WO2021236964A1 - Methods for measuring total cell-free dna using alu fragments - Google Patents

Abstract

This invention relates to methods and compositions for measuring total cell-free DNA in a sample by measuring the amount of Alu fragments in the sample. The methods and compositions provided herein can be used to determine risk of a condition or complication thereof.

Description

METHODS FOR MEASURING TOTAL CELL-FREE DNA

USING ALU FRAGMENTS

RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/027,411, filed May 20, 2020, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to methods and compositions for determining an amount of total cell-free DNA (cf-DNA) by measuring Alu fragments in a sample from a subject. Such amounts can be used to determine risk associated with tissue or cellular injury and various conditions.

SUMMARY OF INVENTION

The present disclosure is based, at least in part on the surprising discovery that the amount of Alu fragments can be used as an accurate and sensitive measure of total cell-free DNA. The amount of total cell-free DNA in the circulation can be a marker of the degree of cellular or tissue injury as well as an indicator of risk for a number of conditions. For example, the amount of total cell-free DNA can be used to assess and/or monitor over time the risk of surgical or transplant complications, infection, cardiac arrest, death, conditions associated with a pro-inflammatory response, inflammation, or inflammatory processes, etc. The amount of total cell-free DNA can also be used to monitor the effectiveness of treatment in a subject.

Therefore, methods of measuring amount(s) of Alu fragments to determine the amount(s) of total cell-free DNA are provided. The methods can also include, in some embodiments, steps for assessing the health of a subject and/or assessing the risk associated with any one of the foregoing based on the amount(s) of total-cell free DNA that is so determined. Any one of the methods provided herein can be used for such purposes.

Provided herein are methods that comprise measuring amount(s) of Alu fragments at one or more points in time to determine the amount(s) of total cell-free DNA at these point(s) in time. Also provided are related reports, kits, databases, compositions, etc. related to such measured amount(s) and/or including such amount(s) alone or in combination with threshold value(s) or other amount(s), such as other amount(s) obtained from other points in time.

What is provided herein includes methods of monitoring the health of a subject over time by measuring the amount of Alu fragments as a measurement of total cell-free DNA.

In one embodiment of any one of the methods provided herein, the subject may be any one of the subjects provided herein.

In one embodiment of any one of the methods provided herein, the threshold is any one of the thresholds provided herein.

In one embodiment of any one of the methods provided, the method further comprises obtaining a sample from the subject.

In one aspect, a report or database comprising one or more of the amounts provided herein is provided.

In one embodiment, any one of the embodiments for the methods provided herein can be an embodiment for any one of the compositions, kits or reports provided. In one embodiment, any one of the embodiments for the compositions, kits or reports provided herein can be an embodiment for any one of the methods provided herein.

In one aspect, any one of the methods provided herein is provided. In one embodiment of any one of the methods provided herein, the amount indicative of a specific risk or complication is any one of the thresholds described herein. In one embodiment of any one of the methods provided herein, the time for obtaining the sample is any one of the times described herein.

In one aspect, a method of treating a subject, determining a treatment regimen for a subject or providing information about a treatment to the subject, based on the amount of the total cell-free DNA as determined by the amount of ALU fragment, or any one of the methods of analysis provided herein, is provided. In one embodiment of any one of such methods, the method comprises a step of treating the subject or providing information about a treatment to the subject. In one embodiment of any one of the methods of treating, the treatment may be any one of the treatments provided herein. In one embodiment of any one of the methods of treating, the treatment is for any one of the conditions or any one of the subjects provided herein.

BRIEF DESCRIPTION OF FIGURES

The accompanying figures are not intended to be drawn to scale. The figures are illustrative only and are not required for enablement of the disclosure. Fig. 1 illustrates an example of a computer system with which some embodiments may operate.

Fig. 2 shows the outcomes related to total cf-DNA concentration determined by RNaseP analysis (left) and total Alu fragment (greater than or equal to 115 bp in length) analysis (right). Death, mechanical circulatory support, and cardiac arrest are indicated in bold.

Fig. 3 shows the outcomes related to total cf-DNA concentration by RNAase P PCR (left) and total Alu fragment (>115 bp) PCR (right). Deaths are indicated in bold.

