RAPID METHOD FOR SUCCESSFUL HLA CLASS-I AND CLASS-II TYPING FROM CADAVERIC BLOOD FOR DIRECT MATCHING IN CORNEA TRANSPLANTATION

Background: The aim of the study was to establish fast methods for pos tmortem HLA class I and II typing of cornea donors using cadaveric blo od. Methods: The commercially available reagents Lymphokwik MN and Dyn abeads were evaluated here to provide an enriched living mononuclear c ell (MNC) population and B-cell population for HLA class I and II typi ng of cadaveric blood by serology. Cadaveric blood was obtained 1-80 h post mortem. After isolation of living B-cells and B-cell-depleted li ving MNC's, cells were serologically typed by double-fluorescence cyto toxicity assay for HLA class I and II antigens. Results: In 373 (81%) of 461 cadaveric blood samples HLA class I typing, and in 36 (62%) of 56 cadaveric blood samples HLA-class II typing, by serology was succes sful and accomplished within 5 h. Results from the serological HLA cla ss I typing were confirmed by the results of HLA class I typing by RNA -based sequencing in seven cases. To improve the HLA class II typing, DNA typing using PCR with sequence-specific primers was performed in 1 48 samples and reverse hybridization of PCR-amplified DNA to immobiliz ed HLA class II specific primers in 270 samples. These data were confi rmed by DNA-based sequencing in five cases and by sequence-specific ol igonucleotide hybridization in all cases. Conclusions. These results l ead to the following typing strategy: HLA class I typing should be per formed by serology, HLA class II typing should be performed by DNA tec hnology because of its relative independence of the quality of the blo od sample. The strategy we have developed is very successful and fast for tissue typing post mortem, thus expanding the time available for i deal HLA matching, increasing the number of available HLA-matched corn eas and therefore reducing the number of graft rejections.