WO2018035340A1 - A novel immunoprobe-based method to assess organ injury status through a biofluid-based cell-free dna (cfdna) assay - Google Patents

A novel immunoprobe-based method to assess organ injury status through a biofluid-based cell-free dna (cfdna) assay Download PDF

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WO2018035340A1
WO2018035340A1 PCT/US2017/047372 US2017047372W WO2018035340A1 WO 2018035340 A1 WO2018035340 A1 WO 2018035340A1 US 2017047372 W US2017047372 W US 2017047372W WO 2018035340 A1 WO2018035340 A1 WO 2018035340A1
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cfdna
method
kidney
sample
probe
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Minnie M. Sarwal
Tara K. SIGDEL
Joshua Y. YANG
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The Regents Of The University Of California
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Abstract

This present disclosure provides methods and compositions that can be used to quantify cfDNA in biofluids using a hybridization approach.

Description

A NOVEL IMMUNOPROBE-BASED METHOD TO ASSESS ORGAN INJURY STATUS THROUGH A BIOFLUID-BASED CELL-FREE DNA

(CFDNA) ASSAY

CROSS-REFERENCES TO RELATED APPLICATIONS

[00011 This application claims priority to U.S. Provisional Application No, 62/376,299, filed August 17, 2016, the disclosure of which % hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002} Twenty-six million Americans suffer from organ injuries,- such as those associated with chronic kidney disease (CKD), and organ transplant rejection and dysfunction, e.g., kidney transplant, o lung transplant. Treatment of these iiiiuries is very costly. For example, twenty-eight percent of annual. Medicare spending, $57.5 billion, is spent on treating CKD. However, for many of these, there is no way to predict when the injury is imminent until clinical symptoms emerge. A l arge proportion of the costs associated wi th these diseases are doe to lack of early de tec tion leading to more severe organ injury and requiring greater arid more expensive therapeutic intervention. Methods for earlier detection of kidney pathologies would enable reductions in medical costs and more effecti ve therapeutic intervention.

[0003} Traditionally, detection of organ injury, e.g. , kidney Injun-', is based on examination of biopsies, which is both costly and i vasive. More recently, cell-tree DMA (CfDNA) from dying celi.s has been discovered in humanurine. Recent efforts have focosed on testing o urine for cfDNA as a marker for allograft rejection rather than organ injury; furthermore, th technique has been limited to using PCR or next-generation sequencing for the detection of donor-specific SNPs or the measurement of Aits elements. These methods have limitations because they are limited to testing for kidney rejection, ar expensi ve, both in consumables and equipment, require relaiively larger quantities of DNA, and are not high-throoghput. Further, ihey may not be able to detect fragment lengths shorter tha 150 bp as it has been found that, although whole, uhfragrnented DNA was optimal for a qPCR based approach for measuring Alu, there was a significant reduction (75% reduction) in DNA quantification for DNA at a fragment. size of < 150 bp (Sedlackova et at, Biol. Proced. Online (2013) 1.5:5. doirlO.l 186/ .1480-9222-1.5-5).

BRIEF SUMMARY OF THE INVENTION

[0ΘΘ4] A novel methodology for quantitative analysis of cell-free, D A in biofluid, such as m ne and bronehoaiveo!ar lavage (B L), is provided. Aspects described herein include -a preservative cocktail of reagents that cao stabilize biofluid cellular DNA , a method of measuring cfDNA by hybridization assay that quantifies human Ala repeats in. the biofluid, and a novel analysis method that factors in clinical assay variables to provide a quantitative risk score for kidney injury. In some cases, the amounts of additional markers in the sample are measured, such as methylat ion markers (e,g„ 5-methyieytosine), tissue inflammation markers (e.g.,

CXCLIO), apoptosis markers (e.g., kidney tubular injury markers, such as elusterin), total protein, and/or creatinine. These aspects can be used to assess the kidney health in the context of kidney transplantation and kidney disease.

[0005] in one aspect, this disclosure provides a solution, e.g., a sterile solution, that comprises a formaldehyde donor, a chelator, aurintoearboxyMe acid, and polyethylene glycol (PEG) in a concentration sufficient to inhibit cell lysis and to. inhibit nucleases in urine. In some

embodiments, th solution further comprises sodium aside and or a buffer, In some

embodiments, the solution further comprises a biofluidic sample. In some embodiments, the biofloidie sample is a urine sample or a BA.L sample, as the effluent of choice for non-invasive measurmeni of kidney or kin organ injury. In some embodiments, the urine sample is from a patient who has received a. kidney transplant or has acute or chronic kidney injury, in others it is the bronchoalveoiar lavage (BAL) fluid. from a patient who has received a lung transplant or has lung injury.

[0006] in one aspect, this disclosure provides a method of detecting ALu copy number in a biofluid sample, the method comprising: obtaining urine or other biofluid sample from a human, extracting cfDNA from the sample, forming a reaction mixture by contacting the cfDNA with a nucleic acid probe under conditions to allow the probe to hybridize to D A in the cfDNA that is complementary to the probe, wherein the nucleic acid probe has a nucleic acid sequence having a 3' and 5' end, wherein the nucleic acid probe is complementary to contiguous 20-292 nucleotides of SEQ ID NO , wherein the' 3* or 5' end is covalently linked to a detectable label; and quantifying die amount of DNA hybridized to the probe. In some embodiments, the probe is complementary to at feast 50 contiguous nucleotides of. SEQ ID NO:2. hi some embodiments,, the probe comprises a sequence .of 50-150, 70-] 00, 80-90, or exactly 8.1 nucleotides

complementary to SEQ ID NO; I. in some embodiments, the detectable label is bi titi and the method comprises contacting the detectable label with a streptavidia-!iaked signal producing agent.

[0007] In some embodiments, before forming there-action mixture, mixing a solution comprising diazolidinyi urea, ED'TA, aurinuiearboxyKe acid, and .polyethylene glycol (PEG) in a concentration sufficient to inhibit cell lysis and. o inhibit nucleases into the urine sample, in some embodiments, the method further comprises quantifying the -amount of creatinine in the reaction .mixture, in some embodiments, the method further comprises normalizing the amount of cfDNA hybridized to the probe against the amount of creatinine in the urine sample to produce a .normalized amount of cfD A. In some embodiments., the method further comprises normalizing the amount of cfDNA hybridized to the probe against the amount of creatinine in the urine sample to produce a normalized amount of hybridized DMA. In some embodiments, the method further comprises determining the patient has kidney injury if the detected amount of target DNA (e.g.., cfDNA) hybridized to the probe or the normalized amount of target DNA (e.g. cfDNA) is greater than the c utoff value.

|00OS| In some embodiments, the human is a patient who has received a kidney or lung transplant and the presence of kidney or lung injury indicates the patient may have acute rejection episodes. In some embodiments, the method farther comprises producing a prediction score for deie iiniag kidney or lung health based on the -normalized Amo nt of target DN A (e.g., cfDNA) and the time post-transplant of the kidney. The patient is determined to have acute rejection episodes when the predictive score is greater than a cutoff value for the predictive scores. In some embodiments, the -urine or BAL- sample is taken 0-400 days, or .10-100 days, or 20-50 days from the patient's receivin a kidney or lung transplant j OO09| In some embodiments , the urine- sample is from an- individual suspected of having, a kidne injury caused by a disease selected from the group consisting of BK viral nephritis, focal segmental glomerulosclerosis (FSGS), kidney stone, acute tubular - necrosis (ATN), IgA nephropathy (IgAN), and diabetic kidney disease or kidney disease from systemie diseases such as hypertension and autoimmune disorders (eg SLY, rheumatoid .arthritis). In these em od ment,, a detennmation of kidney injury indicates the patient has the disease.

{0010} In some embodiments, the method further comprises quantifying the amount CXCLIO in the reaction mixture to add greater specificity and sensitivity to the assa through the addition of a biomarkex reflec ting the .inf$arnn¾atory burden. In some embodiments, the method further comprises quantifying the proportion or absolute quantity of methylated cfDNA and

liydroxymeth lated cfDNA to reflect the methyiation status of the circulating DNA which further defines the presence of intrinsic tissue injury.

{0011} In another aspect, the disclosure provides a reaction mixture comprising non-amplified cell-free DMA (cfDNA) extracted from a -urine sample (e.g., in some embodiments, from a patient who has received a kidney transplant) and ii) a nucleic acid probe having a nucieic acid sequence and having a 3 \and 5' end, wherein the nucleic acid probe is complementary to 20-292 contiguous nucleotides of SEQ ID NO: I and wherein the 3' or 5' end i co va!enily linked to a detectable label. The reaction mixture can comprise any of the components described herein.

{00 2} In another aspect, pro vided herein is a method of detecting Ala copy number in cell- free DNA (cfDNA) in a biofluid sample from an individual having a- lung transplant or mg transplant clinical conditions. The method comprises obtaining a biofluid sample, e.g., a bronchoalveolar' lavage (BAL) fluid sample, from a human; extracting cfDNA from the

bioflnidic sample; forming a reaction mixture by contacting the cfDNA. with a nucleic acid probe under conditions to allow the prob to hybridize to DNA in the cfDNA that is complementary to the probe, wherein the nucleic acid probe has a nucieic acid sequence 'having a .3* and 5' end, wherein the nucleic acid probe is complementary to contiguous 20-292 nucleotides of SEQ ID NOrl and wherein the 3' or 5' end is covaleutly linked detectable label; and quantifying the amount of DNA hybridized to the probe, thereby detecting Alu copy number in cell-free DNA (cfDNA) in the biof!nidie sample. In some- embodiments, the biofluidio sample is a

bronchoalveolar lavage (BAL) fluid sample and wherein the method further comprises

quantifying the total volume of BAL fluid.

[0013] In some embodiments, the method further comprises normalizing the amount of cfDNA hybridized to the probe against the total BAL fluid sample volume to produce a normalized amount of hybridized cfDNA. |tt01 j la some embodiments, the method 'further comprises comparing the normalizedamount of hybridized DMA to a cutoff, value indicati ve of lung status.

|00Ι | In some embodiments, the method f rther comprises the determiniag the pattern has lung injury if the detected amount of target D A (e.g., cfD A) hybridized to the probe or {lie normalized 'amount of target DN A (e.g., efD A) is greater than the -cutoff value, In some embodiments, the human is a patient having received a lung transplant wherein th lnng injury indicates that patient. as rejection episodes.

