116-P: EVALUATION OF ENGRAFTMENT MONITORING FOR ALLOGENEIC AND DOUBLE CORD BLOOD HEMATOPOIETIC STEM CELL TRANSPLANT RECIPIENTS USING REAL-TIME qPCR

Aim To evaluate and validate utilization of the more sensitive engraftment monitoring method, real-time quantitative polymerase chain reaction (qPCR), in detecting chimerism levels during early stages of relapse or rejection. Methods Twenty-four samples from 22 stem cell recipients (14 male; 8 female) were used for this study. The average age of recipients was 54.9 ± 12.6 years. Samples used for analysis were obtained from peripheral blood (n=14) or bone marrow (n=10) and were 274.2 ± 302.7 days post-transplant. Sixteen received allogeneic hematopoietic stem cell transplants and 6 received double cord transplants. Samples were tested for inter- and intra-assay variation. Short tandem repeat (STR) analysis was performed using the AmpFLSTR® Identifiler® PCR Amplification Kit (Life Technologies). qPCR was performed using the AlleleSEQR® Chimerism Assay (Celera Corporation). Results Initial studies with contrived mixtures confirmed the sensitivity of the qPCR to be lower than 0.5%. Based on these studies, we chose to run 2 informative qPCR markers, unique for the minor population, in our clinical assays. These markers should demonstrate the lowest delta Ct values and should not be located on the same chromosome or on chromosomes with known disease-related abnormalities. qPCR results were comparable with STR results for 22 of the 24 samples. Concordance was poor when the pre-determined “minor population” tested at greater than 50%. Reproducibility of the screen and quantitative qPCR assays using informative markers chosen by each method was also demonstrated. Conclusions Validation studies were successful. The sensitivity of the qPCR assay is excellent in the lower ranges of chimerism (below 30-40%), but the accuracy can be reduced when the “minor population” levels increase. The increased sensitivity of the qPCR assay can be useful in evaluating double cord blood recipients, particularly in the early times post-transplant.