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IgG isotype of alloantibodies detected in a flow cytometric antibody screening technique
103
Abstracts
7.1 #121
IgG ISOTYPE OF ALLOANTIBODIES DETECTED IN A FLOW CYTOMETRIC ANTIBODY SCREENING TECHNIQUE. OJ Pidwell, PW Adams and CG Orosz. The Ohio State University Medical Center, Columbus, Ohio. Detection of alloantibodies by flow cytometric methods is thought to be more sensitive than by CDC methods. We have evaluated a flow cytometric antibody detection technique, Flow Pool Analysis, which is based on the method of Shroyer et al (Transplantation 59:626). Flow Pool Analysis detects T cell-binding IgG using a pool of peripheral blood mononuclear cells (PBMC) from six normal donors chosen so as to encompass all the CREG groups (Rodey, ASHI Quarterly, Spring 1993). In our program approximately 13% of patients who have previously had 0% PRA by CDC, either AMOS modified or AHG, demonstrate reactivity by Flow Pool Analysis (flow positive/CDC negative). This indicates that these patients produce T cell-binding IgG which is not detectable in complement-dependent cytotoxic assays. We hypothesized that these patients produce predominantly non-complement binding isotypes of IgG. Using flow pool analysis with isotype specific secondary antibodies, thirteen flow positive/CDC negative serum samples were assessed for the presence of T cell-binding IgG of the isotypes IgG1, IgG2, IgG3, and IgG4. We observed that these thirteen patients did not predominantly produce non-complement binding isotypes, ie IgG2 or IgG4. In fact, the T cell-binding antibodies in these flow positive/CDC negative sera were predominantly IgG 1, which is effective at binding complement. We conclude that lack of complement fixing antibody does not explain the discrepancy between flow pool analysis and CDC results. An alternate possibility is that flow positive/CDC negative sera have low levels of complement-fixing antibody which are detectable by flow cytometry, but are too low to be detected in CDC assays. We are currently investigating this possibility.
7.1 #122
COMPARISON OF FLOWCYTOMETRY AND ELISA CROSSMATCHING. PL Steller. JM Westerkamp, PK Hennessy, PW Adams and CG Orosz. The Ohio State University Medical Center, Columbus, Ohio. To better understand the relationship between alloantibody detection by flow cytometry crossmatching (FXM) and ELISA-based crossmatching (EXM), we compared both techniques for the ability to detect alloantibody in a panel of patient sera with three questions in mind. (1) How often are the results ofthe FXM and the EXM in agreement? (2) What is the incidence of a positive FXM with a negative EXM? (3) Are there antibodies detectable by EXM but not FXM? For this study, we selected 22 sera that had intermediate flow cytometric reactivity (11-30%) with a pool of 6 allogeneic PBMC. We determined the FXM reactivity for each of the 22 serum against each of the 6 members of the cell pool, using a two-color flow cytometric that limited alloantibody detection to T cell-binding IgG. In parallel, EXM was performed, using a commercial kit (Sangstat) that limited antibody detection to anti-MHC Class IlgG. This resulted in 132 matched FXM and EXM tests. Nine EXM results were scored as indeterminant, and excluded from subsequent analyses. When the FXM and EXM resultes were compared, there was agreement in 80/123 (65%) of the tests. The remaining 43/123 tests were all positive by FXM, but negative by EXM. No samples were positive by EXM and negative by FXM. Two alternatives can explain the FXM + /EXM- results: (a) the EXM detects only MHC Class I-reactive IgG, whereas the FXM detects additional T cellreactive IgGs or (b) the FXM is more sensitive at antibody detection than the EXM. This study suggests that EXM detects a subset of alloantibodies detected by FXM, i.e., those alloantibodies that are MHC Class I-reactive. It is these alloantibodies that are thought to be responsible for post-transplant clinical complications. It is not known if the additional alloantibodies detectable by FXM have a clinical relevance in transplant patients. Additional studies are needed to determine which crossmatch methodology correlates best with a favorable post-transplant clinical outcome.