[0016] In some embodiments, the method further comprises generating a KIT score based o the amount of cfDNA hybridized to the probe and. the amount of creatinine in the reaction mixture. In some embodiments, the KIT score is generated using the' ratio of the amount of DNA hybridized to the probe to the amount of creatinine. In some embodiments, the KIT score is generated by use of generalized linear models, such as logistic regression, using the .amount of cfX> A hybridized to the probe, the amount of creatinine, one or more in.fianimat.ion markers (e.g., CXGL10), one or more apoptosis markers (e.g., kidney tubular injury marker such as ciusierin), aad/or one or more DNA methy tion markets present in the ofluid sample. In some embodiments, nonlinear regression models, selected from the group consi stin of neural networks, generalized additive models, similarity least squares, and recursive partitioning methods, may be used to develop KIT Scores. In some embodiments, the KIT score is generated using additional hiomarkers selected from the group of CXCLIO, clusterin and D A -methy!ation markers such that the KIT score results in greater sensitivity and specificity for the. diagnosis and prediction, o f organ injury, than the use of individual markers in the KIT assay . The KIT injury score also detects organ injury with greater -Sensitivity than current markers of kidney fimction such as the serum creatinine or urine protein or current markers of lung function, such as forced expirator volume f 'FEV") and forced vital capacity ("FVC"). In some embodiments, using the ΪΤ score, e.g., a KIT score, a described above can detect kidney injury with a sensitivity of at least 85%,, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, artd or a specificity of at least at least 85%, at least ' 87%, at least 88%, at least 90%, at least 9.1%, at least 92%, at least 93%, at least 94%, at least 95%. in some

embodiments, using the KIT score described above can detect kidney injury with an AUC of at least 85%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96.9%, or at least 99.4%.

{00173 ΐ«. some embodiments, the disclosure provides a method of detecting organ injury comprising measuring the amount of ef H A, and amounts. of one or more of markers in a ioilind sample obtained from ao organ that is suspec ted of havi ng injury or is Hkely to .develop injury, wherei one or more markers is. selected from the group consisting of: i) one or more inflammatio markers, ii) one or more apoptosis markers.. Hi) total protein, and i v) one or more of DNA niethyiation markers; producing an IT score using the amount of cfDNA and the amounts of the one or more markers, and determining the patient having injur in the organ or predict thai the patient will develop injmy in the organ if the if score is above a predetermined cutoff. In some embodiments., the organ injur is kidney injury. I» some embodiments, the IT score is produced by further including creatinine, in some embodiments, the IT score is produced by using a mathematical model using the amount of cfDN A hybridized to the probe, the amount of creatinine, one or more inflammation markers (e.g., QCCLl'O), one or more apoptosis markers (e.g., kidney tubular injury marker such as clusterin), c eatinine, and/car one or more DNA meihylation markers present in tire biofluid sample

BRIEF DESCRIPTION OF THE DRAWINGS

[0018! Fig- t shows eleetropherograrsis that demonstrate in the absence of DN preservative solution the DNA degrades and disintegrates completely over period of 72 hours; whereas DNA preservation solution helps maintain DNA integrity' p to 72 hours.

[0019) Fig, 2 compares using the methods disclosed herein and using the standard PCR based methods in Al detection for cfDNA analysis.

[00201 Fig. 3 shows a dual hiotinyiaied-oiigo ucleotide complementary to the Alit repeats.

{(K>21| Fig. 4. shows the dynamic range of the assay disclosed herein having at least 5 orders of magnitude using a chemiiumineseent assay system. {0022] Figs. 5A-5D show using the assay disclosed herein to distinguish kidney transplant rejection (Figs. 5 -5B), native, kidney disease (Fig. 5C.) and its improved performance over proteinuria in detecting kidney iajury,(Fig. 5D).

[0023J Fig. 6 shows the correlation between, chromosome Y and chromosome i copy number in the urine validates the method in this disclosure.

[0024| Fig. 7. shows an exponential decay curve showing change ofcfDNA creatintne ratio over the .number of days post ransplantation using data from 9 patients who received kidne transplant and did not experience; acute rejection episodes.

{00 5) Fig. 8A shows the change of cfl NA creatinitte ratio over the days of posttransplantation and Fig. 8B shows change of th Kidney inj ury Test (KIT) score over the days of post-transplantatk .

[0026| Figs. 9A and 9B show using the cfDNA/creatinine ratio to predict acute rejection in patient #2 and patient #3. j002?f Figs. IGA and 10B show using the methods disclosed herein to detect IgA nephropathy (a native kidney disease) in patients; i tills study, urine samples from individuals -- 11:7 of which were healthy and 85 individuals had IgA nephropathy, as previously determined by other methods - were analyzed using the methods disclosed herein. Fig, 10A shows the differences in the detected. cfDNA/creatinine ratios between healthy individuals ("normal") and those having IgA nephropathy ("IgA"). The ROC analysis indicates that die ADC of die ROC curve is 0.9651 with a confidence interval ..of 0.9431. to 0.9871 with a P value of <0,0001. Fig. IQB.

(002S| Fig, 11 shows a comparison 'between the cfDNA concentration found in

bronchoaS veolar lavage (BAL) fluid from individuals who had stable lung transplants ("ST A") and from those who underwent occult, acute, or chronic .rejection ("Rejection"), The p-value was

0,0427.

10029] Fig. 12 shows a predietheness curve for the probability of acute kidne rejection in kidney transplant patients as a function of a KIT score, which is generated using, measurements of cfDNA and creatinine from urine. Urine samples from 41 patients having received kidney transplants were collected as disclosed herein. Measurements of cfDN A and creatinine in urine were obtained from each pahent.

{0030} Fig. 13 shows using a KIT score to assess kidne injury based on a datasef of 490 clinical samples with multiple causes of kidney injury.

{0031} Figs, 14A-E show using generalised: linear model and associated ROC curves to predict probability of kidney injury as well as various diseases mat are associated with kidney injury, namely, type ΙΪ diabetes meilrtus, immune response, kidney stones, transplant rejection, and hypertension.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0032 j The terms "a," "a ," or "the" as used herein not only include aspects with one member, httt al so include aspects with more than one. member. For instance, the singular forms "a," "an, " and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "th agent" includes reference to one or more agents known to those skilled, in the .art, and so forth.

{0033} The terms "subject",, "patient" or "indi idual" are .used herein interchangeably to refer to a human or animal. For example, die animal subject may be a mammal, a primate (e.g., monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog,, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird j, an animal of veterinary significance, or an animal of economic significance . 0034} The term "nucleic acid", or "polynucleotide" includes deoxyribonucieotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. The term nucleic acid is used 'interchangeably with gene, cDNA, and mRNA encoded by a gene. {883$} The term "kidney nj r status" refers to whether die patient shows injury in the kidney. For purpose of this disclosure, kidney injury can result .from surgery, such as kidney transplant, or from any kidney disease,

[0036J The term "kidney transplantation or ''kidne transplant:" refers to the organ transplant of a kidney into a patient The source of die donor kidney can be from a deceased or living donor.

(60371 The term "Alu" or "Ala element" or "Alu repeat:" refers to a short stretch, about 300 base pairs Song, of DMA originally characterized by the action of the Arlhwb cier Intern restriction enddnaclease. Alu elements are die most abundant repetitive -elements in die human genome. They are derived from the small cytoplasmic 7SL RMA. SEQ ID NO;} represents an exemplary human Alu repeat

[ 038J The term "hyforidiz©*''' refers to the annealing of one or more probes to a target nucleotide sequence. Hybridization conditions typically include a temperature that is below the melting temperature of the probes but that avoids non-specific hybridization of the probes.

{8039} The term "biofluid" or "biofhiidic sample" refers to a f!uidic composition that is obtained or derived from an individual that is to be characterized and/or identified, for example based on physical biochemical, chemical and/or physiological, characteristics. on-!tmMng examples of biofluid include blood, serun% plasma, saliva, phlegm, gastric juices, semen, tears, and sweat. In one embodiment the biofluid is urine, in another embodiment the biofluid is BAL.

[004u| The term "post-transplantation" refers to a time after the transplantation of an organ, e.g., kidney or lung, into the patient from a donor.

[06411 As used herein, the term "AUC" refers to "area under the curve" or C-statistie, which is examined within the scope of ROC receiver-operating characteristic) curve a alysis, AUC is an indicator that allows representation of the sensitivit and specificity of a test, assay, or method over the entire range of test (or assay) cut points with just a single value. An. AUC of an assay is determined from a diagram in which the sensitivity of the assay on the ordinate is plotted against 1 -specificity on the abscissa, A higher AUC indicates a higher accuracy of the test; an AUC value of 1 means that all 'samples have been assigned correctly (specificit and sensitivity of 1 ,), an AUG value of 50% means thai the samples 'have been assigned with guesswork probability' and the parameter thus has no significance.

{0042} Using AUCs through the ROC' curve analysis to evaluate the accuracy of a diagnostic or prognostic tes are well known in the art, for example, as described in, Pepe et aL,

"Limitations of the Odds Rati ia Gauging the Performan ce of a Diagnostic., Prognostic, or Screening Marker," Am, i. Epidemiol 2004, 159 (9): 882-890, and "ROC Curve Analysis: An Example Showing The Relationships Among Serum Lipid And Apohpoprotein levels In identifying .Subjects With Coronary Artery Disease," Clin, Cfeern., 1 92, 38(8): 1 25-1428. See also, CLSl Document EP24-A2: Assessment of the Diagnostic Accuracy of Laboratory Tests Using Receiver Operating Characteristic Curves; Approved Guideline - Second Edition. Clinical and Laboratory .Standards Institute; 2011: CLSl Document 1/LA.2I-A2: Clinical Evaluation of immunoassays; Approved Guideline - Second Edition. Clinical and Laboratory Standards institute;. 2008. j 043| As used herein, the term "diagnose" means assigning symptoms or phenomena to a disease or injury. For the purpose of this invention, diagnosis means determining the presence of organ injury in a subject

|'004 | As used herein, the term "predict" refers to predicting as to whether organ injury is likely to develop in a subject i- KIDNEY INJURY STATUS

[00 5J Compositions and methods are provided that can be used to assess kidney injury status, e., the presence or absence of kidney injury in an individual. Such an assessment is helpful for diagnosing when an individual is in need of medical intervention, such as being given more medication to address the medical problem or having medication decreased (including cessation) where it is no longer medically necessary. For example, compositions and methods described herein can be used to determine when an individual has kidney injury due to kidney transplant or kidney disease.

