P023 A Novel flow cytometry based cellular crossnatch (Flow-CXM) predicts graft failure and GHVD post bone marrow transplantation

Abstract

Aim Pre-Transplant samples of two bone marrow transplantation(BMT) cases were tested by new developed Flow-CXM assay. The aim of this study is to evaluate the Flow-CXM assay in prediction of graft failure and GVHD in BMT. Methods Short Tandem Repeat; Luminex IgG/C1q SAB assay; Flow-CXM: 0.1×106 irradiated donor PBMCs were incubated with 0.1×106 recipient’s PBMCs and Th1 (IFN γ& IL-2) cytokine capture beads for 16 h. The cells were lysed and the captured cytokines on the beads were detected by PE-labeled 2nd antibodies. The positive PE stained beads were counted to represent the relative number of donor specific responding T cells. Results: Case 1 A 53/F patient was diagnosed with Sezary disease and has received two BMTs. No DSA to 1st and 2nd donors was detected in pre-Tx patient serum by Luminex Ab testing. STR results showed primary graft failure on day 45 after 1st BMT and 100% engraftment on day 28 after 2nd BMT. However, patient deceased 2.5 month after 2nd BMT. The retrospective Flow-CXM results showed that recipient has increased IFN-r and IL-2 response to 1st donor cells(HvG). In addition, there was weaker IFN-r and IL-2 responses in graft vs host (GvH) directions. Decreased IL-2 secretion was found in HvG direction to 2nd donor but strong positive IFN-r response in GvH directions. The patient finally was diagnosed as GI GVHD. Case 2:A 64/ F patient was diagnosed with Mycoses fungoides Sezary disease. The patient got one BMT and maintained a low level engraftment. LMX IgG/C1q DSA results were negative in pre-Tx serum. STR assay has been performed to monitor engraftment from day 28 to day 2264 (2011–2018) post BMT. No graft failure and rejection were reported even though the engraftment was relatively low and varies with large scope (CD3 17–65%, CD15 6–92%, CD19 12–83%, CD56 17–86% and whole blood 9–77%). Flow-CXM results showed that no positive allo response in both HvG and GvH directions. Conclusions The newly developed Flow-CXM may predict graft failure by positive IFN-r and IL-2 response in HvG direction. Positive IFN-r response in GvH direction may indicate the occurrence of GVHD.