Issue link: https://ahint.epubxp.com/i/574474
22 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 Effects of Serum and IgG-FITC Incubation Time on FCXM Results The effects of varying serum incubation time on FCXM results are shown in Figures 2A and 2B Time course experiments revealed a time dependent increase in PC MCFS values across the 3-30 minute incubation interval studied (Fig 2A and 2B) Interestingly, the majority of reactivity occurred within the first 3-5 minutes of incubation with serum for all PC concentrations tested While additional increases in MCFS values were seen as the serum incubation time was extended, the incremental MCFS increases beyond the 15 minute time point were minimal For example, MCFS values for the 1:400 PC dilution were 68 channels for T cell and 161 channels for B cell crossmatches in the first 15 minutes of incubation with serum, while in the subsequent 15 minutes of incubation (15-30 minute time interval) the MCFS values increased only by additional 6 and 17 channels for T cell and B cell crossmatches respectively (Fig 2A and 2B) Time course experiments investigating the effects of IgG-FITC incubation time yielded even more interesting results As shown in Figure 2, the maximal FCXM reactivity for T cells (Fig 2C) and B cells (Fig 2D) was reached in the first five minutes of incubation with IgG-FITC and there was no further increase in PC MCFS values when the incubation was extended beyond the five-minute time point This was true at all concentrations of PC tested Effects of Incubation Temperature, Serum to Cell Volume Ratio and Target Cell Number on FCXM Results Figure 3 shows the effects of altering several assay conditions on FCXM results Raising the incubation temperature from 4 o C to room temperature (RT) led to a slight increase in PC MCFS values (approximately 15-35 channels) for T cell (Fig 3A) and B cell (Fig 3B) crossmatches Similarly, PC MCFS values were slightly enhanced (15-30 channels) when serum-to-cell-volume ratio was increased from 1:1 to 2:1 (Fig 3C and 3D) Finally, there was a step wise enhancement of PC MCFS values as the number of target cells was decreased in two-fold increments from 1x10 6 cells/ well to 1 25x10 5 cells/well (Fig 3E and 3F) The change in cell number within the 2 5x10 5 –1 0x10 6 cells/reaction range, which is used by most HLA laboratories for clinical testing, appeared to have the largest impact on PC MCFS values of all the parameters tested, with up to 120 channel differences observed between the experimental groups Figure 3. Effects of Incubation Temperature, Serum/Cell Volume Ratio, and Cell Number on FCXM Results T cell (A, C, and E) and B cell (B, D, and F) flow cytometric crossmatches were performed with different dilutions of positive control (PC) sera using the tray method. In panel A and B, crossmatches were performed either at 4 o C or at room temperature (RT). In panel C and D, sera were incubated with donor cell either at a 1:1 or 2:1 volume ratio. In panel E and F, varying numbers of donor cells were used. Data are expressed as median channel fluorescence shifts (MCFS) from the negative control. One representative experiment is shown. Incubation Temperature Incubation Temperature Serum/Cell Volume Ratio Serum/Cell Volume Ratio