Methods for non-invasive detection of transplant health or rejection

Abstract

The present invention relates to the technical field of organ and tissue transplantation and specifically to methods allowing rejection of transplanted organs and tissues by a transplant patient. The present invention provides methods for non-invasive and ex vivo or in vitro detection of the presence or absence of transplant rejection, i.e. transplant health or rejection, The present methods allow for effective and continuous monitoring of transplant status with a minimal or absent discomfort for a transplant patent.

1 - 13 . (canceled) 
     
     
         14 . A method for amplifying at least one sample from a recipient of a tissue or organ from a donor, comprising
 (a) amplifying amplicons by nucleic acid amplification of cell-free DNA (cfDNA) wherein each amplicon comprises a Single Nucleotide Polymorphism (SNP), thereby providing amplified amplicons wherein said amplification is performed on:
 (i) at least one sample A of said recipient and at least one sample of said donor; or 
 (ii) at least one sample A of said recipient; 
   (b) determining donor discriminating amplicons;   (c) amplifying cfDNA from a sample B of said recipient; and   (d) determining the presence or amount of donor derived alleles in said amplified cfDNA.   
     
     
         15 . The method of  claim 14 , wherein said amplifying in step (a) comprises a multiplex Polymerase Chain Reaction (PCR). 
     
     
         16 . The method of  claim 14 , wherein said amplifying of cfDNA in step (c) comprises a multiplex Polymerase Chain Reaction (PCR). 
     
     
         17 . The method of  claim 14 , wherein said determining the donor discriminating amplicons in step (b) is performed using massively parallel sequencing. 
     
     
         18 . The method of  claim 14 , wherein said determining the presence or amount of donor derived alleles in step (d) is performed using massively parallel sequencing. 
     
     
         19 . The method of  claim 14 , wherein, in step (d), the amount of donor derived alleles is determined, and said amount is indicative for detection of transplant health or rejection. 
     
     
         20 . The method of  claim 14 , wherein said tissue or organ is selected from the group consisting of heart, blood vessel, gland, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum, endocrine gland, kidney, ureters, bladder, spleen, thymus, spinal cord, ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate, pharynx, larynx, trachea, bronchi, lungs, diaphragm, bone, cartilage, ligament, tendon and parts and tissues thereof. 
     
     
         21 . The method of  claim 14 , wherein said at least one sample of said donor is a biopsy of said transplanted tissue or organ. 
     
     
         22 . The method of  claim 14 , wherein said sample B is derived from blood, urine, faces or sputum. 
     
     
         23 . The method of  claim 22 , wherein said sample is whole blood, serum or plasma. 
     
     
         24 . The method of  claim 14 , wherein said at least one sample A of said recipient was obtained from a patient experiencing acute or chronic transplant rejection. 
     
     
         25 . The method of  claim 14 , wherein steps (c) and (d) are repeated at different time-points after receiving said tissue or organ from said donor. 
     
     
         26 . The method of  claim 14 , wherein step (a) comprises amplifying at least 250 amplicons. 
     
     
         27 . The method of  claim 14 , wherein the amplicons are selected based on amplicons with the least amplification variation, amplicons with the most efficient amplification, and amplicons devoid of allele specific amplification bias. 
     
     
         28 . The method of  claim 14 , wherein step (a) comprises amplifying at least one sample A of said recipient.