[0046J Kidney injury can develop in patients who have undergone a kidney transplant. This can happen because of several immune and non-immune factors such as ischemia reperfusion injury, size disparity, donor related factors, cell-mediated rejection, and antibody-mediated rejection, by way of example. Problems after a transplant may include; transplant rejection (hyperacute, acute or chroiiic), infections and sepsis due to the immunosuppressant drugs that are required to decrease r sk of rejection, post- transplant lymphoproiiferati ve disorder (a form of lymphoma due to the immune suppressants),, imbalances in electrolytes including calcium and phosphate which can lead to bone problems among oilier things, and oilier side effects of medications inclu ding gastrointestinal inflammation and. ulceration of t he stomach and esophagus, hirsutism (excessive hair growth in a male-pattern distribution},, hair loss, obesity, acne, diabetes mell.itus type 2, hypercholesterolemia, and osteoporosis.

(0047| Kidney injury can also develop in patients having kidney disease. Kidney diseases are diverse, but individual with kidney disease frequently display characteristic clinical features. Common clinical conditions involving the kidney include but are not limited to the nephritic and nephrotic syndromes, renal cysts, acute kidney injury , chronic kidney disease, diabetes-induced nephropathy, urinary tract infection, nephrolithiasis, and urinary tract obstruction, glomerular nephritis (GN), focal segmental glomerular .sclerosis. (FSGS), igA nephropathy (IgAN), mesangiocapiliary, lupus and membranous etc, hypertensive nephropathy, and drug induced nephropatliy. Kidney diseases can also include the various cancers of the kidney which exist. For example such cancers- include, but are not limited to, renal ceil carcinoma, urothelial cell carcinoma of the renal pelvis, squamous cell carcinoma, juxtaglomerular cell tumor (reninoma), angiomyoiiporna, renal oncocytoma, bellini duct carcinoma, clear-cell sarcoma of the kidney, mesoblastic nephroma, Wilms' tumor, mixed epithelial stromal tumors, clear cell

adenocarcinoma, transitional cell carcinoma, inverted papilloma, renal lymphoma, teratoma, carcinosarcoma, and carcinoid tumor of the renal pelvis. Kidney disease can also be virally induced and include, but are not limited to BKV nephropathy and nephropathy induced by EBV and CMV. Kidney disease can also be drug-induced as some medications. re nephrotoxic, (they have an elevated risk tor harming the kidneys). In the worst case, the drug causes kidney failure, while in other cases, {lie kidneys are damaged, but do not fail. Common nephrotoxic drugs include, but are not limited to, nonsteroidal anfi-mflarnrnatory drags (NSAIDs), som antibiotics, some painkillers, and radiocontrast dyes used or some imaging procedures. {'0848) la. some embodiments, a urine sample is from am individual having a kidney transplant or one. of the above-listed kidney disorders or kidney transplant -elifiical conditions described above is assayed as described herein.

2. LUNG INJURY STATUS

{8849) Compositions and Methods are provided herein that can be used to assess lung injury status, i.e., the presence or absence of lung injury in aa individual. Such an assessment is helpful for diagnosing when an individual i in need of medical intervention., such as being given more medication to address the medical problem or having medication decreased (includin cessatioti) where it is no longer medically necessary. For example, the compositions and methods described herein can he used to determine when an indi vidiial has long injury due to lung transplant.

{8850} Lung injury can develop i patients who have undergone a lung transplant This can happen because of several immune and non-immune factors such as ischemia reperfusion injury, size disparity, donor-related factors, -cell-mediated rejection, and antibody-mediated rejection, by way of example. Problems after lung transplantation may include hyperacute rejection, acute rejection, several types of chronic rejection or chronic lung allograft dysfunction (CLAD) such as restrictive allograft syndrome (RAS) or bronchiolitis obliterans syndrome (BOS), infections, aid sepsis '-due to the immunosuppressant drugs that are required to decrease risk of rejection,

[0051 in some embodiments, a biofluid. sample, e.g., a bronchoalveolar- lavage (BAL) fluid sample, is from an individual having lung transplant, or lung- transplant clinical conditions described above i assayed as described herein. Thus, in some embodiments, provided herein is a method of detecting Ahs copy number in cell-free MA (efDNA) in a. bioflnid sample from an individual having a lung transplant or lung transplant clinical conditions. The metho comprises obtaining a biofluid sample, e.g., a bronchoalveolar lavage (BAL) fluid sample, from a human; extracting cfDNA from the biofluidic sample; forming a reaction mixture by contacting the cfDNA with a nucleic acid probe under conditions to allow the probe to hybridize to DMA in the cfDNA that is complementary to the probe, wherein the nucleic acid probe has a nucleic acid sequence having a 3' and 5' end, wherein the nucleic acid probe is complementary to contiguous 20-292 nucleotides of SEQ ID'MOrl and wherein the 3' or 5' end is covalently linked detectable label; and quantifying the amount of DNA hybridized to the probe, thereby detecting Aiu copy number in cell-free D A (cfDNA) i the biofluidic sample l'0052'l The amount of DNA hybridized to the; probe may be .normalized against the total biofliiid sample volume to produce a .normalized amount of hybridized DISIA. hi some cases, the normalized amount .of hybridized DNA is compared to a cutoff value indicative of lung status.. In some cases, the method further comprises determining the patient has lung injur if the detected amount of target DNA (e.g., cfDNA) hybridized to the probe or the .normalized amount of target DNA (e.g., cfDNA) is greater than the cutoff value.

3- THE PRESERVATIO COCKTAIL

[60531 in some aspects, a preservative cocktail of reagents that can stabilize biofiuid. ceil-free DNA ("cfDNA") is provided. In some embodiments, the preservative cocktail is a solution comprising a formaldehyde donor and a chelator, such as a calcium chelating agent. Non- limiting examples of formaldehyde donor include dtazolidinyl urea and imida¾oitdinyl urea. Non-limiting examples of chelators include EDI and EGTA. in some embodiments, the solution further comprises one or both of PEG, aur tricarboxylic acid. In some embodiments, the solution further comprises sodium azide, and a buffer. Non- limiting examples of buffers include PBS, TAPS, Bieme, Tris, T/rioke, HEPES, TES, MOPS, PIPES, Cacody!ate, and'MES. In some embodiments, the solution comprises the respective components at a concentration that can preserve the biofiuid (e.g., urine) by preventing degradation of efBNA by nucleases, and/or by inhibitin cell lysis. In addition to stabilizing cfDNA, the solution can also prevent genomic DNA contamination through stabilization of ceils present in. the biofiuid. Various molecular weight of PEG and various forms of EDTA can be used in. this cocktail to provide the desired properties above. In one embodiment, the PEG used in the cocktail is PEG 35,000. I one embodiment, EDTA is Na2EDTA, In one embodiment, EDTA is K2EDTA. In one embodiment, EDTA is KSEDTA.

(0OS4| Typically, the cocktail is prepared as a concentrated stock solution, e.g., a OOx, 20x, lOx, Sx, or 2x concentrated stock solution, and is mixed and dilated with biofluid to be assayed, e.g., a urine sample, to a final working concentration. For example, for a 10x concentration of the cocktail, which will be diluted 1.0 times after mixing with urine, the concentration of diazo.hdinyl urea can be 0, 1 g/L to 50 g/L, e.g., 0.5 g/L to 30 g/L, 1 g/L to 20 g/L, 5 g/L to 20 g/L, 5 g/L to 1.5 g L, or 5 g/L to 10 g/L; the concentration of PEG 35,0000 can be 0.2 g/L to 50 g/L, e.g., 0.5 g/L to 40 g/L, 1 g L to 30 g/L, 20 g/L, or 15 g/L to 25 g/L; the concentration of aimiitacarbox lic acid can be 0.1 raM to 10 mM, e.g., .0,2 raM to 10 mM, ,0.5 mM to 5 mM, 0.5 mM to 2 mM, or 1 mM to 2 mM The 'concentration of EDTA can be 1 mM to 100 mM, e.g., 2 mM to 50 m, 5 mM to 40 ,rnM, .5.raM to.20 mM, or 1.0 mM to 20 mM, The 1 Ox concentrated stock solution may also comprise sodium azide at a concentration of I mM to 100 mM, 2 mM to 50 mM, 5 raM to 20 mM., or 10 raM" to 15 s»M. The cocktail can comprise a buffer, such as phosphate buffer saline (PBS), for example' ί Ox. PBS in a I Ox concentrated stock solution,

(0055} In some eases, the cocktail is provided in a powder or solid table format and is reconstituted to solution by adding water or aqueous buffer, or the biofluid itself

(0056J In certain embodiments, the cocktail comprises about ! g L Dia2oIidiiry! Urea, about 20g/L PolyEtheiyene Glycoeol, .about xM A rmiricarboxylic acid, about 10 mM K2EDTA, about lOraM Sodium Azide, and/or about IX Phosphate Buffered Sa!iue, pH?,4,

4- THE NUCLEEC ACID PROBE

(0057} The Alp gene has been extensively studied for amplification of various regions tor assessin the quantity ofcfDNA in patients with cancer (Biol Proced Online (2013); Park et a!,, Oncol Lett (201.2}}. in one aspect, the present invention provides a meth od of assessing the quantity of efDN in hiofliuds by using -a labeled nucleic acid probe to hybridize to the AIu repeats in cfD A.

[00581 ∞ some embodiments , the nucleic acid probe comprises a nucleotide sequence that comprises, or is fully complementary to, at least 20-300, e.g., 20-292, 20-180, 50-150, 70-100, 80-90 contiguous nucleotides of SEQ ID NO: 1 :

5'GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGG

GCGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCC

GTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCGCGCCTGTAATCCCA

GCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTGCA

GTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTC

TCAAAAAAAA-3 ' .

[0059} in some embodiments, the probe comprises a nucleotide sequence that comprises, or is fully complementary to, at least 20, 30, 40, 50, 60, 70, 80, or 81 contiguous nucleoti des of SEQ ID NO: 2: GCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGA GGCGGAGGTTGCAGTGAGCCGAGAT.

{0060} A nucleotide referred to herein in the context of the nucleic acid probe can be a natural nucleotide, e.g., cytosine, guanine, th mine, adenosine, or modified nucleotide. A modified nucleotide refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced by a different nucleotide thai provides a desired property (e.g., abilit to base pair with a complementary nucleotide in a target nucleic acid) to the oligonucleotide; Non-limiting xam les of modified nucleotide inc lude locked nucleic, acids (LN A), peptide nucleic acids (P Aj, morpho linos, and those described in OS Fat. Pub, No, 2016Ό 177377.

