Issue link: https://ahint.epubxp.com/i/574474
21 ASHI Quarterly Third Quarter 2015 S C I E N T I F I C C O M M U N I C A T I O N S Results FCXM Assay Optimization Phase Experimental Design In the optimization phase of the study, we performed flow cytometric crossmatches (FCXM) to investigate the effects of several assay parameters on FCXM results The parameters studied included: assay platform (tube method vs 96-well tray method), incubation time of cells with serum and with the IgG-FITC secondary antibody, incubation temperature, serum to cell volume ratio, and target cell number All crossmatches were performed using Lympholyte enriched peripheral blood mononuclear cells (PBMC) isolated from volunteer donors, as well as the pooled positive control sera (PC), and the pooled negative control sera (NC) Serial dilutions of PC sera ranging from 1:50 to 1:400 were chosen to span the relevant spectrum of FCXM reactivity ranging from weakly positive (within 50 channels from the cutoff for T cells and 100 channels for B cells) to clearly positive (150-250 channels above cutoff for T cells and 200-300 channels for B cells) All results are represented as median channel fluorescence shift (MCFS) values from the NC serum Effect of Assay Platform on FCXM Results The results of FCXM comparison between the standard tube method vs standard 96-well tray method (Table 1) are summarized in Figure 1 and show that the PC MCFS values obtained with the two methods were essentially equivalent for both T cell (panel A) and B cell (panel B) FCXM at all dilutions of PC tested (1:50- 1:400) There were no significant differences in the NC MCF values obtained with the two methods (data not shown) However, as expected, we observed significant time savings of 25-30 minutes per crossmatch, when FCXM was performed using the standard 96-well tray method This is due to decreased centrifugation time (one minute vs five minutes per spin), more efficient delivery of reagents and wash buffers using a multichannel pipette, as well as more efficient elimination of supernatants (flick technique) during the wash steps afforded by the 96-well tray method Therefore, we used the 96-well tray method for the remainder of this validation study Figure 1. FCXM Assays Performed Using the Tube vs Tray Method T cell (A) and B cell (B) flow cytometric crossmatches were performed with different dilutions of positive control (PC) sera and negative control sera using the tube method vs tray method. Data are expressed as median channel fluorescence shifts (MCFS) from the negative control. Two independent experiments are shown. Figure 2. Time Course Experiments Showing Effects of Serum and Anti-IgG-FITC Incubation Time on FCXM Results T cell (A and C) and B cell (B and D) flow cytometric crossmatches were performed with different dilutions of positive control (PC) sera and negative control sera using the tray method. In panel A and B, donor cells were incubated with sera for either 3, 5, 10, 15, or 30 minutes and then with anti-IgG-FITC for 30 minutes. In panel C and D, donor cells were incubated with sera for 30 minutes and then with anti-IgG-FITC for either 5, 10, 15 , or 30 minutes. Data are expressed as median channel fluorescence shifts (MCFS) from the negative control. One representative experiment is shown.