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Comparative analysis of 4 HLA class II typing methods in prospective organdonor typing
110
Abstracts
C-6.1 #103
HLA CLASS II GENOTYPING, DONOR-SPECIFIC ANTIBODIES AND E A R L Y E P I S O D E S O F A C U T E R E J E C T I O N IN K I D N E Y TRANSPLANT. Adomo D., °PinzTU A., *Papola F., Canossi A., Romano P., °Torlone N., °Valeri M., Ozzella G., Liberatore G., Di Rocco M., °Poggi E. and °Casciani C. U. Istituto C.N.R. Tipi77uzione Tissutale e Problemi della Dialisi - L'Aquila, Italy °Clinica Chirurgica, Universit~ Tor Vergata - Roma, Italy *Centro Interregionale Riferimento Trapianti Abruzzo-Molise - L'Aquila, Italy Genomic typing was retrospectively performed in 32 cadaveric kidney transplant donorrecipient pairs. The occurrence of acute rejection episodes during early post-transplant 6 months was also evaluated. Patients were monitorized for donor-specific anti-HLA class II antibodies during the first year after transplantation, by flow cytometry (FCXM). DRB1 typing was performed by PCR-SSP analysis and PCR-SSO technique with biotinylated probes to define subtypes, DQA1 and DQB1 by SSP and confirmed by p32 labelled SSOs, DPB1 by radioisotopic PCR-SSO method. All episodes of rejection were hystologically confirmed and resolved by pulses of steroids. 9 out of 32 patients showed one episode of rejection. Of these, 5/14 showed o n e DRB1 mismatch (MM) and 4/15 two MMs; no rejection was observed in 3 pairs with identical DRB1, DQA1 and DQBI alleles. Positive post-Tx FCXM was observed in 14/32 recipients. In 1 of these, with z e r o M M for DRBI-DQA1DQB1 loci, the presence of anti-HLA class II antibodies was probably directed to DP antigens. An intriguing result was observed evaluating DRB1 alleles of recipients: 6 showed DRBI*0402; among these patients, 5 had episodes of acute rejection. This evidence seems to correlate DRBI*0402 allele with an higher response versus graft. No correlation was reported between degree of MM and the incidence of acute rejection or the occurrence of donor-specific antibodies. However, the role of HLA class II matching and biological importance of donor-specific antibodies need a greater number of cases and a longer followup.
C-6.1 #104
COMPARATIVE ANALYSIS OF 4 HLA CLASS II TYPING METHODS IN PROSPECTIVE ORGANDONOR TYPING. M.P. Emonds, A. Volckaerts, J. Dendievel, H. Claeys, C. Vermylen.BIoodbank Belgian Red Cross, O.L.Vrouwstraat 42, B-3000 Leuven. We evaluated classicalserology and One Lambda Monoclonal trays (MT) lot #1F in combination with a DRB INN0-LiPA and DRB PCR-SSP (CTS primer mixes) for its applicability in prospective donor typing for organtransplantation. 27 donor class II typings of donors offered during the working day between 5/1993 and ! 1/1993 are considered. Classical TCF on density gradient separated spleen cells was performed in parallel with DynabeadsR isolated spleen cells for MT analysis. Samples were analysed by two different technologists and read independently not knowing about the DNA typing results. A fast DNA extraction method (80 rain starting from whole blood) based on protein precipitation with perchlorate was used for DNA methods (BiozymR).All samples could be typed with a high accuracy in the 4 methods. Only 1 discrepancy in 54 antigens was found for classical serology (DR11 stead of DR1303). One DR 7 was missed in 54 antigens with the MT; extra reactions were obtained with DR18 and DQ4 sera. DR 2 could not be split but recognised correctly all DRI 5 and DR16 as DR2. No errors were obtained for PCR-SSP if weak extra bands with primer mix 5 and 16 were exduded. The higher resolution of INNO-LiPA made the interpretation of the INNO-LiPA difficult for 1 sample having an aberrant association of DR11 with DRB3*0101 but this haplotype typed as a dassical DR11 in serology and PCR-SSP. Conclusions: All four techniques are applicable for prospective donortyping. PCR-SSP (3-4h) and INNO-LiPA (4h) remain more labour intensive than serology. Moreover a double sample (DNA and living cells) has to be prepared as an HLA crossmatch remains to be done on isolated lymphocytes. Classical TCF takes 4h whereas MT can be read after 60 minutes if Dynabead separation is used. INNO-LiPA gives the most accurate results without extra reactions but due to its high resolution remains difficult to interprete if no key oligo's are selected for interpretation. PCR-SSP is a faster oligotyping technique more adapted for the single sample but needs an optimal sample preparation and PCR. The MT gives the fastest results (< 2h) if Dynabeads are used but here also optimalisation of the system is necessary to eliminate extra reaction and to subtype DR2 antigens. MT and genotyping systems have the supplemental advantage of standardisation.
