Issue link: https://ahint.epubxp.com/i/574474
24 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 3SD cutoffs were very similar for both protocols (Table 2) Of the 90 predicted negative crossmatches there was one false positive reaction (T+/B-) with the Halifax FCXM protocol (26 MCFS above the 3SD cutoff) and one false positive reaction (T+/B-) with the SFCXM protocol (34 MCSF above the cutoff; Figure 4C) Both of these false positive reactions would have been interpreted as negative in a clinical setting given they were T+/B- All 54 predicted positive crossmatches resulted in positive reactions with both FCXM protocols Chi-square analysis showed that the two protocols are equivalent with regards to crossmatch interpretation (p<0 001, Figure 4C) Validation of the Enhanced Rapid Optimized FCXM assay, the Halifaster FCXM Protocol An important time limitation of FCXM is the cell isolation step Many HLA labs use either Ficoll or Lympholyte density gradient centrifugation to obtain PBMC This procedure takes approximately 90 minutes to complete (including pronase/ DNase treatment) and yields variable lymphocyte purity ranging approximately from 20% to 85% To improve the isolation time and the purity of lymphocytes, we adopted a recently developed EasySep TM Direct lymphocyte purification method from STEMCELL Technologies Inc This method utilizes an immunomagnetic bead negative selection principle and consistently provides >90% pure lymphocytes with a 60-minute turnaround time (including pronase/DNase treatment) Next, we made minor modifications to the Halifax FCXM protocol to enhance it for use with pure lymphocytes The resulting Halifaster FCXM procedure is detailed in Table 1 The differences between the Halifaster and the Halifax protocol include: decreased cell number and suspension volume (1 5x10 5 vs 2 5x10 5 and 15 µl vs 25 µl), decreased serum volume (30 µl vs 50 µl), decreased antibody cocktail volume (50 µl vs 100 µl) and decreased IgG-FITC incubation time (5 vs 10 minutes) We compared the Halifaster FCXM and Halifax FCXM protocols by performing a total of 101 (85 predicted negative and 16 predicted positive) crossmatches in parallel A total of eight different donor cells (five live and three deceased) were used Figure 5 shows excellent correlation between MCFS values obtained with the Halifaster FCXM and Halifax FCXM protocols for both T cell (r 2 =0 98; Figure 5A) and B cell (r 2 =0 98; Figure 5B) crossmatches Table 2 shows that the mean NC MCF values, mean negative patient crossmatch MCF values, and calculated 3SD cutoffs were similar for both protocols There was 2/85 false positive reactions with the Halifaster FCXM protocol and 1/85 false positive reaction with the Halifax protocol (Figure 5C) All three false positive reactions were T+/B- with less than 10 MCFS above the 3SD cutoff and would have been interpreted as negative in a clinical setting Finally, 2/16 predicted positive crossmatches gave negative reactions with both protocols (Figure 5C) Thus the two protocols give equivalent crossmatch results as determined by the Chi-square analysis (p<0 001, Figure 5C) Discussion The aim of this study was to develop a rapid optimized FCXM protocol to improve turn around time (TAT) while maintaining the quality and sensitivity of the standard FCXM This was accomplished by modifying several parameters that, during the optimization phase of the study, were found to effect the FCXM assay First, we changed the assay platform from test tubes to a 96- well tray This approach saved 25-40 minutes of the FCXM time, depending on the number of tests, by reducing centrifugation times (from five minutes to one minute per spin), promoting more efficient delivery of reagents/wash buffers, and efficiently eliminating supernatants by tray "flicking " Next, we reduced serum and IgG-FITC incubation times, saving an additional 30-35 minutes To compensate for the potential decrease in MCF values from reduced incubation times, we enhanced the antibody/antigen reaction by increasing the incubation temperature and serum volume Finally, in the Halifaster FCXM protocol, designed for use with pure/enriched lymphocytes, we reduced the number of target cells per reaction This approach served two purposes, namely increasing both the sensitivity of the FCXM and the likelihood that there would be a sufficient number of lymphocytes available for crossmatches where cell yield is low The Halifaster FCXM protocol is a true time saver, especially when combined with the EasySep Direct lymphocyte purification procedure 10 Combined, the entire crossmatch, from start to finish can be performed in less than two hours Parallel testing with 144 crossmatches demonstrated that the Halifax FCXM protocol provides virtually identical results to the standard FCXM method with an r 2 value of 0 98 for both T cell and B cell crossmatches Importantly, there was a 98 6% concordance in crossmatch interpretation between the Halifax and the standard FCXM protocol with only two false positive reactions (one with Halifax and one with standard FCXM protocol) noted Both false reactions were borderline T+/B-, and would have been interpreted as negative crossmatches in the clinical setting given the absence of DSA on SAB testing Table 2. Comparison of FCXM Performance Using Different Study Protocols T cell FCXM B cell FCXM Parameter Standard Halifax Halifaster Standard Halifax Halifaster Negative control, mean MCF (SD) 168 (21) 168 (18) 176 (16) 216 (25) 215(25) 259 (26) Negative Crossmatch Patient Sera, mean MCF (SD) 161 (26) 158 (25) 170 (24) 213 (32) 203(34) 237 (33) 3SD cutoff value, MCFS 79 74 71 97 101 98 Mean crossmatch cutoff, MCF 247 242 247 313 316 357