Abstracts
7.1 #121
IgG ISOTYPE OF ALLOANTIBODIES DETECTED IN A FLOW CYTOMETRIC ANTIBODY SCREENING TECHNIQUE. OJ Pidwell, PW Adams and CG Orosz. The Ohio State University Medical Center, Columbus, Ohio. Detection of alloantibodies by flow cytometric methods is thought to be more sensitive than by CDC methods. We have evaluated a flow cytometric antibody detection technique, Flow Pool Analysis, which is based on the method of Shroyer et al (Transplantation 59:626). Flow Pool Analysis detects T cell-binding IgG using a pool of peripheral blood mononuclear cells (PBMC) from six normal donors chosen so as to encompass all the CREG groups (Rodey, ASHI Quarterly, Spring 1993). In our program approximately 13% of patients who have previously had 0% PRA by CDC, either AMOS modified or AHG, demonstrate reactivity by Flow Pool Analysis (flow positive/CDC negative). This indicates that these patients produce T cell-binding IgG which is not detectable in complement-dependent cytotoxic assays. We hypothesized that these patients produce predominantly non-complement binding isotypes of IgG. Using flow pool analysis with isotype specific secondary antibodies, thirteen flow positive/CDC negative serum samples were assessed for the presence of T cell-binding IgG of the isotypes IgG1, IgG2, IgG3, and IgG4. We observed that these thirteen patients did not predominantly produce non-complement binding isotypes, ie IgG2 or IgG4. In fact, the T cell-binding antibodies in these flow positive/CDC negative sera were predominantly IgG 1, which is effective at binding complement. We conclude that lack of complement fixing antibody does not explain the discrepancy between flow pool analysis and CDC results. An alternate possibility is that flow positive/CDC negative sera have low levels of complement-fixing antibody which are detectable by flow cytometry, but are too low to be detected in CDC assays. We are currently investigating this possibility.
7.1 #122
COMPARISON OF FLOWCYTOMETRY AND ELISA CROSSMATCHING. PL Steller. JM Westerkamp, PK Hennessy, PW Adams and CG Orosz. The Ohio State University Medical Center, Columbus, Ohio. To better understand the relationship between alloantibody detection by flow cytometry crossmatching (FXM) and ELISA-based crossmatching (EXM), we compared both techniques for the ability to detect alloantibody in a panel of patient sera with three questions in mind. (1) How often are the results ofthe FXM and the EXM in agreement? (2) What is the incidence of a positive FXM with a negative EXM? (3) Are there antibodies detectable by EXM but not FXM? For this study, we selected 22 sera that had intermediate flow cytometric reactivity (11-30%) with a pool of 6 allogeneic PBMC. We determined the FXM reactivity for each of the 22 serum against each of the 6 members of the cell pool, using a two-color flow cytometric that limited alloantibody detection to T cell-binding IgG. In parallel, EXM was performed, using a commercial kit (Sangstat) that limited antibody detection to anti-MHC Class IlgG. This resulted in 132 matched FXM and EXM tests. Nine EXM results were scored as indeterminant, and excluded from subsequent analyses. When the FXM and EXM resultes were compared, there was agreement in 80/123 (65%) of the tests. The remaining 43/123 tests were all positive by FXM, but negative by EXM. No samples were positive by EXM and negative by FXM. Two alternatives can explain the FXM + /EXM- results: (a) the EXM detects only MHC Class I-reactive IgG, whereas the FXM detects additional T cellreactive IgGs or (b) the FXM is more sensitive at antibody detection than the EXM. This study suggests that EXM detects a subset of alloantibodies detected by FXM, i.e., those alloantibodies that are MHC Class I-reactive. It is these alloantibodies that are thought to be responsible for post-transplant clinical complications. It is not known if the additional alloantibodies detectable by FXM have a clinical relevance in transplant patients. Additional studies are needed to determine which crossmatch methodology correlates best with a favorable post-transplant clinical outcome.