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Treating sera with ethylenediaminetetraacetic acid: A promising technical solution for the complement-mediated prozone effect in anti-hla antibody detection by single antigen bead assay
Abstracts / Human Immunology 76 (2015) 38–167
P040
TREATING SERA WITH ETHYLENEDIAMINETETRAACETIC ACID: A PROMISING TECHNICAL SOLUTION FOR THE COMPLEMENT-MEDIATED PROZONE EFFECT IN ANTI-HLA ANTIBODY DETECTION BY SINGLE ANTIGEN BEAD ASSAY. Angeliki G. Vittoraki, Sofia I. Ioannou, Hellen M. Vallindra, Alexandra A. Siorenta, Antonis G. Milonas, Nikos M. Seimenis, Maria D. Apostolaki, Aliki G. Iniotaki. National Tissue and Typing Center, ‘G.Gennimatas’ Hospital, Athens, Greece. Aim: The Luminex-single antigen bead (SAB) assay is considered the most sensitive method for the identification of anti-HLA antibodies (Ab). However, false negative results can occur due to the prozone effect (PrE) leading to underestimation of some high titer anti-HLA Ab. The aim of this study was to evaluate the ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement in eliminating the PrE in anti-HLA Ab identification by SAB. Method: Ten patients with PRAs>65% were included in the study. Neat and EDTA treated samples were tested using SAB for either HLA class I (n=14) or class II (n = 10) specificities. A mean fluorescence intensity (MFI)P2 fold increase in EDTA treated versus neat sera was considered relevant to PrE. The PrE was confirmed with 1:10 dilution and with dithiothreitol treatment of the sera. To confirm the clinical significance of the new identified Ab, sera with defined PrE and MFI value<1000 in neat sera were further tested with AHG-CDC crossmatch against donor cells expressing the relevant HLA. Results: PrE was found in 7/10 patients concerning either HLA class I (n = 4) or class II (n = 3) Ab specificities. Using an MFI = 1000 as a cut off, six ‘new’ HLA specificities were identified either HLA class I (HLA-A⁄01:01, A⁄34:02, A⁄11:02) or class II (DQB1⁄03:01, DQB1⁄03:02, DQB1⁄03:03). However, a significant shift of MFI value of HLA Ab class I and II was observed. More precisely, 38 HLA-class I Ab directed against HLA-A (n = 31) and HLA-B (n = 7) showed a significant mean MFI value shift from 5162 (266 lower value) to 20207. Regarding HLA class II Ab, 26 directed against HLA-DQ (n = 23) and HLA-DR (n = 3) showed a significant shift from MFI 4419 (113 lower value) to 23795. New HLA Ab specificities detected after EDTA treatment were confirmed with a strong positive AHG-CDC crossmatch. Conclusion: Taking into account that selection of potential recipients pre-Tx as well as the follow up post-Tx is based to HLA Ab identification, these preliminary results highlight the importance of using EDTA treatment of the patient’s sera prior to SAB assay, in order to abolish the prozone effect in HLA antibody detection.
P041
INDICATORS OF RITUXIMAB RESPONSIVENESS IN ANTIBODY-MEDIATED REJECTION AFTER KIDNEY TRANSPLANTATION. Stephan Immenschuh a, Eva Zilian a, Maximilian E. Daemmrich a, Wilfried Gwinner a, Jan Ulrich Becker b, Cornelia A. Blume a. a Hannover Medical School, Hannover, Germany; b University of Cologne, Cologne, Germany. Aim: Treatment of patients with antibody-mediated rejection (AMR) after kidney transplantation with rituximab is ambiguous. Due to its unknown efficiency and serious side-effects, biomarkers, that are predictive for responsiveness to rituximab therapy in AMR patients, are required. Methods: Twenty renal transplant patients were included in this retrospective study. Selection was based on Renal Index Biopsies, classified using Banff, where at least two AMR diagnostic criteria were positive within the three months prior to rituximab therapy. Patients were categorized into responders (R) and non-responders (NR) depending on whether they returned to dialysis within 6 months after initiation of rituximab treatment. Clinical, histopathological (Banff classification) and serological parameters were compared between both groups by t-test, Mann-Whitney-U-test or Likelihood-Ratio-Chi-square-test. Results: In comparisons between the groups, the R group had a 1.5-fold higher level of estimated glomerular filtration rate (eGFR) and a 4-fold lower level of proteinuria. By contrast, there were no differences in the histological scores for chronic transplant lesions between the groups. The t- and i-scores were higher in NRs, whereas Banff-C4d-scores of peritubular capillaries were increased in the Rs. Transplant biopsies in the Rs exhibited more CD138+cell infiltrates. Serological determination of HLA antibodies showed higher positivity for HLA class II donor-specific antibodies in the R group. No significant differences in other clinical criteria were found. Conclusions: Increased proteinuria, decreased graft function and a higher grade of tubulitis and inflammation in AMR are negative predictors for responsiveness to rituximab therapy. Rituximab therapy therefore should be initiated in an early phase of AMR.
