Chromosome Y Detection in Sex-Mismatched Transplant Recipients. Because previous research has reported conflicting results on the possibility of detecting genetic signatures from transplanted organs using conventional PCR (14, 15), we first explored the use of the more sensitive technique of microfluidic digital PCR (21, 22) in sex-mismatched transplants where a female recipient has received a male donor heart. We purified DNA from the plasma of nine patients collected immediately before an endomyocardial biopsy that established a cellular rejection episode (≥grade 3A/2R). Six patients (identified as patients 1–6) were females who had received a heart from a female donor and three patients (identified as patients 7–9) were females who had received a heart from a male donor. Digital PCR was performed on the cell-free DNA using probes for chromosome (Chr) 1 and chromosome Y to establish the % Chr Y signal relative to Chr 1 (Fig. 2A). For the six female patients receiving organs from female donors, either no or a very low level of Chr Y was observed 0.32 ± 0.27% (SD). However, for the three female patients who received male organs, in four unique plasma samples (one patient had two documented rejection events), a greater than tenfold higher average level of Chr Y was observed, 3.93 ± 3.07% (SD), with a range from 1.4 to 8.2%. The signal from the male-donor samples is well separated from that of the female-donor controls (P = 0.018, Student's t test). These data establish that genetic material from the transplanted organ can be detected in the recipient's plasma during rejection episodes.