Clinical importance of pre-morteum blood lymphocytes in cadaver donor tissue typing

We have refined our immunomagnetic bead (IM bead) procedures to isolate pure and viable lymphocyte subpopulation from premorteum (PM) blood for cadaver donor HLA typing, preliminary and final crossmatches (XMs). The results of 1220 XMs were compared using T/B lymphocytes isolated either from PM blood or spleen/lymphnode (SPLN) tissue. IM bead technique was used to isolate T/B cells from PM blood and nylon wool column (NWC) technique was used to isolate T/B cells from SPLN. When we compared the outcome of 800 T-cell crossmatches using T cells from PM blood or SPLN of 5 separate cadaver donors, NWC TXMs tended to be more falsenegative for high PRA (> 10%, total 500 XMs) as well as low PRA (< 10%, total 300 XMs) did not reach statistical significance. In contrast, NW BXM (420 B XM) were found to be far more false negative than IM bead BXM regardless of the PRA of the patients. In order to ensure that NWC BXMs were indeed false negative, 23 sera with known anti-DR antibodies were BXMed where antigen-specific B cells were isolated by both the techniques. Our results showed that IM bead BXM identified the DR specificities greater than 90% of the time, the titers of ab specificities were stronger (1:8). In comparison, NWB cell XMs were weak (titers 1:2), and the false negative rate for some ab was as high as 73%. Using IM bead and NWC techniques we compared our turnaround time (TAT) for cadaver donor typing, preliminary and final XMs. Using PM blood with IM bead, our TAT was less than 5 hours for the entire cadaver donor work-up. In contrast, TAT from SPLN with NWC technique was as long as 8 hours. We also compared graft survival statistics of 153 patients transplanted on a basis of negative T/BXM using either PM blood lymphocytes (98 patients) or SPLN lymphocytes (55 patients). However, lymphocytes in either case were isolated by IM bead assay. We found no adverse outcome due to usage of PM blood lymphocytes in terms of long- or short-term graft survival or incidence of hyperacute rejection. In fact, cold ischemia time (CIT) was much shorter (median 12 hours) in the PM group versus more than 20 hours in SPLN group. In summary, PM blood is an ideal source of T/B lymphocytes for the solid organ pretransplant workup and final XM.