Detection of microchimerism with high sensitivity droplet digital PCR technique and its significance in monitoring of hematopoietic stem cell transplanted patients

Introduction: In patients undergoing allogeneic hematopoietic stem cell transplantation, a disease -specific biomarker is not always available. Such cases can be monitored with a "chimerism test", which provides information about relapse, engraftment and recipient -derived hematopoiesis. Determination of clinical chimerism can be performed by different methods. In case of hematopoietic stem cell transplantation, the detection of "short tandem repeats" (STR) by fragment analysis can detect up to 1-5% recipient ratio, while more sensitive and accurate techniques are capable of measuring microchimerism (<1%). Objective: Introduction of a method based on the detection of deletion insertion polymorphisms (DIP) by droplet digital PCR (ddPCR) and comparison of results measured with STR and DIP methods. Method: The assay was set up with artificial, mixed chimera samples prepared from genomic DNA of volunteers (n = 6), and limit of blank and limit of detection values were calculated. We correlated STR results to those of DIP technique (n = 48 recipients, 146 samples). Informativity values were calculated by using 403 transplantation cases. We used 8 DIP markers and a Y chromosome specific marker in our study. Retrospective studies were performed for early relapse -detection. Results: The results achieved by ddPCR showed strong correlation with STR in the 1-100% mixed chimerism range (R2 = 0.988, n = 146 samples). The average informativity value was 96% in case of transplantation with one donor, i.e., we were able to detect the mixed chimerism state with at least 1 marker with a high probability. The new method shortened the turnaround time and limit of detection is improved by 1-1.5 orders of magnitude compared to STR. Conclusion: With regular ddPCR monitoring, disease relapse can be predicted in an early phase before the onset of clinical relapse. The potential applications of ddPCR-based high -sensitivity chimerism detection are the following: microchimerism during hematopoietic microtransplantation, early detection of graft rejection after solid organ transplantation, and microchimerism studies in autoimmune diseases or pregnancies.