[0061] In some embodiments, the nucleotide sequence of the probe is conjugated to a detectable label. The detectable label can be conjugated onto an nucleotide of the probe so long as it does not inhibit hybridization of the probe to the target, i.e., the Alu repeats i the cfDNA, In some embodiments, the 5 s end of the nucleotide sequence is conjugated to one or more (e.g., two or more) detectable label. In some embodiments, tire detectable label itself is conjugated to tire signal-producing agent. In some embodiments, the detectable label is a molecule that can bind a binding partner and the binding partner is linked to a signal producing agent. For example, in some embodiments, the detectable label is biotin and the binding partner is

streptavidin or vice versa. In some embodiments the detectable label is digoxigenin and its binding partner is anti-digoxigeni or vice versa. In some embodiments, the detectable label is 2, 4-dinitrophenoi (DNP) and the binding partner is anti-DNP or vice versa, Othe labels known to one skilled hi the art can also be used in the nucleic acid probe disclosed herein. The signal- producing agent cau be any agent that produees qnantifiabte signal including hut not limited to, chemilummescenee, color, or fluorescence. Non-limiting examples of signal-producing agents include an enzyme, a fluorescent molecule, or the like. Other non-limiting examples of detectable labels and signal-producing agents can be found i US20.130209990, hereby incorporated by reference. In one preferred embodiment, the signal-producing agent is horseradish peroxidase (HRP),

(0062J In some cases, the 3 " end of the nucleotide sequence of the probe is conj ugated to one or more detectable label In some cases, both the 5' and 3' ends of the nucleotide sequence are conj ugated to one or more detec table label 'The number of detectable labels conj ugated to the probe caa vary, for example, the number can be at least one, two, raree, four, five, six, seven, tea, fifteen, eighteen, or more. in general, a higher number of detectable labels will produce a higher signal in the assa (Division et al.. Nucleic Acids Res. (1 88) 16: 4077-4095)., One o f ordinar skill can readily determine the number of detectable labels to be used based on the amount of signal required to detect die target, e.g., the A u repeats, in the cell-free DN A from biof kt

[0063 In one embodiment,' the 5' or the 3' end of the nucleic acid sequence of the probe is conjugated to one biotin . In one embodiment the total number of biotin conjugated to the nuclei acid sequence is I, or 18, or any number in between. The one or more biotin can be all conjugated at the same 5' or 3' end of the nucleic acid probe. The one or more biotin can also be conjugated at both ends, in any combiiiation. In some embodiments, two biotins -ate conjugated to the nucleic acid sequence of the probe, in one embodiment, the two biotins are conjugated to the 5' end of the nucleic acid sequence of the probe,

{0064} The nucleic acid probes described herein may be produced, b any suitable method 3 known in the art, including for example, by chemical synthesis, isolation from a naturally- occurring source, recombinant production and asymmetric PGR (McCabe, 1990 in; PGR

Protocols: A guide to methods and applications, San Diego, Calif, Academic Press, 76-83). It may e preferred to chemically synthesize the probes in one or more segments and subsequently link the segments. Several chemical synthesis methods are described by Narang et at (1979 Mem. Enzymol, 68:90), Brown et al. (1979 eth, Enzymo!, 68:109) and Camthers et al. (1 85 Mem, Enzymol. 154:287), which are incorporated, herein by reference. Alternatively, cloning methods ma provide a. convenient nucleic acid fragment which can be isolated for use as a promoter primer. A double-stranded DN A probe, can be First rendered single-stranded using, for example, conventional denaturation methods prior to hybridization to the. target in cfDN in biofluids,

5. METHOD OF USE

[0865} In some embodiments, the methods involve linking cfDMA (not previousl amplified) to a solid support:, hybridizing a probe specific for an Alu. repeat to the cfl>N.A,. removing (e.g., washing away ) unbound probe, and men detecting the amount of specifically hybridizing probe, thereb determining the amount of cfDNA in sample. a. Obtaining biofluid samples

{0066} Biofluid- samples, e.g., urine or BAL, from individuals e.g., those suspected of having organ injury, can be collected i any manner recommended by a medical professional, e ., being collected, raid-stream, in sterile containers. In some embodiments, the sample is then processed through centrifttgatton. to remove cellular components, thereby producing a cell-free sample. Optionally, the sam le can be mixed with 'buffer (e.g., PBS or Tris) and/or the above-described preservation cocktail. The sample can be stored at -80 °C until further analysis. In some embodiments, a preservative cocktail as described above is added to the biofluid sample to produce a mixture. In some embodiments, the mixture can be aliquoied for extraction of cil A, b. Extracting cell free DMA

{0067} Methods for extracting cell free DMA are well known. in. the art and commercial kits are readily available, for example, QiAanip® Circulating Nucleic Acids Kit from Qiagen (Valencia, California), in general, the sample can be treated to degrade cell debris and remov DNase and RNase to produce a lysate. DMA in the lysate can be extracted by. e.g., passing the lysate through a NA binding column.' rid the bound DMA 'can be eluted with water or buffer.

Optionally, a aliquot of the elnent can be taken to determine the concentration of the cfDNA recovered. 0068| The cfDNA is -subsequently linked to a solid support. Solid supports can be a containing vessel, a bead or any other solid support. In some embodiments, the cfDNA is placed in a desired vessel, for example, in the wells of a .oiierowe!I plate and incubated for a period of time that, is sufficient to allow the cfDNA to bind surface of the wells. In some embodiments* the incubation occurs over a period of at least one, at least two, at least four hours long, optionally at room temperature. In some embodiments, the incubation occurs at 4 °C for at least 8 hours. After the incubation, the vessel can be washed and blocked with a blocking solution, e.g., solution comprising 5% BSA to mi imize non-specific binding. .The blocking solution can Comprise a bufFer (e.g., PBS). The blocking solution can then be removed before adding the nucleic acid probe for hybridizing with the target, i.e., the Alu repeats. c. Hybridizing a. nucleic acid probe with Alu repeat

{'0069} Hybridizatio assays of the nucleic acid probe with the Alu repeats in cfDNA can be performed in any reaction vessel, including but not limited to a multi-well plate, e.g., a 96-wetl

1? plate, e.g., the 96- weil LUMfTRAC 600 (Gretner Bio-One). The assay of detecting cfDMA using a hybridization assay, ie,, hybridization of a probe conjugated to a detectable label without previous v amplification, of the target nucleic acid is desirable eonip red to a PCR-based approach because hybridization assays are cost-effective and can be multiplexed to a higher grade (e.g., 384/ atcb). I» addition, the hybridization approach allows for more complete and accurate quantification of efDNA as the probe will detect all single and double-stranded ofDNA in the biofiiud, irrespective of fragment length,

[Θ07θ| n an .exemplary hyjbtidizarion assay, a pre-determined volume of cS 'NA extracted from the biafi aids as described above is allowed to bind to a support, e.g., the bottom, of the micropiate wells, in some embodiments, fee surface of the support is previously treated to increase th binding affinit of the efDNA to the surface of the reaction vessel, in some embodiments, a buffer (e.g., PBS) and salts (e.g., MgC¾) is added to facilitate hybridization between the probe and efDNA. The final working concentration of the salt can be 0.O5M-O.5M, e.g., 0.05M- 0.2 , or 0.1 . Optionally, a standard curve can; be created using known quantities of human DNA extract.

[00711 .Hybridization of the fcfDN nd fee nucleic acid probe can be conducted under standard hybridization conditions. Reaction conditions for hybridization of a probe to a nucleic acid sequence vary from probe to probe, depending on factors such as probe length, the number of G and C nucleotides in the sequence, and the composition of the buffer utilized in the hybridization reaction. Moderately stringent hybridization conditions are generally understood by those skilled in the art as conditions .approximately 20° C-5Q0 C, e;g, 25°€-40° C below the melting temperature of a perfectly base-paired double stranded DNA. Higher specificity is generally- chieved by employing incubation conditions having highertem.perat.ures, m ther words more stringent conditions. Chapter .1 1 of fee well-known laboratory manual of Sambrook at al„ MOLECULAR CLONING: A LABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press, New York (1990) (which is incorporated by reference herein), describes hybridizatio conditions for oligonucleotide probes in great detail, including a description of fee factors involved and the level of stringency necessary to guarantee

hybridization wife specificity. Hybridization is typically performed in a buffered aqueous solution, for which conditions such as temperature, salt concentration, and pH are selected to provide sufficient stringency such feat the probes hybridize specifically to their respeciive target nucleic acid sequences but not any other sequence.

{0072} Generally, the efficiency ofhybridization between the oucieic acid probe and target, e.g., the Alu repeals in cfDNA, improves under conditions where the amount of probe added is in molar excess to the template, hi some embodiments, the oucieic acid pmbe of the invention is diluted in 5% BSA at a. concentration of between 10 ag/μΐ- 200 og/μΐ, e.g., 20 ng/ul- 100 ng μΐ, 30 ng/μί- 75 ng/μΐ, 30 ng/ul- 50 ng/μΐ in a particular embodiinent, nucleic acid probe is. the doiible-biotmy iaie and is used at the concentration of 30-40 ng/μΐ. The nucleic acid probe and the cflDNA in the plate can.be incubated to allow the hybridization of the probe and the Alu repeats in the cfDMA. The wells of the plate can then be washed (e.g., with PBS or anothe buffer) and optionall dried before detection. d. Detecting signal

[00731 Methods of detecting signal produced by detectable labels are well-known in the art and the methods vary depend on the nature of the chemical reaction employed to produce th signal. As described above, in some embodiments, the detectable label itself is a signal

producin agent that produce a signal, which can be read directl using appropriate equipment, for example, a plate reader, in some embodiments, the detectable label produces signal indirectly, a solution comprising a binding partner of the label that is conjugated to a signal producing agent is added. to the plate to produc the signal. The -signal producing agent include, tor example, enzyme or enzyme substrates, reactive groups, chromophore such as dyes or colored particles, luminescent moieties including a bioiuminescent, phosphorescent or chemihmiinescent moieties, and fluorescent moieties. In one embodiment, the detectable label is biotift and the binding partner is streptavidin and the signal producing agent is horse radish peroxidase (H P). in this particular embodiment, the signal is a chemiluminescent signal, which can be readily detected and quantified by methods well known in the art. e. Detecting creatinine levels

j00?4| The quantification of cfDNA disclosed herein can be combined with measurement of a urine protein. In one specific embodiment the urin protein is creatinine. Creatinine can be measured using the Jaffe reaction, an absorban.ee based method. Commercial assays for measuring creatinine are readily available, such as the QuantiChrom™ Creatinine Assay Ki (BioAssay Systems), which produces an output in tag of creatinine / deciliter of urine. See, e.g.. w w'.bioam sysveom/I>a{ashe¾ DICT,pd . In some embodiments, urine■creatinine

measurements are taken before or after extracting and qnantifying'ciD A in the urine sample. I some .embodiments, urine creatinine measurements and cfDNA quantification are performed in the same microweli piste (but different wells), by e.g., placing urine samples for measuring creatinine in the same microp!ate as the cfBNAs extracted from these urine samples. Measuring the creatinine and cfDNA in the same plate/assay saves time arid cost as compared to

conventional PCR methods, which respires one assay to read the amount of amplification product of the cfDNA and one assay to read creatinine level. f. Detecting additional markers.,

j 0075] Additional known markers can be measured to further improve the sensitivity and/or specificity of detection of organ injury, in some embodiments, the additional markers compose one or more (issue. inflammatory markers, e.g., CXCLIO, e.g., inflammation in the kidney. In some embodiments, CXCLIO is detected and quantified. Methods for measuring CXCLIO is: well known, for example, using a sandwich ELISA approach, where a capture aatibody adsorbs onto the plate first aid then sample is added to allow the CXCLIO in the sample to be captured.

