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Abstracts section 1B
Transfus Clin Biol 2002 ; 9 : 43-3 © 2002 E´ditions scientifiques et médicales Elsevier SAS Tous droits réservés S1246782001002142/MIS
ABSTRACTS
Section 1B : Rh flow cytometry QUANTITATION OF IGG ANTI-D BOUND TO RED CELLS OF NORMAL AND WEAK D PHENOTYPE BY FLOW CYTOMETRY AND IAGT, USING A HIGH SENSITIVITY SENSITISATION PROTOCOL AND RECOMMENDED WORKSHOP PROTOCOLS B.M. KUMPEL*, D.J. JACKSON
INTERNATIONAL BLOOD GROUP REFERENCE LABORATORY, BRISTOL INSTITUTE OF TRANSFUSION SCIENCES, SOUTHMEAD ROAD, BRISTOL BS10 5ND, UK *Correspondence and reprints : [email protected] A comparison of methods for sensitising cells was made. Using the recommended protocols for sections 1A and 1B of the 4th Workshop, negative reactions were obtained with D category VII cells by the indirect antiglobulin test using an anti-D of low concentration and affinity, 1-50 (JAC10, BRAD-7). This MAb was positive with these cells using a highsensitivity method. By flow cytometry, only the latter method detected antibody bound to weak D type 1 cells. Incorrect assignments of epitope specificity may thus be made unless long incubation times and antibody excess are used for sensitising cells. Standard curves of IgG1 and IgG3 anti-D were constructed by analysing samples by both flow cytometry and SolELISA so that the number of molecules of IgG bound to the test cells could be determined. The amount of antibody bound to weak D and partial D cells by the MAbs submitted to section 1B was determined by flow cytometry using the high-sensitivity protocol. With few exceptions, all MAbs bound to the weak D and D category VII cells supplied, up to a maximum of 2000 (weak D) and 9000 (D category VII), molecules of IgG/cell. There was no correlation between the IgG concentration of the individual Mabs and the amount of IgG bound to cells. © 2002 Éditions scientifiques et médicales Elsevier SAS
RESULTS OF FLOW CYTOMETRY TESTING OF DCS AND TWO OTHER ATYPICAL RHD VARIANTS M. PÍSAÈKA, Y. MARINOV, H. FLÍDROVÁ
INSTITUTE OF HAEMATOLOGY AND BLOOD TRANSFUSION, PRAGUE, CZECH REPUBLIC. Three RhD variants (DCS, D-K and D-W) with a serological and molecular pattern different from published types were tested by flow cytometry with IgG anti-D monoclonal antibodies from the 4th workshop. Results of the median of fluorescence and calculated antigen densities show significant differences in comparison with D weak types 1, 2 and 3 and with D variants category VII and VI – type III. These results show that DCS, D-K and D-W erythrocytes are carrying different RhD variant proteins. © 2002 Éditions scientifiques et médicales Elsevier SAS
WEAK D AND VARIANT D PHENOTYPE STUDIES BY FLOW CYTOMETRY R.R. RUPREHT, M. URBAJS, K. PRETNAR-HARTMAN, P. ROZ{MAN, V. C { URIN-S {ERBEC* BLOOD TRANSFUSION CENTRE OF SLOVENIA, ŠLAJMERJEVA 6, SI1105 LJUBLJANA, SLOVENIA *Correspondence and reprints : [email protected]
In the present work we show results of flow cytometry testing of weak D and variant D erythrocytes. Antigen D densities of weak D type 1, 2 and 3 were calculated and density profiles of 36 anti-RhD monoclonal antibodies were compared. IgG anti-Rh monoclonal antibodies were donated by the participants of the 4th International Workshop on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens. Erhythrocytes of molecularly-defined weak D type 1, 2 and 3, partial DVII, and partial DVItypeIII, as well as control CcDEe RBC sample were supplied by Dr. W. Flegel or found locally. The RBC samples were also directly labelled with FITC-conjugated-BRAD-3 antibody and tested by flow cytometry. © 2002 Éditions scientifiques et médicales Elsevier SAS
RH MONOCLONAL ANTIBODY EVALUATION BY FLOW VYTOMETRY R. FONTÃO-WENDEL*, C. A.DE BRITO, L. C. NOGUEIRA DA SILVA, S. WENDEL HOSPITAL SÍRIO LIBANÊS – BLOOD BANK, R. D. ADMA JAFET, 91 01308050, SÃO PAULO, BRAZIL *Correspondence and reprints : [email protected]
A total of 52 monoclonal antibodies of the RH system (46 anti-D, one anti-C, one anti-D/G, one anti-G, three anti-E) (section 1B) were tested by flow cytometry against eight red cells of the following phenotype : one CcDEe, one ccddee, one DVI type III, one DVII, and four weak Ds (type 1, 2 and 3). For the non-D MoAbs, the following red cells were also tested : two ccddEe G +, one ccddee G + against anti-G ; CCddEe, ccDEwe, CCDEe against anti-E ; and RH 48, c(C)DE(e), and CCDEe against anti-C. For the anti-Ds tested only one (2.2 %) (1-145) failed with all red cells tested and two (4.4 %) reacted only weakly with the positive control. Five (11 %) reacted with all red cells tested. Eight (17.4 %) of the anti-Ds tested reacted against DVI cells while 42 (91 %) reacted against DVII. For the weak Ds tested the reactivity of the MoAbs were similar except for weak D type 1 donor B, where 11 (24 %) failed. The epitope numbers for the red cells tested, based on median fluorescence calculated by an Excel file provided by the Workshop organizers, were similar with previous data except for weak D type 2. For the non-D MoAbs tested a poor discrimination was observed with the red cells tested, except for 1 anti-E (1-126). © 2002 Éditions scientifiques et médicales Elsevier SAS
