140-P: THE OPO EXPERIENCE WITH (ALMOST) REAL TIME HLA TYPING BY REAL-TIME PCR

Abstract

We are one of a few organ procurement organization (OPO)-based laboratories that provide full-service deceased donor testing (HLA, ABO, final crossmatch, infectious diseases). Here we compared the new real-time PCR HLA typing system to the traditional SSP-gel method for deceased donor typing. The real-time PCR method was performed using the Linkage Biosciences LinkSeq HLA-ABCDRDQDP+ 384-well typing kit (210 deceased donors typed to date) and the SSP-gel method was performed using the Invitrogen SSP UniTray low-resolution HLA-A, B, C, DRB1/3/4/5 and DQB1 and high-resolution DPB1 and DQA1 typing kits (256 donors typed). While the reagent cost is comparable between the two methods ($230/donor), the total assay time from setup to resulting is significantly less with the real-time method (2 hrs) than the SSP-gel method (4.5 hrs). To date, no repeat testing has been performed due to lane dropout as more than half of the 245 unique reaction wells on each real-time typing tray are duplicated, whereas additional testing was performed on a donor for resolving a major antigen ambiguity (B61 vs B4005 in presence of B45/50:02). The real-time typing kit also maintains the capability to detect the presence of some common null alleles (e.g. C∗04:09N, DRB4∗01:03N, DRB5∗01:08N). Unfortunately, the need of a costly real-time PCR instrument (or two for redundancy in deceased donor workup) may be prohibitive for many laboratories. Moreover, there has not been a wipe test available specifically for this technology, which uses uracil as one of the nucleobases instead of the standard thymine, and the DPB1 typing employs a vendor-specific nomenclature system (epitype) which requires some conversions for reporting. The real-time HLA typing platform appears to be a good fit for OPO laboratories where time-sensitive deceased donor testing is frequently performed after hours with technologists constantly struggling with multitasking and time constraints.