Introduction: We have previously demonstrated that pre-formed donor specific antibodies (DSA) to human leukocyte antigens (HLA) are associated with reduced islet function and survival but positive (pos) T and/or B cell flow crossmatch (FCXM) alone is not. (1) Thresholds for FCXM are established using HLA un-sensitized normal samples, which may not reflect serum background reactivity in all patient populations. Here we re-evaluated T and B cell FCXM pos thresholds using un-sensitized kidney and islet cell/pancreas transplant patient sera.
Methods: The FCXM method includes incubating pronased treated donor lymphocytes with patient serum followed by detection of IgG antibody on the CD3 labelled donor T and CD19 labelled B cells. (2) Determination of routine positive (pos) threshold was determined by FCXM with 10 cells and 20 sera from un-sensitized normal donors. Patient FCXM data from July 2013 to December 2015 were examined. Calculated panel reactive antibody (cPRA) values were determined using the Canadian cPRA tool. A 0 cPRA was assigned to all patients with no HLA antibody determined by threshold of MFI 1000 and no evidence of reactivity pattern to HLA epitopes. The median channel value (MCV) shift above the negative control serum was determined for all FCXM and results were separated by donor peripheral blood (PBL) or spleen (SPL) source as per routine threshold determination. The mean and standard deviation (SD) were calculated for each patient and cell type.
Results: A total of 1417 FCXM were performed; 367 patients had 0 cPRA. The total number of kidney and islet transplant patients was 166 and 63, respectively. The breakdown of PBL vs SPL cell source is shown Table 1. The FCXM pos cut-offs determined using the kidney sera are very similar to those established from normal controls. The pos thresholds calculated using islet/pancreas patient sera are higher.
Conclusion: Islet and pancreas patient sera may have inherent, non-HLA specific reactivity to T and B cells, which affects the interpretation of FCXM. The current thresholds may result in false positive T and B FCXM in some islet and pancreas transplant patients. Our data suggest that the crossmatch must be interpreted in the context of HLA antibody specificity information. Higher pos thresholds for FCXM may be required in this patient population however an increased threshold requires validation in the setting of known DSA to ensure that false negative FCXM results do not result.
1. Campbell PM, Salam A, Ryan EA, Senior P, Paty BW, Bigam D, et al. Pretransplant HLA Antibodies Are Associated with Reduced Graft Survival After Clinical Islet Transplantation. American Journal of Transplantation. 2007 May;7(5):1242-8.
2. Liwski R, Pochinco D, Tinckam K, Gebel H, Campbell P, Nickerson P. 30-or: Canada-Wide Evaluation of Rapid Optimized Flow Crossmatch (rofcxm) Protocol. Human Immunology. 2012 October 2;73:26-.