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Abstracts / Human Immunology 73 (2012) 49–167
ANTI-HLA ANTIBODY IN A GROUP OF UNRELATED HSCT DONOR/RECIPENT PAIRS MISMATCHED IN THE HLA ALLELE. Urszula Siekiera 1, Alicja Dobrowolska 1, Miroslaw Markiewicz 2, Anna Koclenga 2, Monika DzierzakMietla 2, Slawomira Kyrcz-Krzemien 2. 1 HLA & Immunogenetic Lab, Regional Blood Center and Blood Treatment, Katowice, Poland; 2 Dept. of Hematology and BMT, Medical University of Silesia, Katowice, Poland. Aim: Biology of antibody production and function of immunologically competent cells is closely related to transplant process.This kind of research gives a possibility to observe and estimate patient and donor matching. The most interesting is to appoint some common denominator of HLA mismatches and antibody specificity detected in a serum of mismatched patient. We collected sera from patients both pre and post allo-HSCT transplantation. All the donor/recipient pair mismatches were known. Methods: We tested 130 sera collected from patients there were three periods:before transplantation, 6 month and 12 months after transplantation. DynaChip and LabScreen Luminex methods were used to identify anti-HLA antibody against HLA-A,B,C,DR,DQ and DP. We were able to define specificity of antibodies on the low and high resolution level. Software DynaChip and Fusion were used to perform immunogenetic analyses of our research. Results: In a group of patients 57% people expressed anti-HLA antibodies. We identified anti-HLA antibodies against HLA class I and HLA class II. We were able to define specificity of antibodies on low and high resolution level. In a group of 43% of patients we did not identify anti-HLA antibodies. We devided a patient into two groups mismatched in HLA class I antigen and mismatched in HLA class II antigen. We analysed people produced antibody against one or many antigens. As a second step of our research we analyse donor recipient mismatches on the level of allele. Donor recipient mismatches were connected with HLA allele locus C (6,6%), HLA allele locus B (13,3%), HLA allele locus A (3,3%), HLA allele locus DQB1⁄(10%). When we look at the PRA positive patient 20% of them express presence of antibodies against many specificities, 16,7% against no more than 3 specificity, 20% against single antigen. Conclusions: As a continuation of our research we are going to use the Immport Database. We would like to try compare specificity of anti-HLA antibodies and molecular structure of mismatched alleles. Siekiera: Blood Center: Other: HLA Lab Director. Dobrowolska: Blood Center: Employee. Markiewicz: Medical University of Silesia: Other: Clinician.
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COMPARISON OF METHODOLOGIES TO REDUCE RITUXIMAB INTERFERENCE DURING FLOW CYTOMETRIC CROSSMATCH TESTING. Stephanie Souza 1, Patrick Ching 1, Robert O. Endres 1, Leon L. Su 2, Riccardo Valdez 3. 1 HLA Laboratory, Blood Systems Laboratories, Tempe, AZ, USA; 2 Blood Systems, Inc., Scottsdale, AZ, USA; 3 Mayo Clinic in Arizona, Phoenix, AZ, USA. Aim: Rituximab (RIT) is currently used to treat and prevent allograft rejection in transplant recipients, and is known to interfere with the flow cytometric crossmatch (FCXM). Pre-FCXM pronase treatment reduces, but does not eliminate, background interference. In this study, we employed common red cell serology techniques to reduce interference in the FCXM for five patients on RIT therapy. Methods: Pre- (ACD) and post-administration (CLOT) blood samples were collected from five subjects eligible to receive RIT therapy. Buffy coats were harvested from the ACD samples and cryopreserved. Sera from the CLOT samples was filtered and frozen at < 20 °C until testing. A 3-color FCXM was performed as follows: 1) NEAT–All samples were tested without modification. 2) Autologous Adsorption (AA)–Subject serum was adsorbed against a double volume autologous leukocyte-rich buffy coat aliquot. 3) Competitive Binding (CD20)–Donor cells were incubated with non-humanized monocolonal anti-CD20. 4) Inhibition–Donor cells were incubated with heat-inactivated rabbit serum (HIRS). 5) Enzyme Treatment (PRO)–Donor cells were treated with pronase prior to testing. Median fluorescence intensity (MFI) data from each modification were compared against the NEAT data, within run, for each subject. The results were analyzed for significance using the student’s t-test for unequal variance. Results: All post-RIT samples resulted in strongly positive B cell FCXMs, (mean MFINEAT = 895). CD20 and PRO significantly reduced mean MFI (835.2, p < 0.001; 608.2, p < 0.001), while AA and HIRS showed no significant difference from the neat FCXM (896.2, p = 0.867; 891.2, p = 0.558). The T cell FCXM was unaffected by RIT, however, an artifact was observed during HIRS that resulted in strongly positive T cell FCXMs for all samples (529.6, p < 0.001). Conclusions: Two modifications to the FCXM protocol showed promise in reducing RIT interference: 1) competitive binding using a non-humanized monoclonal anti-CD20, and 2) pronase treatment of the donor cells.