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly found that the amount of Alu fragments may be used to measure the amount of total cf-DNA in a sample (Figs. 2-3). Thus, the amount of Alu fragments can be used to assess the health of a subject, such as a transplant subject, surgical subject, subject with cellular or tissue injury, subject with a pro-inflammatory response, etc. The amount of Alu fragments can also be used to monitor any one of the subject provided herein over time and can also be used to assess the efficacy of treatment. Aspects of the disclosure relate, at least in part, to methods of quantifying the amount of Alu fragments (e.g., in pg/pL) in a sample from a subject. The amounts can be used to assess or determine a condition or risk, respectively, of the subject. In some embodiments, the subject may be on mechanical support (e.g., a ventilator and/or circulatory) and can be assessed or monitored with any one of the methods provided herein. Early detection or monitoring of the state or condition of a subject, such as one with any one of the conditions provided herein or one that has had surgery, including non-transplant surgery, such as heart surgery (e.g., cardiopulmonary bypass), and transplant surgery can facilitate treatment and/or monitoring and improve clinical outcomes. In some embodiments, the subject may show no signs or symptoms of the state or condition or risk associated with the condition or risk. However, in some embodiments, the subject may show symptoms.

Alu fragments are stretches of DNA initially characterized by the action of the Arthrobacter luteus (Alu) restriction endonuclease. Alu repeats (elements) are the most abundant sequences in the human genome, with a copy number of about 1.4 million per genome. Alu sequences are short interspersed nucleotide elements (SINEs), which account for more than 10% of the genome and are primate- specific. They create genetic diversity and can contribute to disease through insertional mutagenesis. They participate in non-allelic homologous recombination events, resulting in copy number variation and disease. Alu fragments impact gene expression, such as through polyadenylation, splicing, and ADAR (adenosine deaminase that acts of RNA) editing. The Alu fragments are DNA fragments from the Alu sequence that can generally be less than or equal to about 170 bps (e.g., between 75 and 170 bps in length), in some embodiments. In one embodiment, the Alu fragments are equal to or greater than 115 bps (Alu 115) and can be measured using forward and reverse primer sets. Such a measurement can be quantified and used as a measure of total cf-DNA.

The methods and compositions provided herein comprise measuring an amount of Alu fragments to determine an amount of total cf-DNA. These amounts can be used to assess a subject’s risk in a variety of instances. For example, the risk can be associated with a surgery. As another example, the risk can be associated with a transplant. As used herein, “transplant” refers to an organ or tissue from a source, such as from a donor, for the purpose of replacing or adding to a subject’s organ or tissue, such as a damaged or absent organ or tissue. In one embodiment, the subject has received an autograft, allograft, xenograft, or has received a donor organ or tissue, such as a heart, or more than one donor organ or tissue, such as a heart and lung.

As another example, the subject is one who has undergone surgery. Post-surgical complications are a major cause of prolonged hospital stays and late mortality. Treatment of post-surgical complications with an appropriate therapy has been shown to improve surgical treatment outcomes, particularly if the complication is detected early. Following surgery, subjects can be monitored for surgery- specific complications with any one of the methods provided herein. Such methods can also include other forms of monitoring, which can include, for example, such as after cardiac surgery, continuous telemetry, measurement of the arterial blood pressure via an arterial catheter, measurement of the cardiac filling pressures via a pulmonary artery catheter (i.e., right heart catheter, Swan Ganz catheter), continuous assessment of the arterial oxygen saturation via pulse oximetry, and the continuous measurement of the mixed venous oxygen saturation via an oximetric pulmonary artery catheter. Invasive procedures, however, are associated with risks and discomfort for a patient, and may be particularly disadvantageous for pediatric patients. Accordingly, the methods provided herein are sensitive, specific, cost effective, and non-invasive for the surveillance of subjects, such as surgical patients.

In another embodiment of any one of the methods provided herein, the subject is one in which inflammation is occurring or one that has or is suspected of having an inflammatory disease or disorder. As used herein, an “inflammatory disease or disorder” is any one in which the disease or disorder occurs, or symptoms thereof, are at least in part due to inflammation or an inflammatory process, such as an undesirable pro-inflammatory response. Examples of such diseases or disorders include Alzheimer's, ankylosing spondylitis, arthritis (e.g., osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis), asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis, Parkinson's disease, ulcerative colitis, sepsis, acute respiratory disorder, acute inflammatory disorder, cytokine storm, blunt trauma, penetrating trauma, etc.

Such a subject may be one that has been or is being administered an anti inflammatory. Anti-inflammatories include aspirin, celecoxib, diclofenac, ibuprofen, indomethacin, naproxen, oxaprozin, piroxicam, etc. Anti-inflammatories also include corticosteroids.

As a still further example, the subject is one that has or is suspected of having an infection or infectious disease. An “infection” or “infectious disease” is any condition or disease caused by a microorganism, pathogen or other agent, such as a bacterium, fungus, prion or vims.

