Projects

The Virtual Crossmatch and Definition of Epitopes Using Single Antigen Beads

Study Outline

Advancements in HLA antibody methodologies for the detection and characterization of HLA specific antibody over the last few years have dramatically enhanced our ability to dissect the nature of humoral responses to HLA antigens. Perhaps the most intriguing possibility these technologies have offered is an enhanced ability to accurately predict a crossmatch. Since crossmatch prediction does not require donor specimens to be on site, crossmatch prediction could be applied to many more donors, offering the possibility of addressing the continuing problem of transplanting the sensitized transplant candidate by increasing the donor pool for these disadvantaged patients. The major obstacle for this has been the high frequency of a positive crossmatch after shipping an organ, a situation that these new technologies may well address. They also may provide the tools for a greater understanding of the epitopes involved in the highly sensitized patient. We hope to be able to shed some light as to the relative involvement of the different HLA loci in crossmatching, and attempt to resolve which epitopes may be involved.

The opportunity for participation will include two approaches. The first will involve testing a small number of well characterized sera from highly sensitized patients in a large number of flow cytometric crossmatches. The second approach will involve the laboratories examining a greater number of sera for antibody reactivity, and using that information to re-examine previous crossmatches for the patients as well as to perform crossmatches against selected target cells to further test the possibility of predicting a crossmatch. In the hope of maximizing the interpretation of results, common methodologies will be utilized.

Study 1. Flow cytometric crossmatching using well characterized sera.

Basic Design: A small number (~6) of sera will be provided to participating laboratories for study. These sera will be from highly sensitized patients who have been fully HLA typed. The sera will be characterized using all available single antigen beads for class I & II reactivities. Laboratories will include these sera in routine flow cytometric crossmatches performed at their laboratory (living or deceased donors), reporting fluorescence values for T and B reactions and the HLA types of the donors. In cases where complete typings including HLA-C and DP might be helpful in the analyses, molecular typing for these loci will be available.
Requirements: Laboratories must be utilizing a three-color flow crossmatch measuring IgG. Calibration of the instruments using fluorescent beads will also be necessary. Assistance for this aspect will be provided if needed.

Study 2. Antibody characterization and crossmatch prediction in high PRA patients.

Basic Design: This study will require a bit more effort on the part of the participating labs. We would like to study high PRA patients with a history of positive crossmatches (5+) who ultimately had a negative crossmatch against a donor with mismatched antigens. The laboratory would test a peri-transplant serum against a full battery of class I&II antigen beads using the flow cytometer (Analyses using the Luminex platform may be possible depending on bead availability). Again, the emphasis will be on obtaining results of both flow crossmatches and bead analyses as fluorescence. The bead results would be examined in conjunction with the previous crossmatches. In addition, attempts will be made to find cells in house with single antigen mismatches with the patients where there was reactivity found in the bead analyses against that mismatched antigen (It is likely that this will involve some shipping of sera between labs). This should allow additional evaluation of the virtual crossmatch concept as well as provide some insight into the relative influence of individual antigens and loci in the crossmatch. And since we will know which alleles are used in the single antigen beads, this should allow a unique opportunity to discern epitopes that are involved in these reactions.
Requirements: Laboratories must be utilizing a three-color flow crossmatch measuring IgG. Calibration of the instruments (including Luminex if used) using fluorescent beads will also be necessary. Assistance for this aspect will be provided if needed. Full HLA typings of both patients and donors would be very valuable as well, although we may be able to obtain this if DNA is available.

If interested in participating please contact Dan Cook at djc@tt.ccf.org.

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