Abstract
The importance of cell purity to obtain a reliable FCXM result is critical and long established. The aim of this study was to improve efficiency and implement automated cell preparation for FCXM due to the increased workload in the histocompatibility lab after the new Kidney Allocation System (KAS) was implemented.
The use of RobosepTM Instrument along with EasySepTM Direct Human Total Lymphocyte Isolation Kit (RLI) starting from buffy coat was evaluated in parallel with our traditional ficoll hypaque-based lymphocyte isolation method (FLI) that includes CD15 Dynabeads, lysing buffer and soft spin washes to achieve target purity criteria when applicable. FCXM was performed for all cells (n=20) isolated in parallel using FLI & RLI. Spiked Positive Controls were used for FCXM and tested in parallel to verify sensitivity of the assay. The costs associated with both methods were evaluated by comparing labor-based expenses and reagent price.
The purity of the cell preparations obtained by the RLI method was superior (CD45-95.4%, myeloid -0.9%, plt-0.8∗103/μL) when compared with the traditional FLI technique (CD45-84.7%, myeloid -4.9%, plt-7.9∗10 textsuperscript3/μL). Lymphocyte yield and viability was equivalent by both methods. There was no significant change in the Negative Control MFI values for 90% of the isolated cell preparations, however two Negative Controls obtained using RLI showed high MFI for B cell crossmatch. The sensitivity of the FCXM proved to be unmodified when lymphocytes were isolated by RLI. The use of RLI for FCXM reduced the cost of reagents/cell isolation by 75% compared to the FLI method. The total isolation protocol time was reduced by 25% when using RLI in comparison with FLI, and the total tech hands-on time was dramatically reduced by 55%.
Our study revealed that automation using EasySepTM kit on RobosepTM markedly improves the purity of the cells with identical results and sensitivity in the FCXM. The use of RLI proved to be more efficient and cost effective, reducing not only the reagent cost but also more importantly ”hands-on” tech time.