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P040
TREATING SERA WITH ETHYLENEDIAMINETETRAACETIC ACID: A PROMISING TECHNICAL SOLUTION FOR THE COMPLEMENT-MEDIATED PROZONE EFFECT IN ANTI-HLA ANTIBODY DETECTION BY SINGLE ANTIGEN BEAD ASSAY. Angeliki G. Vittoraki, Sofia I. Ioannou, Hellen M. Vallindra, Alexandra A. Siorenta, Antonis G. Milonas, Nikos M. Seimenis, Maria D. Apostolaki, Aliki G. Iniotaki. National Tissue and Typing Center, ‘G.Gennimatas’ Hospital, Athens, Greece. Aim: The Luminex-single antigen bead (SAB) assay is considered the most sensitive method for the identification of anti-HLA antibodies (Ab). However, false negative results can occur due to the prozone effect (PrE) leading to underestimation of some high titer anti-HLA Ab. The aim of this study was to evaluate the ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement in eliminating the PrE in anti-HLA Ab identification by SAB. Method: Ten patients with PRAs>65% were included in the study. Neat and EDTA treated samples were tested using SAB for either HLA class I (n=14) or class II (n = 10) specificities. A mean fluorescence intensity (MFI)P2 fold increase in EDTA treated versus neat sera was considered relevant to PrE. The PrE was confirmed with 1:10 dilution and with dithiothreitol treatment of the sera. To confirm the clinical significance of the new identified Ab, sera with defined PrE and MFI value<1000 in neat sera were further tested with AHG-CDC crossmatch against donor cells expressing the relevant HLA. Results: PrE was found in 7/10 patients concerning either HLA class I (n = 4) or class II (n = 3) Ab specificities. Using an MFI = 1000 as a cut off, six ‘new’ HLA specificities were identified either HLA class I (HLA-A⁄01:01, A⁄34:02, A⁄11:02) or class II (DQB1⁄03:01, DQB1⁄03:02, DQB1⁄03:03). However, a significant shift of MFI value of HLA Ab class I and II was observed. More precisely, 38 HLA-class I Ab directed against HLA-A (n = 31) and HLA-B (n = 7) showed a significant mean MFI value shift from 5162 (266 lower value) to 20207. Regarding HLA class II Ab, 26 directed against HLA-DQ (n = 23) and HLA-DR (n = 3) showed a significant shift from MFI 4419 (113 lower value) to 23795. New HLA Ab specificities detected after EDTA treatment were confirmed with a strong positive AHG-CDC crossmatch. Conclusion: Taking into account that selection of potential recipients pre-Tx as well as the follow up post-Tx is based to HLA Ab identification, these preliminary results highlight the importance of using EDTA treatment of the patient’s sera prior to SAB assay, in order to abolish the prozone effect in HLA antibody detection.
P041
INDICATORS OF RITUXIMAB RESPONSIVENESS IN ANTIBODY-MEDIATED REJECTION AFTER KIDNEY TRANSPLANTATION. Stephan Immenschuh a, Eva Zilian a, Maximilian E. Daemmrich a, Wilfried Gwinner a, Jan Ulrich Becker b, Cornelia A. Blume a. a Hannover Medical School, Hannover, Germany; b University of Cologne, Cologne, Germany. Aim: Treatment of patients with antibody-mediated rejection (AMR) after kidney transplantation with rituximab is ambiguous. Due to its unknown efficiency and serious side-effects, biomarkers, that are predictive for responsiveness to rituximab therapy in AMR patients, are required. Methods: Twenty renal transplant patients were included in this retrospective study. Selection was based on Renal Index Biopsies, classified using Banff, where at least two AMR diagnostic criteria were positive within the three months prior to rituximab therapy. Patients were categorized into responders (R) and non-responders (NR) depending on whether they returned to dialysis within 6 months after initiation of rituximab treatment. Clinical, histopathological (Banff classification) and serological parameters were compared between both groups by t-test, Mann-Whitney-U-test or Likelihood-Ratio-Chi-square-test. Results: In comparisons between the groups, the R group had a 1.5-fold higher level of estimated glomerular filtration rate (eGFR) and a 4-fold lower level of proteinuria. By contrast, there were no differences in the histological scores for chronic transplant lesions between the groups. The t- and i-scores were higher in NRs, whereas Banff-C4d-scores of peritubular capillaries were increased in the Rs. Transplant biopsies in the Rs exhibited more CD138+cell infiltrates. Serological determination of HLA antibodies showed higher positivity for HLA class II donor-specific antibodies in the R group. No significant differences in other clinical criteria were found. Conclusions: Increased proteinuria, decreased graft function and a higher grade of tubulitis and inflammation in AMR are negative predictors for responsiveness to rituximab therapy. Rituximab therapy therefore should be initiated in an early phase of AMR.
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