Afterwards, a detection antibody is added that will bind the captured CXCL IO. This can be detected using the usual secondary antibody - HRP Conjugation: approaches well known in the literature. A standard curve would be generated with standard concentrations of purified

CXCLIO protein. Commercial kits are readily available for measuring CXCLIO, for example.

Human CXCLlWJF-iO Quantikme ELISA Kit from R & D systems. Urine CXCLIO can be measured before or after the cfDNA measurement. Urine CXCLIO can also be measured in the same microweli plate as cfDNA, by e.g., coating CXCL 10 capture antibody in designated wells in the mieroplate first and placing urine samples for measuring CXCLI O in these designated wells.

[0076] hi addition, differential methyktion status of the cfDN A in urine adds additional significance for kidne injury and markers correlated with methyla ion stains ca be measured and added to the assa described above for detecting kidney injury. Thus, in some embodiments, the additional markers comprise one or more BN meihy!aiion markers, hi one particular embodiment, the DMA methylation marker is 5-emthy.lcytosine that is incorporated into the nucleic acid in the sample. {'0077} DMA methylation markets can be detected and quantified using an ELISA-based approach, .Commercial kits that are used to measure the DMA methylation. markers are readil available, .for example, the MethylFlash Urine S-Methylcytosirse (5-mC) Quantification Kit from EpiGeittek thai is used to measure die amount of 5-raemylcytosine incorporated in the DNA, Briefly, a plate that has methylated DNA is incubated, with the sample and an andbody that recognize the methylation marker. This solution is allowed to incubate and is then washed arid further detected with a detection antibody and/or substrate.

[0078] In some embodiments, the additional markers comprise total protein in die biofhnd sample. Th amount of total protein can be measured using any methods known in the art that can be used to measure the protein, in one embodiment, the assay to measure total protein is a colorimetric assay , e.g., the Bradford protein assay. f0079f In some embodiments, the additional markers comprise cl sterin. Clusterin is a protein that is associated with the clearance of cellular debris and apoptosis. The presence of clusterin can also be detected and measured using a ELISA-based assay, such as the Human Clusterin DuoSet ELISA. Like othe sandwich ELISAs, a plate with capture antibod against Clusterin. bound to it is incubated with urine samples, optionally diluted in sample diluent. A detection antibody also against Clusterin is then incubated with the plate,- and an HRP-detection system is used to measure absorbance. g. Optional reagents and devices .

[0080] The methods may be used in a variety of assay devices and/or format. The assay devices may include, e.g., assay plates, cartridges, mahi-well assay plates, reaction vessels, test tubes, cuvettes, 'flow cells, assay chips, lateral Sow devices, etc., having assay reagents (which may include targeting agents or other binding reagents) added as the assay progresses or preloaded in the wells, chambers, or assay regions of the assay module, lliese devices may employ a variety of assay formats for specific binding assays, e.g., immunoassay or

ira imoehromatograpMc assays. Illustrative assay devices, e.g., microweli plates and formats, 96-welI plate format, are described herein below,

[0081} In certain embodiments, the methods can employ assay reagents that are stored in a dry state and the assay devices/kits may further comprise or be supplied with desiccant materials for maintaining the assay reagents in a dry state. The assay devices preloaded with the assay reagents can greatly improve the. speed rid reduce die complexity of assay measurements while maintaining excellent stability during storage. The assay reagents may also include' substances mat are not directly involved in the mechanism of 'detection but pla an auxiliary rote in an assay including, but not limited to, blocking agents, stabilizing agents, detergents, salts, pH buffers, preservatives, etc. Reagents ma be present in free form or supported on solid pbases including the surfaces of compartments (e.g., chambers, channels, flo cells, wells, etc.) in. the assay modules © the sur faces of col loids, beads, or other particulate supports .

[60821 in some embodiments, the methods described above can be performed in a lateral flow assay ("LFA"). LFA depends on the capillary action of a fluid sample drawn across a pad that contains capture reagents to the antigen of interest In some embodiments, the biofluid sample is mixed upon application to a device, e.g., a dipstick or a test strip, with reagents that are either purified antigen of interest bound to a visible marker (e.g. colloidal gold) i a competitive format or an antibody that is bound to a visible marker and recognize an antigen of interest in the ofiitid sample in a non-competitive format. The antigen of interest can be any molecule in the biofluid sample, for example, any of the markers disclosed in this application, such as creatinine, cfDNA, methylation markers, C CL10, and/or clusterin. in some embodiments, a competitive format of LFA is used to measure the creatinine and/or methylation markers. In some

embodiments, a non-competitive format LFA is used to measure cfDMA, CXCLIO, and or clusterin.;

6, .DETERMINATION. OF ORG AN INJURY STATUS

a. Determining organ health based on the amount of cfDNA

[0083] In some embodiments, the determination organ health of an individual comprises comparing the amount of cfDNA in the biofltikl sample to a cutoff value or predictive probability estimate indicative of organ Injury status. The cutoff value, can be .a pre-determined value or predictive probability estimate, e.g., a value recommended by medical professionals.

Depending on circumstances, it may be necessary in some cases to establish a cutoff value for the determination. To establish such a cutoff value for practicing methods disclosed herein., a group of healthy indi viduals, such as a group of individuals who -do not have organ injury after a organ transplantation is selected. These individuals are within the appropriate parameters, if applicable, for the purpose of determining organ injury status using the methods of the present in ention. For instance, the individuals may be of similar age, gender, and comparable health status.

(0084] lift some embodiments, to assess kidney injury, the detected amount ofefDNA, i.e., the amount of D A hybridized to the nucleic acid probe, in urine is first normalized to the amount of urine creatinine to produce a normalized amount of cfDNA, hi some cases, the normalized amount of cfDNA is a ratio of the detected amount of cfDNA to the amount of creatinine in urine. To determine the kidney injury status, the normalized amount of cfDNA. is compared to a cutoff valise, which is also a relative ratio of the cfDNA amount to the creatinine- amount in ur ine, as determined to be indicative of kidney inj ury status by medical professionals or established as described above. j0085| If the amount of cfDNA or the normalized aniount of cfDNA in urine is higher man its respective cutoff value, the patient, is determined to have kidne injury; in general, a higher value indicates a higher degree of kidne injury. In some cases, if the amount of cfDNA or normalized amount of cfDNA is higher than but near the cutoff value, the patient is determined to have subclinical injury. If the amount of cfDNA or the .normalized amount of cfDNA in urine is equal to or lower than its respective cutoff value, the patient is determined to have no kidney injury, i.e., good kidney health. For patients who have received kidney transplants, detection of kidney injury from urine indicates they are likely to have acute rejection episodes. Fo patients who are suspected of having certain kidney disease, in some embodiments, detection of kidney injury indicates the patients -are likely to have the kidney disease.

|MM| In contrast to conventional methods of detecting cfDNA in the blood, which would be contaminated with cfDNA mostly from the recipient of the kidney transplant, cfDNA detected, in the urine specifically reflects donor-derived DNA, even when testing for total cfDNA, Figure 6 shows the correlation between chromosome Y and chromosome 1 copy number in the urine. The strong linear correlation (R~ - 0 9253) indicates quantification of total cfDNA reflects the donor- derived burden and correctly reflects the kidney injury status due to the kidney transplantation, b. Determining organ injury status for organ transplantation patients using a I score j00S7| in certain embodiments, a predictive score, i.e., IT score, is used to diagnose whether a patient who has received an organ transplant may have organ injury, or to predict the likelihood a patient will develop organ injury ia the future. Organ .injury developed after organ transplant is typically associated with acute rejection episodes to the transplanted organ,

[00881 The IT score can be a composite value that can be calculated based on the amount of cfDNA in a biotluid sample from the organ and a factor {'aorniaUztag factor") that can be used to normalize- the amount of cfDNA ia the biofiiiid sample. In one embodiment, the amount of creatinine is used to normalize the cfD A in urine. In another embodiment the sample volume of the BAL is used to normalize the cfDNA in BAL from lung. The ST score may also include the time point, i.e., days post-transplantation, when the biotluid sample is taken. The time post- transplant can be a confounder of organ injury because patients often experience injuries that are not necessarily doe to the rejection to the transplanted organ, for example, the ongoing

nephrotojctc. injury to the transplanted organ from ischemia reperfuston and nephrotoxic damage due to infections and c&leineurin inhibitor drug exposure. These injuries are not indicative of acute rejection and the extent of such injuries may vary at different time points post

transplantation. Thus, the ΓΓ score, taking the time includes time point post transplantation, can accurately predict whether the patient has acute rejection episodes to th transplanted organ,

[00891 in some embodiments, measurements. f cfPNA in the bioiluid sample ma be combined with other bior tarkers in the biotluid sample to form the IT score that can be used to diagnose and/or predict organ injury: In some embodiments, multivariate methods can be used to incorporate these other biomarkers, e.g., to CXCL10 and DMA methylation. markers, to calculate the IT score, in some embodiments, cfDNA concentrations are normalized relative to the amount of creatinine in the sample by taking the ratio of cfDNA to creatinine or scaling the logarithmic measurements of CfDNA and creatinine proportionately (e.g.. through regression analysis). In .some embodiments, cfDN concentrations are normalized relative to the sample volume of BAL by taking the ratio of cfDNA to the volume or scaling the logarithmic measurements of cfDNA and volume proportionately (e.g. through regression analysis). In. some cases, the generated values are regressed using generalized linear models incorporating a quasibinormal distribution and a logistic link function. In some cases, the resulting model probability estimates for acute rejection are reseated from 0 - 100 t form the IT score and used to generate predictiveness curve. Figure 12 shows an illustrative embodiment, which displays the probability of acute kidne rejection in kidney transplant patients as a function of the resulting a IT score for assessing kidney injury ("KIT").

