Detection and quantification of donor cell-free DNA in the circulation of organ transplant recipients
This invention provides methods, compositions, and kits relating to detecting donor cell-free DNA in the circulation of a transplant recipient for the early identification of transplant rejection or for detection of damage to transplant cells.
1. A method of quantifying the amount of donor DNA in a cell-free blood sample of a recipient of a transplanted organ from a donor without genotyping a sample obtained from said donor, the method comprising:
(a) identifying that one or more SNPs of a panel of SNPs preselected to have a minor allele frequency of 0.4 or greater across human populations are homozygous in the recipient;
(b) performing a reaction comprising digital PCR to quantify the one or more SNPs identified in (a), in a cfDNA preparation from a blood sample from which cells have been removed obtained from the recipient following transplantation of donor tissue to the recipient, to detect the level of an alternative allele to the allele present in the recipient, wherein detecting the level of the alternative allele comprises counting the DNA molecules that comprise the alternative allele compared to those that comprise the allele present in the recipient;
wherein the amount of donor DNA in the cell-free cell sample is quantified without genotyping a donor specific sample.
2. The method of claim 1 , further comprising a step of amplifying cfDNA from the blood sample to generate a cf library.
3. The method of claim 1 , wherein step (a) is performed using DNA isolated from peripheral blood leukocytes obtained from the recipient.
4. The method of claim 1 , wherein step (b) comprises performing droplet digital PCR.
5. The method of claim 1 , wherein the level of the alternative allele is determined as a percentage of total cfDNA.
6. The method of claim 1 , wherein the at least one SNP having an alternative allele quantified in (b) is homozygous in the donor.
7. The method of claim 1 , wherein the level of the alternative allele is determined as a concentration.
8. The method of claim 1 , further comprising obtaining cfDNA from a further blood sample from which cells have been removed from the recipient following transplantation of donor tissue; and
quantifying the level of the alternative allele for the at least one SNP of (b) in a cfDNA preparation from the further blood sample.
9. The method of claim 8 , wherein the quantifying step comprises determining a concentration of the alternative allele in the cfDNA preparation from the further blood sample.
10. The method of claim 8 , wherein the quantifying step comprises determining the percentage of the alternative allele in the cfDNA preparation from the further blood sample.
11. The method of claim 8 , wherein the transplanted material is a marginal organ.
12. The method of claim 8 , wherein the further blood sample is obtained at least seven days following transplant.
13. The method of claim 8 , wherein the further blood sample is obtained a year or longer following transplant.
14. The method of claim 12 , further comprising adjusting an administration schedule or dosage of an immunosuppressive drug.
15. The method of claim 8 , further comprising detecting donor-specific antibodies in the blood of the recipient.
16. The method of claim 1 , wherein the blood sample is obtained at least five days following transplant.
17. The method of claim 1 , wherein the transplanted organ is a marginal organ.
18. The method of claim 1 , wherein the transplanted organ is a heart, liver, or kidney.
19. The method of claim 1 , wherein the blood sample is serum.
20. The method of claim 1 , wherein the blood sample is plasma.
21. The method of claim 8 , wherein the further blood sample is serum.
22. The method of claim 8 , wherein the further blood sample is plasma.