Issue link: https://ahint.epubxp.com/i/574474
23 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 FCXM Assay Validation Phase Validation of the Rapid Optimized FCXM assay, the Halifax FCXM Protocol Based on the above results we developed the rapid optimized FCXM assay, the Halifax FCXM protocol, and compared its performance to the standard FCXM (SFCXM) protocol in a validation study The differences in assay parameters between the SFCXM and the Halifax protocol are listed in Table 1 and include: decreased serum incubation time (20 vs 30 minutes), decreased IgG-FITC incubation time (10 vs 30 minutes), increased incubation temperature (RT vs 4 o C), and increased serum to cell volume ratio (2:1 vs 1:1) The absolute Halifax FCXM protocol assay time is 35 minutes, which represents an approximately 60% time reduction compared to the standard tube method FCXM (Table 1) To validate the Halifax FCXM protocol, a total of 144 crossmatches were performed in parallel with the SFCXM protocol Ninety of these crossmatches were predicted to be negative (no donor specific HLA antibodies in patient sera by SAB assay) and 54 were predicted to be positive (presence of one or more donor specific HLA antibodies, >5000 MFI, in patient sera by SAB assay) A total of 15 different donor cells were used (12 live and three deceased) in this validation study Linear regression analysis of MCFS values showed an excellent correlation between the protocols for both T cell (r 2 =0 977; Figure 4A) and B cell (r 2 =0 975; Figure 4B) crossmatches In addition, the mean NC MCF values, mean negative patient crossmatch MCF values, and calculated Figure 4. Comparison of the Halifax FCXM Protocol vs Standard FCXM Protocol T cell (A and C) and B cell (B and C) flow cytometric crossmatches were performed in parallel using the standard FCXM protocol and the Halifax FCXM protocol. Patient sera predicted to give a negative FCXM (n=90) or a positive FCXM (n=54) against selected donor cells were used. In panel A and B the data are expressed as median channel fluorescence shifts (MCFS) from the 3SD cutoff. In panel C, chi-square analysis of FCXM interpretation is shown. Figure 5. Comparison of the Halifaster FCXM Protocol vs the Halifax FCXM Protocol T cell (A and C) and B cell (B and C) flow cytometric crossmatches were performed in parallel using the Halifax FCXM protocol and the Halifaster FCXM protocol. Patient sera predicted to give a negative FCXM (n=85) or a positive FCXM (n=16) against selected donor cells were used. In panels A and B the data are expressed as median channel fluorescence shifts (MCFS) from the 3SD cutoff. In panel C, a chi-square analysis of FCXM interpretation is shown.