Abstracts
C-6.1 #103
HLA CLASS II GENOTYPING, DONOR-SPECIFIC ANTIBODIES AND E A R L Y E P I S O D E S O F A C U T E R E J E C T I O N IN K I D N E Y TRANSPLANT. Adomo D., °PinzTU A., *Papola F., Canossi A., Romano P., °Torlone N., °Valeri M., Ozzella G., Liberatore G., Di Rocco M., °Poggi E. and °Casciani C. U. Istituto C.N.R. Tipi77uzione Tissutale e Problemi della Dialisi - L'Aquila, Italy °Clinica Chirurgica, Universit~ Tor Vergata - Roma, Italy *Centro Interregionale Riferimento Trapianti Abruzzo-Molise - L'Aquila, Italy Genomic typing was retrospectively performed in 32 cadaveric kidney transplant donorrecipient pairs. The occurrence of acute rejection episodes during early post-transplant 6 months was also evaluated. Patients were monitorized for donor-specific anti-HLA class II antibodies during the first year after transplantation, by flow cytometry (FCXM). DRB1 typing was performed by PCR-SSP analysis and PCR-SSO technique with biotinylated probes to define subtypes, DQA1 and DQB1 by SSP and confirmed by p32 labelled SSOs, DPB1 by radioisotopic PCR-SSO method. All episodes of rejection were hystologically confirmed and resolved by pulses of steroids. 9 out of 32 patients showed one episode of rejection. Of these, 5/14 showed o n e DRB1 mismatch (MM) and 4/15 two MMs; no rejection was observed in 3 pairs with identical DRB1, DQA1 and DQBI alleles. Positive post-Tx FCXM was observed in 14/32 recipients. In 1 of these, with z e r o M M for DRBI-DQA1DQB1 loci, the presence of anti-HLA class II antibodies was probably directed to DP antigens. An intriguing result was observed evaluating DRB1 alleles of recipients: 6 showed DRBI*0402; among these patients, 5 had episodes of acute rejection. This evidence seems to correlate DRBI*0402 allele with an higher response versus graft. No correlation was reported between degree of MM and the incidence of acute rejection or the occurrence of donor-specific antibodies. However, the role of HLA class II matching and biological importance of donor-specific antibodies need a greater number of cases and a longer followup.
C-6.1 #104
COMPARATIVE ANALYSIS OF 4 HLA CLASS II TYPING METHODS IN PROSPECTIVE ORGANDONOR TYPING. M.P. Emonds, A. Volckaerts, J. Dendievel, H. Claeys, C. Vermylen.BIoodbank Belgian Red Cross, O.L.Vrouwstraat 42, B-3000 Leuven. We evaluated classicalserology and One Lambda Monoclonal trays (MT) lot #1F in combination with a DRB INN0-LiPA and DRB PCR-SSP (CTS primer mixes) for its applicability in prospective donor typing for organtransplantation. 27 donor class II typings of donors offered during the working day between 5/1993 and ! 1/1993 are considered. Classical TCF on density gradient separated spleen cells was performed in parallel with DynabeadsR isolated spleen cells for MT analysis. Samples were analysed by two different technologists and read independently not knowing about the DNA typing results. A fast DNA extraction method (80 rain starting from whole blood) based on protein precipitation with perchlorate was used for DNA methods (BiozymR).All samples could be typed with a high accuracy in the 4 methods. Only 1 discrepancy in 54 antigens was found for classical serology (DR11 stead of DR1303). One DR 7 was missed in 54 antigens with the MT; extra reactions were obtained with DR18 and DQ4 sera. DR 2 could not be split but recognised correctly all DRI 5 and DR16 as DR2. No errors were obtained for PCR-SSP if weak extra bands with primer mix 5 and 16 were exduded. The higher resolution of INNO-LiPA made the interpretation of the INNO-LiPA difficult for 1 sample having an aberrant association of DR11 with DRB3*0101 but this haplotype typed as a dassical DR11 in serology and PCR-SSP. Conclusions: All four techniques are applicable for prospective donortyping. PCR-SSP (3-4h) and INNO-LiPA (4h) remain more labour intensive than serology. Moreover a double sample (DNA and living cells) has to be prepared as an HLA crossmatch remains to be done on isolated lymphocytes. Classical TCF takes 4h whereas MT can be read after 60 minutes if Dynabead separation is used. INNO-LiPA gives the most accurate results without extra reactions but due to its high resolution remains difficult to interprete if no key oligo's are selected for interpretation. PCR-SSP is a faster oligotyping technique more adapted for the single sample but needs an optimal sample preparation and PCR. The MT gives the fastest results (< 2h) if Dynabeads are used but here also optimalisation of the system is necessary to eliminate extra reaction and to subtype DR2 antigens. MT and genotyping systems have the supplemental advantage of standardisation.