METHODS FOR ASSESSING GRAFT SUITABILITY FOR TRANSPLANTATION
This invention relates to methods and compositions for assessing the suitability of a graft for transplantation by measuring total and/or specific cell-free nucleic acids (such as cf-DNA) and/or cell lysis. Specifically, the method comprising obtaining an amount of total short fragment cf-DNA and/or graft-specific short fragment cf-DNA released from a potential graft (e.g., ex vivo), e.g., prior to contacting of the potential graft with blood cells of a potential recipient, and/or subsequent to contacting of the potential graft or cells thereof with blood cells from a potential recipient, and assessing the amount(s) to determine the suitability of the potential graft for transplantation.
1 . A method of assessing the suitability of a potential graft for transplantation, comprising:
a. obtaining an amount of total short fragment cf-DNA and/or graft-specific short-fragment cf-DNA released from a graft prior to contacting the graft with blood cells from a potential recipient, and
b. assessing the amount(s) to determine the suitability of the graft for transplantation.
2 . The method of claim 1 , wherein obtaining the amount comprises performing a method to quantify the amount of total short fragment cf-DNA and/or graft-specific short fragment cf-DNA released by the graft.
3 . The method of claim 1 or 2 , wherein the method further comprises performing a method to quantify the amount of total short fragment cf-DNA and/or graft-specific short-fragment cf-DNA released by the graft subsequent to contacting the graft or cells thereof with blood cells from a potential recipient.
4 . A method of assessing the suitability of a graft for transplantation, comprising:
a. obtaining an amount of total short fragment cf-DNA and/or graft-specific short fragment cf-DNA released by the graft subsequent to contacting of the graft or cells thereof with blood cells from a potential recipient, and
b. assessing the amount(s) to determine the suitability of the potential graft for transplantation.
5 . The method of claim 4 , wherein obtaining the amount comprises performing a method to quantify the amount(s) of total short fragment cell-free DNA and/or graft-specific short fragment cf-DNA in order to obtain the amount.
6 . The method of any one of claims 1-5 , wherein the method further comprises determining an amount of cell lysis.
7 . The method of claim 6 , wherein the determining the amount of cell lysis comprises performing fragment analysis.
8 . The method of claim 7 , wherein the fragment analysis comprising quantifying long and/or short fragments.
9 . The method of any one of the preceding claims , wherein the method further comprises one, two, three or more centrifugation steps of the sample(s) prior to obtaining the amount of total short fragment cf-DNA and/or graft-specific short-fragment cf-DNA and/or cell lysis.
10 . The method of any one of the preceding claims , wherein the method further comprises obtaining the potential graft or cells thereof.
11 . The method of any one of the preceding claims , wherein the method further comprises obtaining an amount of total short fragment cf-DNA and/or graft-specific short-fragment cf-DNA and/or determining the amount of cell lysis at one or more additional time points.
12 . The method of any one of the preceding claims , wherein the method further comprises comparing the amount(s) of the total short fragment cf-DNA and/or graft-specific cf-DNA and/or amount of cell lysis with threshold value(s) or amount(s) obtained from additional time point(s).
13 . The method of any one of the preceding claims , wherein the amount(s) of the total short fragment cf-DNA and/or graft-specific cf-DNA and/or amount of cell lysis are obtained in a sample of media in which the potential graft or cells therefrom are contained or are in contact with.
14 . The method of claim 13 , wherein the media is a storage, perfusion, or preservation media in which the graft is contained or in contact with.
15 . The method of claim 13 or 14 , wherein the cf-DNA is released from the graft into the media.
16 . The method of any one of the preceding claims , wherein the amount(s) is/are obtained in a perfusate sample.
17 . The method of any one of the preceding claims , wherein the amount(s) is/are obtained in an EVLP sample.
18 . The method of any one of the preceding claims , wherein the method further comprises obtaining the blood cells (e.g., blood) from the potential recipient.
19 . The method of any one of the preceding claims , wherein the potential graft or cells thereof are from a potential donor of the same species as the potential recipient.
20 . The method of any one of the preceding claims , wherein the potential graft is an organ or organs.
21 . The method of claim 20 , wherein the organ or organs comprise a heart, a lung, or a heart and a lung.
22 . The method any one of the preceding claims , wherein the method further comprises providing the values for the amount(s) of total cell-free DNA and/or graft-specific cf-DNA and/or cell lysis in a report.
23 . The method of any one of the preceding claims , wherein the method further comprises providing a determination about the suitability of the graft.
24 . The method of any one of the preceding claims , wherein the graft is monitored over time.
25 . The method of claim 24 , wherein the graft is assessed every 15 minutes, every 30 minutes, hourly, daily, weekly, bimonthly or monthly prior to transplantation.
26 . The method of any one of the preceding claims , wherein the total short fragment cell-free DNA and/or graft-specific short fragment cf-DNA and/or cell lysis is measured using a quantitative amplification method.
27 . The method of claim 26 , wherein the quantitation amplification method is selected from real-time PCR, digital PCR, and mismatch PCR.
28 . The method of any one of claims 1-25 , wherein the amount of total short fragment cell-free DNA and/or graft-specific short fragment cf-DNA and/or cell lysis is measured using next-generation sequencing.
29 . The method of any one of the preceding claims , wherein when the value for the amount(s) are above a threshold value or a value from a prior point in time, decreasing suitability of the graft or unsuitability of the graft for transplantation is indicated.
30 . The method of any one of the preceding claims , wherein when the value for the amount(s) are below a threshold value or a value from a prior point in time, increasing suitability of the graft or suitability of the graft for transplantation is indicated.
31 . The method of any one of the preceding claims , further comprising performing treatment of the graft, potential donor, and/or recipient, or providing information regarding such a treatment.
32 . A report comprising the value(s) or amount(s) of total short fragment cell-free DNA, specific short fragment cell-free DNA, and/or cell lysis obtained by the method of any one of the preceding claims .
33 . The report of claim 32 , further comprising one or more threshold values and/or values from one or more additional time points.