Such a subject may be one that has been or is being administered an anti-infection treatment. Anti-infection treatments include therapies for treating a bacterial, fungal and/or viral infections. Such therapies include antibiotics. Other examples include, but are not limited to, amebicides, aminoglycosides, anthelmintics, antifungals, azole antifungals, echinocandins, polyenes, diary lquinolines, hydrazide derivatives, nicotinic acid derivatives, rifamycin derivatives, streptomyces derivatives, antiviral agents, chemokine receptor antagonist, integrase strand transfer inhibitor, neuraminidase inhibitors, NNRTIs, NS5A inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors, purine nucleosides, carbapenems, cephalosporins, glycylcyclines, leprostatics, lincomycin derivatives, macrolide derivatives, ketolides, macrolides, oxazolidinone antibiotics, penicillins, beta-lactamase inhibitors, quinolones, sulfonamides, and tetracyclines. Other such therapies are known to those of ordinary skill in the art. Other anti-infection treatments are known to those of ordinary skill in the art.

As an even further example, the subject is one who is on mechanical support (e.g., a ventilator and/or circulatory). In one embodiment, the mechanical support is a device for extracorporeal membrane oxygenation (ECMO) or cardiopulmonary bypass.

The amount of total cf-DNA can be used to assess the risk in any one of these subjects. The risk in some embodiments may be of cardiac arrest and/or death. As provided herein, any one of the methods can be used to assess a subject that has or is suspected of having a complication or risk associated with any one of the conditions provided here.

In another embodiment of any one of the methods provided herein, the subject is one in which any organ injury, stress or dysfunction is occurring or is suspected of occurring. Such subjects include those with cardiac cardiomyopathy, congestive or other heart failure, congestive heart disease, ischemia, cardiomyopathy or other conditions of an organ, such as the heart. Such subjects also include subject with organ failure. Such subjects include heart failure, renal failure or hepatic failure. Subjects that fall within this category may also be one with pulmonary arterial hypertension. Such a subject may be one that has been or is being administered treatment for any one of the foregoing.

In another embodiment of any one of the methods provided herein, the subject is one with sepsis or shock or is suspected of having sepsis or shock. Such a subject may be one that has been or is being administered treatment for sepsis or shock.

The subject of any one of the methods provided herein may be any subject receiving a treatment, such as for any one of conditions provided herein. The amount(s) of total cf-DNA as determined by measuring the amount of Alu fragments may be used to assess the effectiveness of the treatment. The treatment may be any one of the treatments provided herein or known in the art.

In an embodiment of any one of the methods provided herein, the subject may have or be suspected of having any one of the disease or conditions provided herein. As used herein, “suspected of having” refers to a subject whereby a clinician believes there is a likelihood the subject has a specific condition or any one of the complications provided herein. Subjects may be suspected of having, determined to have had, or determined to have a likelihood or risk of having a complication based on symptoms (and/or lack thereof). However, in some embodiments, the subject is suspected of having, determined to have had, or determined to have a likelihood or risk of having a complication based on one or more other tests. In such an embodiment, the methods provided herein can be used to confirm such a finding or monitor such a subject for worsening or improving condition or complication.

An amount of Alu fragments may be determined with experimental techniques, such as those provided elsewhere herein. “Obtaining” as used herein refers to any method by which the respective information or materials can be acquired. Thus, the respective information can be acquired by experimental methods. Respective materials can be created, designed, etc. with various experimental or laboratory methods, in some embodiments. The respective information or materials can also be acquired by being given or provided with the information, such as in a report, or materials. Materials may be given or provided through commercial means (i.e., by purchasing), in some embodiments.

A risk of improving or worsening condition can be determined in any one of the subjects provided herein. A “risk” as provided herein, refers to the presence or absence or progression of any undesirable condition or complication in a subject, or an increased likelihood of the presence or absence or progression of such a condition or complication. As provided herein “increased risk” refers to the presence or progression of any undesirable condition or complication in a subject or an increased likelihood of the presence or progression of such a condition or complication. As provided herein, “decreased risk” refers to the absence of any undesirable condition or complication or progression in a subject or a decreased likelihood of the presence or progression (or increased likelihood of the absence or non-progression) of such a condition or complication. The risk in any one of the methods provided herein may be the presence or progression of any one of the conditions, complications or states provided herein.

Such techniques can also be used to monitor subject recovery and in the selection and monitoring of a treatment or therapy, thus improving a subject’s chance of recovery, increasing survival rates and/or improving clinical outcomes. Any one of the methods provided can be used to monitor a subject over time, with or without treatment. Further, such methods can aid in the selection, administration and/or monitoring of a treatment or therapy. Accordingly, the methods provided herein can be used to determine a treatment or monitoring regimen. In an embodiment, the method can be used to assess treatment efficacy in any one of the subjects provided herein.

