Research Qpcr Method for Engraftment Monitoring: A 6-Year Review of Proficiency Testing Results

A total of 40 samples were tested from 11 proficiency surveys from 2006 through 2012 using samples from the American Society for Histocompatibility and Immunogenetics (ASHI) and the Post-SCT Chimerism Monitoring Programme of the United Kingdom National External Quality Assessment Schemes (UK NEQAS) for Leucocyte Immunophenotyping.

Sample percentage breakdown by reported means for the 39 ASHI samples is as follows:  3 did not consist of target cells (0% samples), 5 consisted of target percentages from 5.7 to 22.28%, 8 consisted of target percentages from 26.2 to 49.2%, 20 consisted of target percentages from 51.2 to 95.93%, and 3 consisted only of target cells (100% samples). The single UK NEQAS sample reported a median percentage of 94.70%. Thirty-two of the forty samples (80%) consisted of target percentages above 25%.

All 40 samples tested to date have received passing results with our research qPCR method. For the three 0% samples from 3 separate surveys, our qPCR method gave a result of 0% for each sample while the STR PCR method gave a result other than 0% in 14 instances. For the three 100% samples from 3 separate surveys, our qPCR method gave a result of 100% for each sample while the STR PCR method gave a result other than 100% in 10 instances.

It is not until recently (2012), that more than 1 or 2 testing sites in the ASHI proficiency testing program have used a qPCR method. The last ASHI survey of 2012 and the first and current UK NEQAS trial of the 2012-2013 schedule both included 4 sites using qPCR. The qPCR method is a valuable tool capable of studying chimerism far below the levels attained by STR PCR (e.g. microchimerism < 1%). As the number of sites using qPCR potentially increases, proficiency testing organizations should include lower percentage samples in order to adequately test the lower sensitivities attainable with the qPCR method.

While ASHI participation began in 2006, it was not until 2010 that specific mention of our qPCR approach appeared in the summary report. In the latest ASHI survey (2012 EMO-2), it was recognized in the summary report that both conventional (STR PCR) and qPCR assays can be used successfully based on the comparable performance of the methods. Six years of proficiency testing participation lends concrete evidence that our research qPCR method is a viable alternative to the STR PCR approach offering unmatched sensitivity while maintaining high accuracy and reproducibility with low to high percentage samples.