{0090} In some embodiments, an IT score, e.g., a KIT score, can be used to predict or diagnose organ injur wi th high specificity arid sensitivity. In some embodiments, the IT score is generated b including the amount of cfD A, the amount of DNAmeth.yla&on markers, and o infiammation markers present in the biofluid sample from the organ that is suspected of having injur or being likely to develop injury in the future, in some embodiments, the IT score is a KIT score for assessin kidneyinjury, in some embodiments, the KIT score is generated by further including the amount of creatinine and/or the amount of a kidney tubular injury marker (e.g., clusterin), in some embodiments, the IT score produced by including multiple markers as described above can detect organ injury with a sensitivity of a least 85%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, and/or a specificity of at least at least 85%, at least 87%, at least 88%, at least 90%, at least 1 , at least 92%, at least 93%, at least 94%, at least 95%. In some embodiments, using the KIT score described above can detect kidney injury with a AUG of at least 85%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96.9%, or at least 99,4%. Figure 13 provides an. illustrative embodiment, which displays a IT score for assessing kidney injury ("KIT") based on dataset of 490 clinical patient samples with multiple causes of kidney injury. The resulting KIT score is based on a subset of 299 clinical patient samples that had complete measurements of cfDNA, creatine, CXCL10. Clusterin, Protein, and DMA methylation markets; 11 1 type II diabetes melHtus, 71 immune, 50 kidney stones, 20 transplant rejections, 19 h erte sion and 28 controls. The resulting KIT score has over 91% sensitivity and specificit for detection of kidney injury (AUC ;~ 96.9%).

{0091} In some embodiments, mathematical models/algorithms are used to develop the IT scores using the amount of cfDNA. and one or more markers as described above. Such .models may include generalized linear models, such as logistic regression; and nonlinear regression models, such as neural networks, generalized additive models, similarity least squares, and recursive partitioning methods. In some embodiments, the mathematical models/algorithm that are used to generate the IT score are executed by one or more computer processors. Preferably, the mathematical models used to develop the IT scores are based on a database comprising sufficient sample size. In some eiribodimeais, die database comprises at least 50, at least 60, at least 70, at least 100, at least 200, at least 300, or at least 400 samples. Figures 14a --- 14e provide an illustrative embodiment of using generalized linea model fits and associated ROC" curves to provide the probability of kidney injury, as well as the probability of kidney injur due to each disease cause; namely, type II diabetes melltos, immune response, kidney stones, transplant rejection, and hypertension. In one specific embodiment as shown in Figures 14A-E, the resulting algorithm has over 92% sensitivity and specifici ty (AUG > 99.4%) for detection of each cause of kidney injury.

[0092] In some embodiments, a cutoff value for d e IT scores can foe established by measuring markers present in bioffoid samples from die same or similar types of organs from a group of healthy indi viduals, such as a group of individuals who do not have organ injury after an organ transplantation is selected. These individuals are within the appropriate parameters, if applicable, for the purpose of determining organ injury status using the methods of the present invention. For instance, the individuals may be of similar age, : gender, and comparable health status. The cutoff value of the IT scores is then, produced using mathematical models and or markers that are the same as those used to generate die IT scores for patients to he tested,

[6093] In. some embodiments, multivariate methods have been used to incorporate multiple biomarkers, e.g., to CXCL10 and DMA inediylation markers, with cfD A and creatinine to further refine the KIT score and the KIT score can be used to diagnose and/or predict kidney injury that has bee induced by multiple clinical conditions such as diabetes 1, diabetes 0, kidney stones, cancer, and immune complexes such as IgA nephropathy.

[0094| A number of ways can be used to produce the KIT score . In some embodime nts, a cutoff value of the cfDNA/ereatimne ratios k first determined biased on normalized cfDNA. values (e.g., normalized to creatinine levels) from urine samples from kidney transplant patients who have not shown rejection. These uri ne samples can be collected 'at predetermined t ime points over a post-transplant ioii period. KJT scores for a patient can then be determined baseti on the patient's normalized cfBNA values. Such normalized values can then be compared 'to cutoff values for those lime points to determine whether the normalized values are predictive of healthy or diseased kidney function. Such comparisons can be performed on computer if desired. W9S In some embodiments, the 'cutoff value is the-cfDNA/ereatraine ratios in patients who received kidney transplantation but who have not shown organ injury at respective time points in a post-transptantatkm period, in some embodiments, a prediction band of normalized cfDNA levels can be generated and a patient's actual normalized cfGN A value can be com ared to the value in the prediction band corresponding to the same time point posttransplantation. The prediction band can be established in a variety of ways, i some embodiments, number of stable, non-rejecting patients were examined over time and an exponential decay curve was fit to the cfD A^creatinine values with respect to time post-transplant. The prediction band was subsequently generated based on a prescribed probability' to cover the. values .of future observations from the same grou that was sampled. For example, a 95% prediction band consists- of upper limits of cf NA/creatinine ratios from 95% of stable non-rejecting patients examined at various time points posMnrasplantation, The KIT score at a particular time posttransplantation is then determined based on the actual measurements of erDNA/ereatmine and the prediction band value corresponding to that time point.

[0096) In some approaches, the cfDNA values relative to the creatinine values are processed into other forms of information, e.g., by using either common mathematical transformations such as logarithmic transforms, or statistical models, such as logistic or generalized linear models. Other data processing approaches, such as normalization of the results in reference to a population's mean values, etc. are also well known to those skilled in the art and can be used,

[0097] The KIT score is typically a numerical score o a defined scale or withi -a defined, range o values. The KIT score can be compared with a cutoff value of the KIT scores that is predictive of whether the patient has acute rejection episodes. If the KIT score is above the cutoff value of the KIT scores, a patient is predicted to have kidney injury, indicating he or she is likely to have acute rejection (AR) episodes. In some cases, a higher KIT score indicates a higher degree of injury due to the AR episodes. For example, in the ease where the KIT score is the logarithm of the patent's efDNA/creatinine ratio divided by the cutoff value of the ratio, the c utoff val ue of the KIT score is 0.

(0098J The KIT score is highly predictive of organ injuries associated with acute rejection and can be used to determine if the patient has acute rejection episodes . See Figure 5 A. Receiver operating -characteristic curves (ROC) analysis of showed that AUG of ROC curve for detecting

2? acute rejection using the KIT score was 0,9649, with a p- value of 0.0001 and a 95% confidence interval of 0.9346-0.9952, indicating the method is highl sensitive and specific Figure SB. hi contrast, the conventional kidney injury prediction method, i.e., detecting proteinuria, had muc lower AUC of 0.6498, with a p- value of 0,0747 and a confidence interval of 0.5032- 0.7963. The significance of difference was tested using McNemar's Faired e i-square from results from the same set of patients. The p- alue was 0,021 , suggesting that KIT is providing informatio above and beyond protein and 'that they are significantly different with respective to sensitivity. This comparison suggests that KI T method is more acute than the proteinuria method i determining acnte rejection

McNe ar Test results :

Figure imgf000030_0001

Test Statisti **

Figure imgf000030_0002

a. McNemsf Tesi

b. Continuity Corrected

|0099| In some embodiments, the KIT score is generated y further including measurements of other known kidney injury markers, in addition to the ciDNA/ereatmine ratios, at various time point post-transplantation increase assay sensitivity. These markers include but not limited to CXCLl 0 and DMA methy!ation markers, as described above. For any biom&rker of interest., a longitudinal trend curve can be generated for each of these biomarkers and a exponential decay curve i.e. a 90% or 95% predication band, can be established in a manner similar to what is- used to generate the prediction band for the cfDNA/creatinine ratios. jOlOOj Approaches similar to what is disclosed above, i.e., methods of generating a KIT score and using the generated KIT score to assess kidney injury, can be used to produce KIT scores to assess injury -of any other organ, e.g., injury developed after an organ transplant,

[01011 Thus,. the present invention can be used to conveniently .monitor organ transplant patient for organ injuries associated with acute .rejection episodes by eithe using the cflDNA normalized amounts (e.g., normalized to creatinine levels) or the IT scores corresponding to designated time points over a period of time post-transplantation. The period can be of any length -as. deemed necessary by the treating physician, e.g., at least 20 days, at least 50 days, at least 100 days, at least .150 days, at least 200 days, or at least 400 days, at least 500 days, or the life time of the transplanted kidney. Measuring cfDNA and creatinine can be performed at any frequency as deemed necessary, e.g., at least once year, at least twice a year, at least every three months, at least every two months, or at least every one month, or at least every- 20 days, or at least every 10 days. Patients who are so determined to have acute rejection episode can be treated as soon as possible, for example, by administering immunosuppressant drugs. Non- limiting examples of immunosuppressant drugs include calcuieurin inhibitors, such as

Tacrolimus and Cyclosporine; anfi-proliferative agents, such as Mycophenoiate ofetil, Mycophenolate Sodium and Azathioprine;- mXQR inhibitors, .such as Sirolimus, steroids, such as Prednisone, and induction agents such as thymoglobulin, IL2R blockade or belatacept. On the other hand, if the IT score is equal to or below the cutoff value, the patient is determined to have no kidney injury and thus no intervention is needed if a patient has a IT score near the cutoff value, the patient is determined to have subclinical injury. The IT scores can assist the treating

Figure imgf000031_0001

when intervention is needed. In addition, a patient specific trend of the IT score can also be analyzed to determine whether any clinical intervention is needed. For example, a trend of increase m the IT scores suggests that the patient is developing acute rejection episodes and therefore the clinical intervention may be necessary. c. Detecting kidney di seases

[01021 the cases of kidney injury associated with kidney diseases, the KIT score is not applicable, and the determination is based on ώδ eiDNA/eteatinine ratio as described above. In some cases, the addition of other hiomarfcers, e.g., CXCLiG that can be multiplexed onto the cfDNA plate may be applicable in creating a predictive model for the detection. The methods can be used to detect kidney diseases such as focal segmental glomerulosclerosis (FSGS), IgA nephropathy, and early diabetic kidney disease. BK viral nephritis (BK.VN), focal segmental glomerulosclerosis (FSGS), glomerulonephritis (GN), acttte tubular necrosis ¾AT ),. IgA disease and diabetic kidney disease. Fig, 4C shows the use of eiDNA½eati»ine ratio to separate patterns Slaving B VN from those not having the disease. d. Computer a»d smart phone devices

0163! & some embodiments, signals from live one or more markers described herein, e.g., cfDNA, creat ne, e.g., as detected from a lateral Sow assay, are tiaosmitted to a computer device, a camera, or a smart phone. In some embodiments, the signals are tratimitted to a smart- phone app for processing.

