors carried the FY*X allele at the FY locus and that the Fyx antigen corresponds to highly reduced expression of a grossly normal Fyb polypeptide caused by the Arg89Cys substitution. Because FY is a single copy gene, this defect should also affect DARC expression in nonerythroid cells. Because the Fyx phenotype is not associated with apparent clinical consequences, we discussed these findings in the light of the putative roles of DARC in various tissues. Finally, we developed a Fyx DNA typing assay that should be useful for genetic studies and clinical transfusion medicine., (Copyright 1998 by The American Society of Hematology.)

The Kidd (JK) blood group is carried by an integral membrane glycoprotein which transports urea through the red cell membrane and is also present on endothelial cells of the vasa recta in the kidney. The exon-intron structure of the human blood group Kidd/urea transporter gene has been determined. It is organized into 11 exons distributed over 30 kilobase pairs. The mature protein is encoded by exons 4-11. The transcription initiation site was identified by 5'-rapid amplification of cDNA ends-polymerase chain reaction at 335 base pairs upstream of the translation start point located in exon 4. The 5'-flanking region, from nucleotide -837 to -336, contains TATA and inverted CAAT boxes as well as GATA-1/SP1 erythroid-specific cis-acting regulatory elements. Analysis of the 3'-untranslated region reveals that the two equally abundant erythroid transcripts of 4.4 and 2.0 kilobase pairs arise from usage of different alternative polyadenylation signals. No obvious abnormality of the Kidd/urea transporter gene, including the 5'- and 3'-untranslated regions, has been detected by Southern blot analysis of the blood of two unrelated Jknull individuals (B.S. and L.P.), which lacks all Jk antigens and Jk proteins on red cells, but was genotyped as homozygous for a "silent" Jkb allele. Further analysis indicated that different splice site mutations occurred in each variant. The first mutation affected the invariant G residue of the 3'-acceptor splice site of intron 5 (variant B.S.), while the second mutation affected the invariant G residue of the 5'-donor splice site of intron 7 (variant L.P.). These mutations caused the skipping of exon 6 and 7, respectively, as seen by sequence analysis of the Jk transcripts present in reticulocytes. Expression studies in Xenopus oocytes demonstrated that the truncated proteins encoded by the spliced transcripts did not mediate a facilitated urea transport compared with the wild type Kidd/urea transporter protein and were not expressed on the oocyte's plasma membrane. These findings provide a rational explanation for the lack of Kidd/urea transporter protein and defect in urea transport of Jknull cells.

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 RH blood group locus is composed of two sequence-related genes, RHD and RHCE, encoding the D, Cc, and Ee antigens in common Rh-positive phenotypes. In this report, we have analyzed the molecular basis of Rh antigens expression in weak D (Du) and RN donors, in whom there is a severe reduction of the D and C/e antigens, respectively. Genomic and transcript analysis of three unrelated low-grade weak D (Du) variants indicated that the very low expression of the D antigen is not the result of rearrangement or mutation in the coding sequence of the RHO gone. Accordingly, weak D (Du) erythrocytes should carry a normal RhD polypeptide, which is in agreement with the observation that these variants never produce anti-D antibodies. Comparative polymerase chain reaction analysis showed a lower steady-state level of RhD transcripts in weak D (Du) reticulocytes, as compared with normal RhD-positive controls, thus providing direct evidence that the difference between the D antigen of D-positive and weak D (Du) red blood cells is quantitative only. Conversely, analysis of the molecular genetic basis of the RN phenotype Indicated that the severely decreased expression of the RhC and Rhe antigens in three variants is associated with a qualitative alteration identified as a segmental DNA exchange between the RHCE and RHD genes. These genomic rearrangements, which resulted in hybrid RhCe-D-Ce proteins expressing the low frequency Rh32 but not the high incidence Rh46 antigens, involved either axon 4 alone or both exons 3 and 4. These findings show that an identical phenotypical alteration of Rh antigens (reduced expression) may result either from a quantitative or a qualitative alteration of the RH genes expression.

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.

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.

The sensitivity of the low ionic strength solution antiglobulin test (LISS-AGT), polyethylene glycol antiglobulin test (PEG-AGT), low ionic strength solution solid-phase antiglobulin test (LISS-SPAT), gel low ionic strength solution antiglobulin test (GEL-LISS), and gel papain test (GEL-PAP) was compared in titration studies of 460 sera containing identified IgG alloantibodies. The GEL-PAP was 100% sensitive to detect Rh antibodies, whereas the PEG-AGT was the most sensitive to detect Kell, Duffy, Kidd, Ss, and rare blood group antibodies. The better performance of PEG-AGT was especially obvious with Kell, Duffy, and Ss antibodies (S = 100%). When the sensitivity of the LISS-AGT, PEG-AGT, GEL-LISS, and GEL-PAP was evaluated in different routines, the GEL-LISS showed to be more sensitive than PEG-AGT in the detection of clinically significant antibodies. These discrepant results showed that the performance of a technique may change when it is applied as a routine.

The Rh blood group antigens are encoded by two highly related genes, RHD and RHCE, and the sequence of the common alleles (D, Ce, CE, ce, and cE) of these genes has been previously elucidated. In this report, Rh transcripts and gene fragments have been amplified using polymerase chain reaction from the blood of donors with the CW+ andCX+ phenotypes. Sequence analysis indicated that the expression of the CW (Rh8) and CX (Rh9) antigens are associated with point mutations in the RHCE gene, which provides the definitive evidence that the CW and CX specificities are encoded by the same gene as the Cc and Ee antigens. As compared with the common (CW- and CX-) transcripts of the RHCE gene, the CW+ and CX+ cDNAs exhibited A122G and G106A transitions that resulted in Gln41Arg and Ala36Thr amino acid substitutions in the CW+ and CX+ polypeptides, respectively. Therefore, although the CW and CX specificities behave serologically as if they were allelic, they cannot not be considered, stricto sensu, as the products of antithetical allelic forms of the RHCE gene. Based on the CW-/CW+ nucleotide polymorphism, a polymerase chain reaction assay useful for diagnosis purposes has been developed that detects the presence of the CW+ allele by the use of an allele-specific primer.

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 Rh (Rhesus) blood group antigens, D, Cc, and Ee, are carried by three unglycosylated membrane proteins of the human erythrocytes encoded by two highly related genes, D and CcEe. The major antigen, D, is a mosaic composed of at least nine determinants (epD1 through epD9). The lack of expression of some of these D epitopes at the surface of variant red blood cells defines the so-called D category phenotypes. In this report, we have determined the molecular basis of the DVI category phenotype characterized by the lack of epitopes D1, D2, D5, D6/7, and D8. Southern blot analysis and mRNA sequencing showed that the DVI phenotype is associated with two types of rearrangement of the D gene. Of 10 DVI genomes investigated, 8 exhibited a segmental DNA replacement (gene conversion) between the D fragment encompassing exons 4, 5, and 6 and the equivalent region of the CcEe gene. In the two other variants, these three exons are deleted. In both cases, the genomic rearrangement did not alter the reading frame of the variant RhD transcripts that are translated in 417 and 266 amino acid polypeptides, respectively. A heterogeneity of category DVI samples based on variable reactivity of the red blood cells with anti-D antibodies was previously found to be associated with the CDVIe or cDVIE haplotypes. Interestingly, our present results indicated that this serologic subdivision of the DVI category is correlated to two types of genomic rearrangements of the D gene.

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.

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.