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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.

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.

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.

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.