19-P : A SAFE AND EFFECTIVE WAY TO PERFORM WASH STEPS IN THE TRAY BASED FCXM ASSAY

Aim Our laboratory is moving from a tube to tray based flow cytometry crossmatch (FCXM). We investigated the use of a vacuum manifold rather than a flick wash (FW) to perform the wash steps. Methods Crossmatches were performed as previously described using the rapid optimized FCXM method (Liwski et al. ASHI 2011, abstract 45-P). Our laboratory adopted this method but used a vacuum manifold to remove the supernatant. This manifold has 96 height adjustable probes and is attached to a vacuum source, in this case the hospital’s central vacuum system. The probes were lowered into the wells and the supernatant was removed. The cell button was vortexed before adding the next wash. We tested this vacuum manifold wash FCXM method in parallel with the FW FCXM method with 2 different cells. Negative serum (NS) samples as well as 4 dilutions of positive controls (PC) were included in these FCXM. The lymphocytes were isolated using the StemCell™ EasySep™. Reproducibility was evaluated based on the standard deviation (SD) of 14 duplicates for each wash method. The median channel values (MVC) shifts for T and B cell FCXM were also determined for each PC dilution in each wash method by subtracting out the NS value. Results The SD of the vacuum washed duplicates was 4.4 vs. 8.9 in the FW. The MCV shift was 1.8-2.0% more sensitive in the vacuum washed FCXM.[figure1]Fig. 1: T and B cell MCV shifts in vacuum vs wash FCXM trays. Conclusions The vacuum manifold wash method for tray based FCXM assay was as least as effective and reproducible as the FW method. The vacuum manifold also avoids the creation of potentially hazardous aerosols in the laboratory. We have adopted this method for performing FCXM wash steps.