Director, HLA Laboratory Children's Hospital Los Angeles Los Angeles, California, United States
Abstract Text: Background Accurate and sensitive quantitation of donor and recipient DNA is clinically important in both hematopoietic stem cell and solid-organ transplantation. Next-generation sequencing can simultaneously measure thousands of targets however has a relativity long turnaround time and high cost. Real-time and digital PCR have offered faster and less expensive alternatives but are practically limited by number of targets per assay. Objectives Our aim was to develop an easy-to-use multiplex digital PCR (mdPCR) method that can detect more than 10 targets in a single reaction while achieving rapid, accurate, sensitive, and absolute DNA quantitation. Methods A novel prototype assay (multiplex PCR chemistry, Luminex, Austin TX) detecting 12 alleles in a single well was evaluated using a microfluidic digital PCR platform (Thermo Fisher Scientific Absolute Q Digital PCR). Performance characteristics were assessed for samples with 1,000 and 10,000 human genome equivalents. Results Each run (up to 16 samples) was completed in < 3 hours. With a sample input of 10,000 copies of genomic DNA, the LoB for 7 probes was 0 – 0.02% and the analytical LoD was 0 – 0.03%. When testing 1,000 copies of genomic DNA, for 7 probes LoB was 0 – 0.18% and the analytical was LoD 0 – 0.30%. The remaining probes require optimization. Conclusions Initial designs for the prototype mdPCR assay show high promise for providing accurate, sensitive, cost-effective, same-day quantitative DNA testing. This methodology offers exciting opportunities for chimerism and cell-free DNA testing after transplantation and will be useful in cancer and infectious diseases.
