General scheme for this study. Cell-free DNA collected in plasma contains a majority of molecules from the recipient (in gray) but may also include some from the transplanted organ (green). Due to increased cell death in the organ during a rejection episode, more donor molecules are expected to be present in the blood at these times. Shotgun sequencing of the purified DNA allows for counting recipient versus donor molecules by looking at single nucleotide polymorphisms (SNPs) that vary between donor and recipient. Very high levels of donor DNA, particularly changes from past measurements, will indicate the onset of rejection.

Donor DNA levels determined by digital PCR using a chromosome Y marker. (A) Ten plasma samples from patients with biopsy-determined rejection events with grade ≥3A–2R were analyzed, including six female patients (1–6) receiving female organs (six total events) and three female patients (7–9) receiving male organs (four total events). Group averages are marked by black lines. (B and C) Time-course graphs of chromosome Y levels (% donor DNA) in patients 7 (B) and 8 (C). Both patients had grade 3A–2R rejections as determined by biopsy at the indicated time point.

SNP-based detection of % donor DNA in control HapMap samples. There is a linear response (R² = 0.998) of calculated % donor DNA compared with the % donor in the mock sequencing libraries. The trendline is given for all SNPs, including both heterozygous and homozygous donor SNPs, but the calculated percentages are similar in both subsets.

Donor DNA levels determined by sequencing. (A–C) Time-course graphs for patients 7 (A) and 11 (B), female patients receiving male hearts also analyzed by digital PCR, and for patient 14 (C), a male patient receiving a male heart. Patients 7 and 14 had grade 3A–2R rejections as determined by biopsy at the indicated time point, whereas patient 11 was a negative control with no rejection events. The calculated donor SNP signal is graphed in purple using both homozygous and heterozygous donor SNP positions. The matched SNP background is graphed in green, demonstrating the error for the assay arising from sequencing errors, genotyping errors, or sample contamination. The difference between the green and purple trend lines represents the changing level of donor-specific DNA. (D) Collected sequencing results for all patients analyzed by sequencing (7, 8, 11, 14–17) using the corrected % donor DNA values. Samples coincident with a biopsy determining an acute cellular rejection event are grouped together in red. Samples within 3 mo of a biopsy-determined rejection are shown in orange. Samples greater than 3 mo from any biopsy-determined rejection, or from a patient without any rejection events, are considered “healthy” normal readings and are shown in green. Group averages are marked by black lines.