The flow cytometry crossmatch (FCXM) is the standard method to prevent hyperacute rejection after transplantation. Surprisingly, few technical improvements have been introduced to the FCXM since its inception. In this study we aimed to optimize the FCXM sensitivity and specificity. Our protocol differs from the traditional FCXM in two ways: 1) The use of magnetic beads (EasySep, STEMCELL Technologies) for lymphocyte isolation instead of the Ficoll-Hypaque method and 2) Substitution of the secondary goat anti-human IgG polyclonal antibody (polyAb) with a mouse anti-human IgG monoclonal antibody (mAb) (clone G18-145, BD Biosciences).

Higher sensitivity was observed in the FCXM performed with EasySep as compared to Ficoll. Eighty percent (8/10) of samples with weak DSA (MFI=1000-3000) displayed a positive T-cell FCXM with EasySep as compared to 60% (6/10) with Ficoll (p<0.05). Additionally, 100% (10/10) of these samples displayed a positive B-cell FCXM with EasySep as compared to 60% (6/10) with Ficoll (p<0.05). Subsequently, we substituted the secondary polyAb with a mAb and performed the modified FCXM in 48 samples from 0% PRA type 1 diabetes patients that showed a high rate of false-positive FCXM. Replacing the polyAb with the mAb resulted in a lower rate of false-positive T-cell FCXM. Four percent (2/48) of these samples displayed a positive T-cell FCXM with the mAb as compared to 48% (23/48) with the polyAb (p<0.05). On the contrary, 42% (20/48) of these samples displayed a positive B-cell FCXM with the mAb as compared to 40% (19/48) with the polyAb. Additionally, 100 samples with weak DSA were crossmatched with both antibodies. Higher sensitivity was observed in the B-cell FCXM performed with the mAb as compared to the polyAb. Eighty-four percent (84/100) of samples displayed a positive B-cell FCXM with the mAb as compared to 61% (61/100) with the polyAb (p<0.05). On the contrary, 73% (73/100) of these samples displayed a positive T-cell FCXM with both the mAb and the polyAb.

These results show that FCXM optimization is especially important when analyzing samples from individuals with autoimmune diseases and/or weak DSA. Introduction of a better cell isolation method and a secondary mAb significantly improves the FCXM sensitivity and specificity resulting in better correlation with the solid-phase HLA antibody assays.

CITATION INFORMATION: Barrios K, Lunz J, Labuda B, Magas D, Freedom D, Szewczyk K, Jendrisak M, Jaramillo A. Optimized Flow Cytometry Crossmatch with Increased Sensitivity and Specificity. Am J Transplant. 2016;16 (suppl 3).

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