Methods for detection of donor-derived cell-free dna

Abstract

The present disclosure provides methods for determining the status of an allograft within a transplant recipient from genotypic data measured from a mixed sample of DNA comprising DNA from both the transplant recipient and from the donor. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

What is claimed is: 
     
         1 . A method of preparing a blood sample for assessment of organ transplant injury or failure in a transplant recipient that has received an organ from a donor, comprising
 receiving a first blood sample from the transplant recipient, the first blood sample being extracted from the transplant recipient at a first time;   extracting a first portion from the first blood sample, wherein the first portion includes DNA comprising cell-free DNA derived from the transplant recipient (bg-cfDNA) and DNA derived from the donor of the transplanted organ (dd-cfDNA); and   performing amplification of 50-50,000 target loci in the first portion in a single reaction volume using 50-50,000 primer pairs, wherein the target loci comprise polymorphic loci and   non-polymorphic loci of up to 100 base pairs.   
     
     
         2 . The method of  claim 1 , wherein the transplanted organ is a kidney, and wherein the method further includes extracting a second portion from the blood sample and measuring serum creatinine level in the second portion. 
     
     
         3 . The method of  claim 2 , wherein the serum creatinine level is not greater than 20% above a steady-state baseline level. 
     
     
         4 . The method of  claim 3 , comprising enriching the dd-cfDNA relative to bg-cfDNA in the first extracted sample, amplifying the enriched DNA, and preferentially enriching specific loci in the amplified DNA. 
     
     
         5 . The method of  claim 4 , wherein the amplification occurs simultaneously for all primers of the target loci. 
     
     
         6 . The method of  claim 4 , wherein the target loci include greater than 10,000 single-nucleotide polymorphisms (SNPs). 
     
     
         7 . The method of  claim 4 , comprising:
 a) receiving a second blood sample taken from the transplant recipient at a second time, the second time being after the first time.   b) extracting a second portion from the second blood sample, wherein the second portion includes DNA comprising bg-cfDNA and dd-cfDNA;   c) performing amplification of 50-50,000 target loci in the second portion in a single reaction volume using 50-50,000 primer pairs, wherein the target loci in the second portion comprise polymorphic loci and non-polymorphic loci of up to 100 base pairs; and   d) inserting the amplification products into a high-throughput sequencing machine.   
     
     
         8 . The method of  claim 1 , wherein the transplant recipient has experienced a physical injury. 
     
     
         9 . The method of  claim 1 , further comprising:
 inserting the amplification products into a high-throughput sequencing machine.   
     
     
         10 . A method of treating a patient that has received a kidney transplant from a donor, wherein the patient has experienced organ injury, comprising
 receiving, from a lab, results of testing of a patient blood sample, the results indicative of first and second component levels in the blood sample, wherein the first component level comprises serum creatinine at first percentage level in the blood sample, the second component level comprises dd-cfDNA derived from the kidney donor at a second percentage level, the dd-cfDNA having been sampled from an extracted portion of the blood sample and enriched relative to background cell-free DNA of the patient; and   when the second percentage level of dd-cfDNA exceeds 1%, or the first percentage level of serum creatinine exceeds 20% of a steady-state baseline level, applying a course of immunosuppressant therapy to the patient during a first time period.