EXAMPLES

EXAMPLE I . THE PROBE TARGETING THE ALU ELEMENT

[010 j To provide more complete coverage of the cfDNA- repertoire in the bioflu , a length of <] 00 bp were selected for the nucleic acid probe targeting the Alu repeats so that both long and very short fragment lengths of cfDNA could be captured. In this particular assay, the probe length was 81 bp (Figure 2), although a shorter length, such as that commonly employed y.P R primers of 18-22 bp should work as well

|010S| Reference AL U sequence (SEQ ID NO; 3 ) is listed below and the 81 bp site (SEQ ID NQ:2) targeted for the Alu probe is highlighted in bold.

5 OGCCGGGCGCGGTGGCTCACGCCIG'rAATCCCAGCACT'rrGGGAGGCCGAGGCGG

GCGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCC

GTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCGCGCCTGTAATCCC

AGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGG

TTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGAC

TCCGTCTC AAAAAAAA-3 '

[0106] Biotin was selected due to wide availability of reagents that are compatible. However , any number of amplification schemes, such as digioxigeniii-aiiti-digioxi eriiii antibodies or even direct HRP conjugation would work. (ΌΙ.07| Dual biotiaykted-oUgo«ucleotide'coin iemen.tary to the Alu element was desigaed aad synthesized (52-B ilB/GCCTGTAATCX:CAO<:TACTC:CiGGiAGCiCTCA

GGCAGGAGAATCGCTTCAACCCGGGAGGCGGAGGTTC^

This was used to quantif cfDNA -using a chemEumiatscence based detection system using str¾0 svidiji-ilRP and 'chsaailumiaescent■ substrate (S perSigftal™ EL1SA Fentto Substrate) solutions (Figure 3).

(0.108J Giemiiuminescence was chosen because it is the most sensitive method available in a microweil for 'HRP detection (Figure 4), although colon metric and tluoronietric methods will work. Coiorimetric methods have also proved satisfactory. However, coiorimetric methods also take longer to incubate and produce results.

EXAMPLE 2. TESTING THE EFFECTIVENESS OF THE DNA STABILIZATION

SOLUTION

(0109| In order to test: the' effectiveness of DNA stabilization solution in urine containing sonicated genomic DNA, we purified genomic DMA from whole blood using QiaAMP DNA Blood Mini Kit (Qiagen), and sonicated it for 10 min using a Model 550 probe Sonic

Bismembrator (ThermoFisher Scientific) on intensity setting 3. Cycles were set at 10 seconds on and 20 seconds off. Samples were run on 1% gel electrophoresis to establish fragment sizes appro*. 150 to 250 base pairs obtained. With semi cation, we achieved DNA fragments of approximately 150 to 250 base pairs in size, a range that best represents cfDNA in transplant rejection (Figure 1.}, Next, we formulated a preservation solution containing l g/L Diazo dirtyl Urea, 20g L polyethelyene glycol, ini.M atirintriearboxylic acid, 1.0 aiM &2EDTA, lOm sodium azide, and IX phosphate buffered saline, pH7.4, An experiment was designed to test the effectiveness of DNA preservation solution in preserving the integrit of DNA. Fragmented genomic DNA (approx, size- 150 to 250 base pairs) was added to 1000 L tube's containing urine with and without preservation solution. The experiment was ran in parallel at 4°C and at room temperature. Figure 1 shows electropherograms that demonstrate how in the absence of DNA preservative solution the DNA degrades -and disintegrates completely over a period of 72 hours, whereas DNA preservation solution helps maintain DNA integrity up to 72 hours. EXAMPLE 3. DETERMINE THE ACUTE REJECTION STATUS FOR KIDNEY

TRANSPLANT PATIENTS

a. Sample collection

{0110} Urine samples from three patients (patient #1 - 3) having received kidney transplant were collected, raid-sttearo, hi sterile containers at different time points during the period post transplantation. The mine samples from patient #! were taken at day 1 , 16, 22, 51, -and 180; urine samples from patient #2 were taken at day 23, da 1 , and day 103; and urine samples from patient #3 were taken at da 100, day 132, da 185, and day 337. At a listed time point, in addition to collection of urine, a biopsy is taken to confirm the acute rejection. For example, the listed time point for patient #1 in this study is day 22, The urine Sample was aliqwoted into tnL for extraction with the QiaAnip® Circulatin Nucleic Acids Kit (Qiagen) following the manufacturer's instructions with an elution volume of 20 μί in ¾0,

[01111 Jsing a white, opaque EL1SA plate such -as the 96-welI LUMITRAC 600 (Greine Bio- One), 5 microliters of the elnted cfPNA were plated onto die plate, either in singUcate, duplicate, or triplicate as needed. To these sample wells, .10 microliters of a 5X. PBS and 0.5 M MgCI? buffer were added and then 35 microliters of molecular grade ¾0 was added for total of 50 microliters per well. To create a standard curve, known quantities of human DN A extract were added in duplicate in a titration series of 1 ; 3 from 200,000 to ~12 GE/niL, where a GE (genomic equivalent) is defined as 6,6 pg of human NA. This was also in the same buffer , a final working concentration of 1 X PBS and 0 J M MgCI*.

[01121 The efDN A plate was incubated -overnight at 4€ or for a minimum of 2 hours at RT shafeing at 300 RPM The liquid was then discarded an the plate was dried via patting against absorbent paper towels. The plate wras then blocked in 5% BSA i PBS with 300 niicroliters per well, for a minimum of 1 hour at. RT shaking at 300 RPM The liquid was then discarded and the plate was dried via patting against absorbent paper towels. The plate- was then, incubated in 50 microliters of our double-bioiinytated Ala oligonucleotide probe diluted i the 5% BSA at a concentratio of 35.56 ng microliters. This was allowed to incubate for a minimum of 1 hour at RT shaking at 300 RPM, The liquid- if-then, discarded and the the wells were washed with 300 microliters of 1 PBS three times. The wash was then discarded and the plate was dried via patting against absorbent paper towels. The plate was then incubated in 50 microliter -of streptavidin-i ¾¾P diluted 1 :200 per manufacturer' instructions in 5% BSA and allowed to incubate for no more than 1 hour at RT shaking at 300 RPM. The liquid is then discarded and then the wells were washed with 300 microliters of I X PBS three times. The wash was then discarded and the plate was dried very tkoroiighly via -patting against absorbent paper towels..; 150 μΐ of SuperSigaal ELISA Pernio chemiiuminescent substrate solution (ThermoFisher) was then added to each well The plate was analyzed upon I minute of mixing based on total luminescence.

(OKI 3] The generated values were then. egressed using a 5 " aram ter sigmoid curve fit, such as. that provided in GraphPad Prism and the resultant cell- free concentration values were corrected for the dilution done in the microweli as well as the concentration done from 2 mL of urine to 20 μΐ of eluate,

{0114} Creatinine was measured using the QuantiChrom Creatinine Assay Kit (BioAssay Systems) according to manufacturer's instructions, see

httpsi yw'Ww.bioassaysys.com Datasheet DICT.pdf. b. Determining the cutoff value of cfDNA/creatiniae ratio

{0115} Prior to live study involving patients # ! ~#3, cutoff values of ctTJNA/ereatinine ratios were established based on dat from nine (9) patients, who received kidney transplant and did not have kidney injury as confirmed b biopsy. cfDNA and creatinine amounts from urine samples from these 9 patients were determined as described herein. The urine creatinine values were first converted to mg.½L and the cell-free DNA values are divided by ibis creatinine measurement to produce cfDNA/creatinine wit uni ts of [GE/rng], wherein a GE (genomic equivalent) is defined a 15.6 pg of human Q A. The efD A'crearinine ratio vs. the days post transplantation was plotted as shown in Figure 7 and a 93% prediction hand (the dotted line) as dependent on time-post-transplant was modeled as an exponential decay curve with following equation. cfDNA''Creatmme [GE/rng] = I A((5.612-4.007)*EXP(-0.05977*(Days Post- Transp!arrt))+4.007)/I00.

{0116} Each data point in this one-sided 95% prediction interval corresponds to the estimate of the upper limit of the ciTJMA creatinine ratios that with 95% .confidence, is predicted to be higher than cfDNA/creatinine ratios from 95% offtiture non-rejecting patients at the same time point: post-transplantation. The urin creatinine values were first converted to .rag/mL and the cell-free DMA values are divided by this creatinine measurement to produce efD A crea&nine with units of [GE/rrtgJ.: The ctDNA/ereatinine ratio was plotted against days post

transplantation. Figure 8A. KIT prediction scores were calculated as described above, and plotted against days post-transplantation. Figure SB. Although between the first and second points the cfDNA/creatinine ratio drops (Figure 8A)„ the prediction score showed that that relative risk actually 'increases prior to the biepsy-confsrnied acute rejection episode (Figure SB) KIT was above zero at time point 22, indicating that patient #1 had kidney injury and acute rejection episodes. The acute rejection status was confirmed by the examining the biopsy take at the listed time point. Patients #I-#3 were all given a imr mosuppressafit after the listed time points: Tacrolimus, MMF, steroids to patient #1, Tacrolimus MMF, Steroids to patient #2, and Tacrolimus and Sirolimus to patient #3,

{'ill I7| cflDNA/creatiniae ratios of patients 2-#3 for urine samples were plotted against days post-transpiaiMation. Figures 9A and 9B. For both patients, acute rejections were predicted using the method disclosed herein at a time point prior to listed time point, when they were confirmed by biopsy. Additionally as shown in Figure 9B, the administration of an immunosuppressant from the listed time point thereo caused the cfDNA ratio to decreas into the stable region, indicating successfiii treatment Clinical determinants of graft injury can be made on the basis of a absolute elevation in the cfDNA value above the determined, threshold as well as an increase in the ciDN burden over time using patient specific threshold data. A grail injury is presumed to result from sub-optimal immunosuppression exposure, an abnormal, elevated cfDNA result could trigger the following clinical actions :1 ) .return of patient to clinic for closer follow-up; 2) consider an earlier protocol biopsy in a patient scheduled to have one; 3} consider an indication biopsy to evaluate for sub-clinical acute rejection and/or other cause of. graft injury; 4) a change in immunosuppression drug type or dosing. Conversely, a low, stable cfDNA result could trigger the following clinical: actions: 1) reduce clinic follow-up frequency;' 2} avoid unnecessary protocol biopsies that are done to look for sab-clinical graft injury; 3} change in

immunosuppression: drug typ or dosing. EXAMPLE 4. OF DETERMINING THE REJECTIO STATUS FOR LUNG TRANSPLANT

PATIENTS - jOllSj Broach oaiveolar lavage (BAL) .fluid samples from 76 patients having received lung transplants were collected in sterile containers either during stable period or during rejection episodes. The BAL fluid was aliquoted into 400 Ε for extraction with the Ql Aarap Circulating Nucleic Acids Kit (Qiagen) following the manufacturer's instructions with an ehition volume of 20 L in HjO. The cfDNA was measured as disclosed in Example 2.