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Abstracts / Human Immunology 73 (2012) 49–167
ANTI-HLA ANTIBODY IN A GROUP OF UNRELATED HSCT DONOR/RECIPENT PAIRS MISMATCHED IN THE HLA ALLELE. Urszula Siekiera 1, Alicja Dobrowolska 1, Miroslaw Markiewicz 2, Anna Koclenga 2, Monika DzierzakMietla 2, Slawomira Kyrcz-Krzemien 2. 1 HLA & Immunogenetic Lab, Regional Blood Center and Blood Treatment, Katowice, Poland; 2 Dept. of Hematology and BMT, Medical University of Silesia, Katowice, Poland. Aim: Biology of antibody production and function of immunologically competent cells is closely related to transplant process.This kind of research gives a possibility to observe and estimate patient and donor matching. The most interesting is to appoint some common denominator of HLA mismatches and antibody specificity detected in a serum of mismatched patient. We collected sera from patients both pre and post allo-HSCT transplantation. All the donor/recipient pair mismatches were known. Methods: We tested 130 sera collected from patients there were three periods:before transplantation, 6 month and 12 months after transplantation. DynaChip and LabScreen Luminex methods were used to identify anti-HLA antibody against HLA-A,B,C,DR,DQ and DP. We were able to define specificity of antibodies on the low and high resolution level. Software DynaChip and Fusion were used to perform immunogenetic analyses of our research. Results: In a group of patients 57% people expressed anti-HLA antibodies. We identified anti-HLA antibodies against HLA class I and HLA class II. We were able to define specificity of antibodies on low and high resolution level. In a group of 43% of patients we did not identify anti-HLA antibodies. We devided a patient into two groups mismatched in HLA class I antigen and mismatched in HLA class II antigen. We analysed people produced antibody against one or many antigens. As a second step of our research we analyse donor recipient mismatches on the level of allele. Donor recipient mismatches were connected with HLA allele locus C (6,6%), HLA allele locus B (13,3%), HLA allele locus A (3,3%), HLA allele locus DQB1⁄(10%). When we look at the PRA positive patient 20% of them express presence of antibodies against many specificities, 16,7% against no more than 3 specificity, 20% against single antigen. Conclusions: As a continuation of our research we are going to use the Immport Database. We would like to try compare specificity of anti-HLA antibodies and molecular structure of mismatched alleles. Siekiera: Blood Center: Other: HLA Lab Director. Dobrowolska: Blood Center: Employee. Markiewicz: Medical University of Silesia: Other: Clinician.
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COMPARISON OF METHODOLOGIES TO REDUCE RITUXIMAB INTERFERENCE DURING FLOW CYTOMETRIC CROSSMATCH TESTING. Stephanie Souza 1, Patrick Ching 1, Robert O. Endres 1, Leon L. Su 2, Riccardo Valdez 3. 1 HLA Laboratory, Blood Systems Laboratories, Tempe, AZ, USA; 2 Blood Systems, Inc., Scottsdale, AZ, USA; 3 Mayo Clinic in Arizona, Phoenix, AZ, USA. Aim: Rituximab (RIT) is currently used to treat and prevent allograft rejection in transplant recipients, and is known to interfere with the flow cytometric crossmatch (FCXM). Pre-FCXM pronase treatment reduces, but does not eliminate, background interference. In this study, we employed common red cell serology techniques to reduce interference in the FCXM for five patients on RIT therapy. Methods: Pre- (ACD) and post-administration (CLOT) blood samples were collected from five subjects eligible to receive RIT therapy. Buffy coats were harvested from the ACD samples and cryopreserved. Sera from the CLOT samples was filtered and frozen at < 20 °C until testing. A 3-color FCXM was performed as follows: 1) NEAT–All samples were tested without modification. 2) Autologous Adsorption (AA)–Subject serum was adsorbed against a double volume autologous leukocyte-rich buffy coat aliquot. 3) Competitive Binding (CD20)–Donor cells were incubated with non-humanized monocolonal anti-CD20. 4) Inhibition–Donor cells were incubated with heat-inactivated rabbit serum (HIRS). 5) Enzyme Treatment (PRO)–Donor cells were treated with pronase prior to testing. Median fluorescence intensity (MFI) data from each modification were compared against the NEAT data, within run, for each subject. The results were analyzed for significance using the student’s t-test for unequal variance. Results: All post-RIT samples resulted in strongly positive B cell FCXMs, (mean MFINEAT = 895). CD20 and PRO significantly reduced mean MFI (835.2, p < 0.001; 608.2, p < 0.001), while AA and HIRS showed no significant difference from the neat FCXM (896.2, p = 0.867; 891.2, p = 0.558). The T cell FCXM was unaffected by RIT, however, an artifact was observed during HIRS that resulted in strongly positive T cell FCXMs for all samples (529.6, p < 0.001). Conclusions: Two modifications to the FCXM protocol showed promise in reducing RIT interference: 1) competitive binding using a non-humanized monoclonal anti-CD20, and 2) pronase treatment of the donor cells.