(513) FBS Adsorption Cannot Fix Everything

Aim: Identify non-specificity in anti-HLA antibody identification panels (LSA) and determine the capacity of heat inactivated fetal bovine serum (FBS) adsorption to consistently decrease spurious binding.

Methods: Clinical sera were consecutively assayed 3/27/2024-7/18/2024 (Thermo Fisher LSA2 Lot 016+EXPLEX Lot 006) and 10/14/2024-1/31/2025 (Thermo Fisher LSA2 Lot 017+EXPLEX Lot 007). All were AdsorbOUT (Thermo Fisher) and EDTA background and prozone reduced. Beads were analyzed for binding frequency above 1,000 MFI and compared between LSA2 Lot 016 + EXPLEX Lot 006 (LSA216+EX6) and LSA2 Lot 017 + EXPLEX Lot 007 (LSA217+EX7). Bead specific average and median normalized MFI values were compared. Parallel native antigen tests (NA) (Luminex PRA2, Flow PRA2 or surrogate flow cytometric crossmatch) were applied for initial work-up or questionable LSA specificity. MatchMaker eplet analysis was applied. Selected sera with supported spurious reactivity were treated with FBS (Breitenbach et al. 2013) or FBS+EDTA and LSA217+EX7 tested. Results were compared to original data, focusing on suspected spurious LSA217+EX7 beads.

Results: LSA216+EX6 and LSA217+EX7 tests of 803 and 1203 samples, respectively, were analyzed. Both lots were applied to 406 same-patient samples. Frequency analysis indicated distinct LSA216+EX6 and LSA217+EX7 bead reactivity patterns (Figure 1 and 2). NA was applied in 56% of samples tested by each LSA lot to rule out spurious reactivities. High frequency >1,000 MFI bead reactivities were supported as spurious in most samples. LSA216+EX6 and LSA217+EX7 yielded 4.2% and 12.5%, respective, panel bead frequent spurious reactivities. Some frequent reacting and frequent spurious beads are shared between both lots, while others are unique. HLA antigens corresponding to frequently reactive and spurious beads, especially those of LSA217+EX7, are CIWD 3.0 common and impactful to waitlist cPRA values. FBS serum adsorption of 5 patient serum samples displaying LSA217+EX7 spurious reactivity was observed. FBS adsorption did not reduce non-specific bead binding, with few exceptions (Figure 3). Efficacy of FBS was not enhanced significantly by EDTA serum treatment.

Conclusion: LSA bead binding often cannot be substantiated as specific. FBS adsorption does not reliably reduce spurious binding. Use of universal, pre-determined positive cut-offs without supportive specificity testing is ill-advised when utilizing LSA due to frequency of spurious reactivities.