Quantification of Donor-Derived Cell-Free DNA
Venous blood samples were collected into 10 mL Streck cell-free DNA BCT tubes (Streck, Omaha, NE) and examined within 7 days. Plasma was separated by centrifugation at 1,600 × g for 20 min, followed by a second centrifugation at 16,000 × g for 10 min, and either plasma was stored at −80°C or cfDNA was extracted immediately using the Circulating Nucleic Acid kit (Qiagen, Redwood City, CA). cfDNA was then amplified using the AlloSeq cfDNA assay (CareDX, Brisbane, CA), a multiplex PCR including PCR primers for 202 single nucleotide polymorphisms (SNPs). Variations in the SNPs loci are used to determine the proportion of donor-derived (dd)-cfDNA in relation to the total cfDNA present in the plasma sample. The PCR products were subsequently sequenced on a MiniSeq (Illumina, Inc.). Sequence data was analyzed using the CareDx AlloSeq cfDNA software. All steps were performed according to the manufacturers’ instructions and as described previously [