ABSTRACTS
Section 1B : Rh flow cytometry QUANTITATION OF IGG ANTI-D BOUND TO RED CELLS OF NORMAL AND WEAK D PHENOTYPE BY FLOW CYTOMETRY AND IAGT, USING A HIGH SENSITIVITY SENSITISATION PROTOCOL AND RECOMMENDED WORKSHOP PROTOCOLS B.M. KUMPEL*, D.J. JACKSON
INTERNATIONAL BLOOD GROUP REFERENCE LABORATORY, BRISTOL INSTITUTE OF TRANSFUSION SCIENCES, SOUTHMEAD ROAD, BRISTOL BS10 5ND, UK *Correspondence and reprints : [email protected] A comparison of methods for sensitising cells was made. Using the recommended protocols for sections 1A and 1B of the 4th Workshop, negative reactions were obtained with D category VII cells by the indirect antiglobulin test using an anti-D of low concentration and affinity, 1-50 (JAC10, BRAD-7). This MAb was positive with these cells using a highsensitivity method. By flow cytometry, only the latter method detected antibody bound to weak D type 1 cells. Incorrect assignments of epitope specificity may thus be made unless long incubation times and antibody excess are used for sensitising cells. Standard curves of IgG1 and IgG3 anti-D were constructed by analysing samples by both flow cytometry and SolELISA so that the number of molecules of IgG bound to the test cells could be determined. The amount of antibody bound to weak D and partial D cells by the MAbs submitted to section 1B was determined by flow cytometry using the high-sensitivity protocol. With few exceptions, all MAbs bound to the weak D and D category VII cells supplied, up to a maximum of 2000 (weak D) and 9000 (D category VII), molecules of IgG/cell. There was no correlation between the IgG concentration of the individual Mabs and the amount of IgG bound to cells. © 2002 Éditions scientifiques et médicales Elsevier SAS
RESULTS OF FLOW CYTOMETRY TESTING OF DCS AND TWO OTHER ATYPICAL RHD VARIANTS M. PÍSAÈKA, Y. MARINOV, H. FLÍDROVÁ
INSTITUTE OF HAEMATOLOGY AND BLOOD TRANSFUSION, PRAGUE, CZECH REPUBLIC. Three RhD variants (DCS, D-K and D-W) with a serological and molecular pattern different from published types were tested by flow cytometry with IgG anti-D monoclonal antibodies from the 4th workshop. Results of the median of fluorescence and calculated antigen densities show significant differences in comparison with D weak types 1, 2 and 3 and with D variants category VII and VI – type III. These results show that DCS, D-K and D-W erythrocytes are carrying different RhD variant proteins. © 2002 Éditions scientifiques et médicales Elsevier SAS
WEAK D AND VARIANT D PHENOTYPE STUDIES BY FLOW CYTOMETRY R.R. RUPREHT, M. URBAJS, K. PRETNAR-HARTMAN, P. ROZ{MAN, V. C { URIN-S {ERBEC* BLOOD TRANSFUSION CENTRE OF SLOVENIA, ŠLAJMERJEVA 6, SI1105 LJUBLJANA, SLOVENIA *Correspondence and reprints : [email protected]
In the present work we show results of flow cytometry testing of weak D and variant D erythrocytes. Antigen D densities of weak D type 1, 2 and 3 were calculated and density profiles of 36 anti-RhD monoclonal antibodies were compared. IgG anti-Rh monoclonal antibodies were donated by the participants of the 4th International Workshop on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens. Erhythrocytes of molecularly-defined weak D type 1, 2 and 3, partial DVII, and partial DVItypeIII, as well as control CcDEe RBC sample were supplied by Dr. W. Flegel or found locally. The RBC samples were also directly labelled with FITC-conjugated-BRAD-3 antibody and tested by flow cytometry. © 2002 Éditions scientifiques et médicales Elsevier SAS
RH MONOCLONAL ANTIBODY EVALUATION BY FLOW VYTOMETRY R. FONTÃO-WENDEL*, C. A.DE BRITO, L. C. NOGUEIRA DA SILVA, S. WENDEL HOSPITAL SÍRIO LIBANÊS – BLOOD BANK, R. D. ADMA JAFET, 91 01308050, SÃO PAULO, BRAZIL *Correspondence and reprints : [email protected]
A total of 52 monoclonal antibodies of the RH system (46 anti-D, one anti-C, one anti-D/G, one anti-G, three anti-E) (section 1B) were tested by flow cytometry against eight red cells of the following phenotype : one CcDEe, one ccddee, one DVI type III, one DVII, and four weak Ds (type 1, 2 and 3). For the non-D MoAbs, the following red cells were also tested : two ccddEe G +, one ccddee G + against anti-G ; CCddEe, ccDEwe, CCDEe against anti-E ; and RH 48, c(C)DE(e), and CCDEe against anti-C. For the anti-Ds tested only one (2.2 %) (1-145) failed with all red cells tested and two (4.4 %) reacted only weakly with the positive control. Five (11 %) reacted with all red cells tested. Eight (17.4 %) of the anti-Ds tested reacted against DVI cells while 42 (91 %) reacted against DVII. For the weak Ds tested the reactivity of the MoAbs were similar except for weak D type 1 donor B, where 11 (24 %) failed. The epitope numbers for the red cells tested, based on median fluorescence calculated by an Excel file provided by the Workshop organizers, were similar with previous data except for weak D type 2. For the non-D MoAbs tested a poor discrimination was observed with the red cells tested, except for 1 anti-E (1-126). © 2002 Éditions scientifiques et médicales Elsevier SAS