Ultra-high sensitive monitoring of early transplantation failure

Abstract

The present invention provides a method for detecting transplantation failure of a transplanted organ or cells which comprises detecting a donor-positive but recipient-negative DNA marker in the recipient's plasma using pyrophosphorolysis activated polymerization. Because of the high sensitivity, specificity and selectivity of pyrophosphorolysis activated polymerization, transplantation failure can be detected at early stages and treatment can be initiate earlier.

What is claimed is: 
     
         1 . A method of detecting transplantation failure of a transplanted organ or transplanted cells in a recipient which comprises:
 (a) screening one or more DNA polymorphisms to identify one or more donor-positive, recipient-negative DNA polymorphisms and   (b) tracking the presence or increased level of one or more donor-positive, recipient-negative DNA polymorphisms in recipient samples over time,   wherein the presence or increased level of one or more donor-positive, recipient-negative DNA polymorphisms in recipient samples over time indicates transplantation failure.   
     
     
         2 . The method of  claim 1 , wherein the screening and tracking are done using bi-directional pyrophosphorolysis activated polymerization. 
     
     
         3 . The method of  claim 1 , wherein the one or more donor-positive, recipient-negative DNA polymorphisms are selected from a panel of polymorphisms. 
     
     
         4 . The method of  claim 3 , wherein the panel of polymorphisms is universal to multiple donors and multiple recipients. 
     
     
         5 . The method of  claim 1 , which further comprises before step (a) the step:
 selecting a panel comprising more than one DNA polymorphism.   
     
     
         6 . The method of  claim 5 , wherein the panel comprises 11 or more DNA polymorphisms. 
     
     
         7 . The method of  claim 5 , wherein the DNA polymorphisms are single nucleotide polymorphisms. 
     
     
         8 . The method of  claim 7 , wherein each of the polymorphisms (i) is a validated polymorphism, (ii) is selected from the group consisting of C/G, G/C, T/A and A/T polymorphisms and (iii) has a frequency of greater than or equal to about 30% and less than or equal to about 70%, preferably about 50%. 
     
     
         9 . The method of  claim 8 , wherein the polymorphisms are those set forth in Table 2. 
     
     
         10 . The method of  claim 9 , wherein primers for each polymorphism are those set forth in Table 2. 
     
     
         11 . The method of  claim 1 , wherein a homozygous and/or heterozygous genotype of a biallelic polymorphism is selected as a DNA polymorphism for the donor and a homozygous genotype is selected as a DNA polymorphism for the recipient. 
     
     
         12 . A method of detecting transplantation failure of a transplanted organ or transplanted cells in a recipient which comprises:
 (a) isolating a donor sample and a recipient sample,   (b) screening one or more DNA polymorphisms in the donor and recipient samples to identify one or more donor-positive, recipient-negative DNA polymorphisms and   (c) tracking the presence or increased level of one or more donor-positive, recipient-negative DNA polymorphisms in recipient samples over time,   wherein the presence or increased level of one or more donor-positive, recipient-negative DNA polymorphisms in recipient samples over time indicates transplantation failure.   
     
     
         13 . The method of  claim 12 , wherein the tracking step comprises:
 (i) isolating a recipient sample,   (ii) detecting the presence or increased level of one or more donor-positive, recipient-negative DNA polymorphisms in the recipient sample, and   (iii) repeating steps (i) and (ii) over time.   
     
     
         14 . The method of  claim 12 , wherein the screening and tracking are done using bi-directional pyrophosphorolysis activated polymerization. 
     
     
         15 . The method of  claim 12 , wherein the one or more donor-positive, recipient-negative DNA polymorphisms are selected from a panel of polymorphisms. 
     
     
         16 . The method of  claim 15 , wherein the panel of polymorphisms is universal to multiple donors and multiple recipients. 
     
     
         17 . The method of  claim 12 , which further comprises before step (a) the step:
 selecting a panel comprising more than one DNA polymorphism.   
     
     
         18 . The method of  claim 17 , wherein the panel comprises 11 or more DNA polymorphisms. 
     
     
         19 . The method of  claim 17 , wherein the DNA polymorphisms are single nucleotide polymorphisms. 
     
     
         20 . The method of  claim 19 , wherein each of the polymorphisms (i) is a validated polymorphism, (ii) is selected from the group consisting of C/G, G/C, T/A and A/T polymorphisms and (iii) has a frequency of greater than or equal to about 30% and less than or equal to about 70%, preferably about 50%. 
     
     
         21 . The method of  claim 20 , wherein the polymorphisms are those set forth in Table 2. 
     
     
         22 . The method of  claim 21 , wherein primers for each polymorphism are those set forth in Table 2. 
     
     
         23 . The method of  claim 12 , wherein a homozygous and/or heterozygous genotype of a biallelic polymorphism is selected as a DNA polymorphism for the donor and a homozygous genotype is selected as a DNA polymorphism for the recipient. 
     
     
         24 . A method of treating transplantation failure of a transplanted organ or transplanted cells in a subject which comprises:
 (a) detecting transplantation failure in a subject according to the method of  claim 1  and   (b) initiating treatment of transplantation failure in the subject if transplantation failure is detected in step (a).   
     
     
         25 . A method of detecting cell death or cellular damage in a recipient receiving transplanted cells which comprises:
 (a) screening one or more DNA polymorphisms to identify one or more recipient-positive, donor-negative DNA polymorphisms and   (b) tracking the presence or increased level of one or more recipient-positive, donor-negative DNA polymorphisms in recipient samples over time,   wherein the presence or increased level of one or more recipient-positive, donor-negative DNA polymorphisms in recipient samples over time indicates cell death or cellular damage in the recipient.   
     
     
         26 . The method of  claim 15 , wherein the screening and tracking are done using bi-directional pyrophosphorolysis activated polymerization. 
     
     
         27 . The method of  claim 25 , wherein a homozygous genotype of a biallelic polymorphism is selected as a DNA polymorphism for the donor and a homozygous and/or heterozygous genotype is selected as a DNA polymorphism for the recipient. 
     
     
         28 . A method of detecting cell death or cellular damage in a recipient receiving transplanted cells which comprises:
 (a) isolating a donor sample and a recipient sample,   (b) screening one or more DNA polymorphisms in the donor and recipient samples to identify one or more recipient-positive, donor-negative DNA polymorphisms and   (c) tracking the presence or increased level of one or more recipient-positive, donor-negative DNA polymorphisms in recipient samples over time,   wherein the presence or increased level of one or more recipient-positive, donor-negative DNA polymorphisms in recipient samples over time indicates cell death or cellular damage in the recipient.   
     
     
         29 . The method of  claim 28 , wherein the tracking step comprises:
 (i) isolating a recipient sample,   (ii) detecting the presence or increased level of one or more recipient-positive, donor-negative DNA polymorphisms in the recipient sample, and   (iii) repeating steps (i) and (ii) over time.   
     
     
         30 . The method of  claim 28 , wherein the screening and tracking are done using bi-directional pyrophosphorolysis activated polymerization. 
     
     
         31 . The method of  claim 28 , wherein a homozygous genotype of a biallelic polymorphism is selected as a DNA polymorphism for the donor and a homozygous and/or heterozygous genotype is selected as a DNA polymorphism for the recipient.