Aim The introduction of Luminex-Single Antigen bead (LSA) assay significantly improved identification of HLA antibodies in sera from highly sensitized transplant candidates. Variants of this assay allowed to detect IgM antibodies and complement-fixing antibodies. Recently, the LSA assay was shown to be prone to the complement-mediated prozone effect leading to false-negative results. Methods In this study we performed a comprehensive analysis of the HLA class I and class II antibodies present in sera from 16 highly sensitized kidney transplant candidates (%FlowPRA class I and II: 96 ± 4 and 95 ± 5 respectively) using four variants of the LSA technique: IgG-LSA, IgM-LSA, C1q-LSA and also EDTA-IgG-LSA to overcome the complement-mediated prozone effect. Results In 61% of EDTA-IgG-LSA tests we evidenced a strong increment of several antibody row-value (13,709 ± 5,863) respect to IgG-LSA results. This method was also able to reveal additional HLA class I and II antibodies (A2, A25, A68, A69, B42, B57, B59, B67, B77,DR17, DQ7, DQA1*05:01) in 33% of the performed tests. Correlating EDTA-IgG-LSA and C1q-LSA results we found that all but one sera contained both complement-fixing and non complement-fixing antibodies; in the remaining serum we only detected anti-DP antibodies unable to fix complement. In one case, EDTA-IgG-LSA assay revealed the presence of a wide antibody pattern (anti-DQ2, -DQ4, -DQ7, -DQ8, -DQ9) specific for 84-87QLEL/89-90TT epitopes while C1q-LSA assay identified a similar but smaller antibody pattern (anti-DQ7, -DQ8, -DQ9) due to the recognition of 55P epitope. In 5 sera, IgM-LSA assay was able to detect HLA antibodies not revealed by IgG-LSA technique; unexpectedly several of these antibodies were HLA-Cw specific. Conclusions In conclusion our findings evidenced the importance to make use of all LSA potentiality for characterization of HLA pre-sensitization status of transplant candidates in order to assess the real immunological risk of a potential kidney transplant.
HLA ANTIBODY TESTING BY CLASSICAL AND MODIFIED LUMINEX-SINGLE ANTIGEN BEADS ASSAYS.
Antonina Piazza;Elvira Poggi;Giuseppina Ozzella;
2013
Abstract
Aim The introduction of Luminex-Single Antigen bead (LSA) assay significantly improved identification of HLA antibodies in sera from highly sensitized transplant candidates. Variants of this assay allowed to detect IgM antibodies and complement-fixing antibodies. Recently, the LSA assay was shown to be prone to the complement-mediated prozone effect leading to false-negative results. Methods In this study we performed a comprehensive analysis of the HLA class I and class II antibodies present in sera from 16 highly sensitized kidney transplant candidates (%FlowPRA class I and II: 96 ± 4 and 95 ± 5 respectively) using four variants of the LSA technique: IgG-LSA, IgM-LSA, C1q-LSA and also EDTA-IgG-LSA to overcome the complement-mediated prozone effect. Results In 61% of EDTA-IgG-LSA tests we evidenced a strong increment of several antibody row-value (13,709 ± 5,863) respect to IgG-LSA results. This method was also able to reveal additional HLA class I and II antibodies (A2, A25, A68, A69, B42, B57, B59, B67, B77,DR17, DQ7, DQA1*05:01) in 33% of the performed tests. Correlating EDTA-IgG-LSA and C1q-LSA results we found that all but one sera contained both complement-fixing and non complement-fixing antibodies; in the remaining serum we only detected anti-DP antibodies unable to fix complement. In one case, EDTA-IgG-LSA assay revealed the presence of a wide antibody pattern (anti-DQ2, -DQ4, -DQ7, -DQ8, -DQ9) specific for 84-87QLEL/89-90TT epitopes while C1q-LSA assay identified a similar but smaller antibody pattern (anti-DQ7, -DQ8, -DQ9) due to the recognition of 55P epitope. In 5 sera, IgM-LSA assay was able to detect HLA antibodies not revealed by IgG-LSA technique; unexpectedly several of these antibodies were HLA-Cw specific. Conclusions In conclusion our findings evidenced the importance to make use of all LSA potentiality for characterization of HLA pre-sensitization status of transplant candidates in order to assess the real immunological risk of a potential kidney transplant.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