[0119\ The generated values were regressed usin a 5-parameter sigm id curve fit, such as that provided in GraphPad Prism an the resultant cell-free concentration values were corrected for the dilution done in the microwel! as well as the concentration done from 400 μί, of B AL fluid to 20 μΤ of eluate. fO120f The normalized cfDNA concentrations from BAL fluid were compared betwee stable and rejection patients. Figure 10. The mea level of cfDNA in the rejection patients was significantl higher than in the stable patients. A abnormal, elevated cfDNA result could trigger similar clinical actions as disclosed in Example 2.

Example 5, determining kidney injury status using a KIT score j0121| Figure 13 provides an illustrative embodimen t, which displays a IT score for assessing kidney injus ("KIT") based on a dataset of 490 clinical patient samples with multiple causes o kidney injury. The resulting KIT score was based on subset of 299 clinical patient samples that, had complete measurements of cfDNA, creatinine, CXCL10, Clustertn, Protein, and DNA methylation markers; 3 3 3 type 11 diabetes meilitus, 71 immune, 50 kidney stones, 20 transplant rejections,, 19 hypertension and.28 controls. The resulting KIT score had over 91% sensitivity and specificity for detection of kidney injury (AUC ~ 96,9%), Figures 14 -E provide results of generalized, linear model fits and associated RO curves. These results illustrate that in additio to displaying the probability of kidney injury, the underlying algorithm can accurately provide the probability of kidney injury due to each disease cause; namely , type II diabetes meilitus, immune response, kidney stones, transplant rejection, and hypertension. For these data, the resulting algorithm showed a over 92% sensitivity and specificity (AUC > 99.4%) for detection of each cause of kidnev injury. |M 22| It is understood that the examples and erabodirnents described herein are for illustrative purposes nly and that various modifications or changes in Sight thereof will be suggested to persons skilled in tile art and are to be included within the spir t arid urview of this application and scope of the appended claims. All publications, patents,, and patent applicatio s cited herein are hereb incorporated by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS: 1. A solution comprising a formaldehyde donor, a chelator, aurmfcricarboxyhc acid, and polyethylene glycol (PEG) ili a concentration sufficient: to inhibit cell lysis and to inhibit nucleases in a biofluid, 2. The solution of claim 1, wherein the formaldehyde donor is diazolidinyl urea. 3. The solution of claim 1 , wherein the chela tor i s EDTA - 4. The solution of claim 1 , wherein the solution further comprises sodium azide. 5. The solution of claim 1 or 4, comprising a buffer. 6. The solution of any of claims 1-5, further comprising a biofluid sample. 7, The solution of any of claims 1 -6, wherein the biofluid sample is from a patient who has recei ved a organ transplant or has a kidney disease. '8 , The solution of claim 7, wherein the organ transplant is a kidney transplant. 9. A method of detecting Alu copy number in cell-free DMA (cfDNA) in a bioftiiid sample, the method compri sing: obtaining a bioilnidic sample from a human; extracting cfDNA from the biof!uidic sample; forming a reaction mi xture by contacting the cfDNA with a nucleic acid probe under conditions to allow the probe to hybridize to the cfDNA that is comptementary to the probe, wherein the nucleic acid probe has a nucleic acid sequence having a 3* and 5' end, wherein the nucleic acid probe is complementary to contiguous 20-292 nucleotides of SEQ ID NO:i and wherein the 3' or 5' end is covalentlv linked detectable label; 10 qualifying the amount' of cfDNA hybridized to the probe, thereby detecting Alu ϊ 3 copy number in cell-free DNA (cfDNA) in the biofiuidic sample. 1 10. The method of claim 9, wherein the probe is complementary to at least 502 contiguous nucleotides of SEQ ID O:2. 1 1 1. The method of claim 9 of 10, furtlier comprising, before forming the2 reaction mixture, mixing a solution of claim I with the biofiuidic sample. 1 12. The method of claim 9, wherein the biotluidie sample is a urine sample2 and wherein the method further comprises quantifying the amount of creatinine in the reaction3 mixture, J 13. The method of claim 12, comprising nonnaJizing the amount of cfDNA2 hybridized to the probe against the amount of creatinine in the urine sample to produce a3 normalized amount o hybridized DNA. 1 14. The method of claim 13, farther comprising comparing the normalized2 amount of hybridized efDNA to a cutoff value indicative of kidney status,. 1 15. The method of claim 14, wherein the method ferther comprises 2 determining- the patient .-has kidney injury if the detected amount of cfDNA hybridized to the3 probe or the normalized amount of cfD A is greater than the cutoff value. J 1. . The method of claim 15, wherein the human is a. patient having received a2 kidne transplant wherein the kidney injury indicates that patient has acute rejection episodes. 1 17, The method of claim 15, wherein the human is a patient having received a2 kidney transplant and whereiii the method further -comprises generating a predictive score for3 determining kidney health based on the normalized amoun of cfDNA and the time post-4 transplant of the kidney when the urine sample is taken from the patient. 1 18. Th method of any of claims 17, wherein the patient is determined to have2 acute rejec tion episodes when the predictive score is greater than a c utoff val ue for the predictive3 scores.
1 . The method of claim 14, wherein the urine sample is from an individual suspected of having a kidney-injury caused by a disease selected from the grou , consisting of BK viral nephritis, focal segmental g!omemlosclerosas, acute tubular necrosis, IgA disease, and diabetic kidney disease. 20. The method of claim 9, further comprising quantifying the amoun CXCL10. 21. The method of claim 9, wherein th probe comprises a nucleotide sequence of 50-150 nucleotides' complementary to SEQ ID NO: 1. 22. The method of claim 9, wherein the probe comprises a nucleotide sequence of 70-100 nucleotides complementary to SEQ ID NO: 1. 23. The method of claim 9, wherein the probe comprises a nucleotide sequence of 80-90 nucleotides complementary to SEQ ID NO: I . 24, The -method of claim 23, wherein a nucleotide sequence has exactly 81 nucleotides. 25. The method of any of claims 9-24, wherein the detectable label is biotin and the method comprises contacting the detectable label with a streptavidin-Hnked signal producing agent. 26. The method of claim 9, wherein the signal-producing agent produces cbemiluminescence, color, or fluorescence, 27. The method of any of claims 9-26, wherein the urine sample is taken 0- 400 days, or 10-ί 00 days, or 20-50 days, or over the lifetime of the transplanted kidney from the patient's receiving a 'kidney transplant. 28. A reaction mixture comprising i) non-amplified ceil-free DMA (cfONA) extracted from a urine sample and ii) a nucleic acid probe having a nucleic acid sequence and having a 3' and 5' end, wherein the nucieic acid probe is complementary to 20-292 contiguous nucleotides of SEQ ID NO; I and wherein the T or 5 ' end is covalentty Halted to a detectable label
29 The solution of claim 7, where in the organ transplant is a lung transplant. 30. The method of claim 9, wherein, the biofluidic sample is a bronc oalveoiar lavage (BAL) fluid sample and wherein the method further comprises quantifying the total volume of BAL fluid. 31. The method of claim 30, comprising ormalising the amount of cfDNA. hybridized to the probe against the total BAL fluid sample volume to produce a normalized amount of hybridized D A. 32. The method of claim 31, further comprising comparing the normalized amount of hybridized cfDNA to a cutoff value indicative of lung status. 33. The method of claim 32, wherein the method further comprises the determining the patient has lung injury if the detected amount of cfDNA hybridized to the probe or the nomwilized amount of cfDN is greater than the cutoff value. 34. The method of claim 33, wherein the human is a patient having received a lung transplant where in the lung■■injury indicates that patient has rejection episodes. 35. A method of detecting organ injury comprising measuring the amount of cfDNA, and amounts of one or more of markers in a bioflnid sample obtained from' an organ that is suspected of having njur)-' or is likely to develop injury, wherein one or more markers is selected from the group consisting of:
i) one or more inflammation markers,
ii) one or more apoptosis markers,
iii) total protein, and
iv) one or more of DNA methylation markers;
producing an IT score using the amount of cfDNA and the amounts of the one or more markers. deienaiaiag the patient having injury in the organ or predict that the patient ml develo injury in the organ if the IT score is above a predetermined cutoff.
PCT/US2017/047372 2016-08-17 2017-08-17 A novel immunoprobe-based method to assess organ injury status through a biofluid-based cell-free dna (cfdna) assay WO2018035340A1 (en)

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JP2019508929A JP2019528063A (en) 2016-08-17 2017-08-17 Novel immunoprobe based method for assessing organ damage status through a body fluid based cell free DNA (cfDNA) assay
CN201780063968.0A CN109890977A (en) 2016-08-17 2017-08-17 The new method based on immunological probe of organ damage state is measured and evaluated by the Cell-free DNA (CFDNA) based on biofluid
US16/325,385 US20190211385A1 (en) 2016-08-17 2017-08-17 Novel immunoprobe-based method to assess organ injury status through a biofluid-based cell-free dna (cfdna) assay
CA3033650A CA3033650A1 (en) 2016-08-17 2017-08-17 A novel immunoprobe-based method to assess organ injury status through a biofluid-based cell-free dna (cfdna) assay
EP17842131.9A EP3500687A1 (en) 2016-08-17 2017-08-17 A novel immunoprobe-based method to assess organ injury status through a biofluid-based cell-free dna (cfdna) assay
AU2017313138A AU2017313138A1 (en) 2016-08-17 2017-08-17 A novel immunoprobe-based method to assess organ injury status through a biofluid-based cell-free DNA (cfDNA) assay
US16/597,782 US20200032331A1 (en) 2016-08-17 2019-10-09 Novel immunoprobe-based method to assess organ injury status through a biofluid-based cell-free dna (cfdna) assay

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Citations (2)

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US20110111410A1 (en) * 2009-11-09 2011-05-12 Streck, Inc. Stabilization of rna in intact cells within a blood sample
WO2014146780A1 (en) * 2013-03-18 2014-09-25 Qiagen Gmbh Stabilization and isolation of extracellular nucleic acids

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110111410A1 (en) * 2009-11-09 2011-05-12 Streck, Inc. Stabilization of rna in intact cells within a blood sample
WO2014146780A1 (en) * 2013-03-18 2014-09-25 Qiagen Gmbh Stabilization and isolation of extracellular nucleic acids

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