“Determining a treatment regimen”, as used herein, refers to the determination of a course of action for treatment of the subject. In one embodiment of any one of the methods provided herein, determining a treatment regimen includes determining an appropriate therapy or information regarding an appropriate therapy to provide to a subject. In some embodiments of any one of the methods provided herein, the determining includes providing an appropriate therapy or information regarding an appropriate therapy to a subject. As used herein, information regarding a treatment or therapy or monitoring may be provided in written form or electronic form. In some embodiments, the information may be provided as computer-readable instructions. In some embodiments, the information may be provided orally. The therapies or treatments can be, for example, for treating any one of the conditions or states provided herein. Suitable therapies are provided or are known to those of ordinary skill in the art. Treatments include any treatment that is indicated based on the risk that is determined. In one embodiment, the treatment is a cardiac arrest treatment. Cardiac arrest treatments include, for example, blood pressure medications, involuntary nervous system blockers, and anti-arrhythmic agents. Further, a subject may be treated with coronary catheterization and/or a cardioverter-defibrillator may be implanted.

In another embodiment, the treatment can be a treatment for infection. In some embodiments, therapies for treating infection include therapies for treating a bacterial, fungal and/or viral infection. Such therapies include antibiotics. Other examples include, but are not limited to, amebicides, aminoglycosides, anthelmintics, antifungals, azole antifungals, echinocandins, polyenes, diary lquinolines, hydrazide derivatives, nicotinic acid derivatives, rifamycin derivatives, streptomyces derivatives, antiviral agents, chemokine receptor antagonist, integrase strand transfer inhibitor, neuraminidase inhibitors, NNRTIs, NS5A inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors, purine nucleosides, carbapenems, cephalosporins, glycylcyclines, leprostatics, lincomycin derivatives, macrolide derivatives, ketolides, macrolides, oxazolidinone antibiotics, penicillins, beta-lactamase inhibitors, quinolones, sulfonamides, and tetracyclines.

Anti-rejection therapies include, for example, immunosuppressives. Immunosuppressives include, but are not limited to, corticosteroids (e.g., prednisolone or hydrocortisone), glucocorticoids, cytostatics, alkylating agents (e.g., nitrogen mustards (cyclophosphamide), nitrosoureas, platinum compounds, cyclophosphamide (Cytoxan)), antimetabolites (e.g., folic acid analogues, such as methotrexate, purine analogues, such as azathioprine and mercaptopurine, pyrimidine analogues, and protein synthesis inhibitors), cytotoxic antibiotics (e.g., dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin), antibodies (e.g., anti-CD20, anti-IL-1, anti-IL-2Ralpha, anti-T-cell or anti-CD- 3 monoclonals and polyclonals, such as Atgam, and Thymoglobuline), drugs acting on immunophilins, ciclosporin, tacrolimus, sirolimus, interferons, opiods, TNF-binding proteins, mycophenolate, fingolimod and myriocin. In some embodiments, anti-rejection therapy comprises blood transfer or marrow transplant. Therapies can also include intravenous fluids, antibiotics, surgical drainage, early goal directed therapy (EGDT), vasopressors, steroids, activated protein C, drotrecogin alfa (activated), oxygen and appropriate support for organ dysfunction. This may include hemodialysis in kidney failure, mechanical ventilation in pulmonary dysfunction, transfusion of blood products, and drug and fluid therapy for circulatory failure. Ensuring adequate nutrition — preferably by enteral feeding, but if necessary, by parenteral nutrition — can also be included particularly during prolonged illness. Other associated therapies can include insulin and medication to prevent deep vein thrombosis and gastric ulcers.

Other therapies are known to those of ordinary skill in the art.

Administration of a treatment or therapy may be accomplished by any method known in the art (see, e.g., Harrison’s Principle of Internal Medicine, McGraw Hill Inc.). Preferably, administration of a treatment or therapy occurs in a therapeutically effective amount. Administration may be local or systemic. Administration may be parenteral (e.g., intravenous, subcutaneous, or intradermal) or oral. Compositions for different routes of administration are known in the art (see, e.g., Remington's Pharmaceutical Sciences by E. W. Martin).

“Determining a monitoring regimen”, as used herein, refers to determining a course of action to monitor a state or condition in the subject over time. In one embodiment of any one of the methods provided herein, determining a monitoring regimen includes determining an appropriate course of action for determining the amount of total cf-DNA in the subject over time or at a subsequent point in time, or suggesting such monitoring to the subject. This can allow for the measurement of variations in a clinical state and/or permit calculation of normal values or baseline levels (as well as comparisons thereto). In some embodiments of any one of the methods provided herein determining a monitoring regimen includes determining the timing and/or frequency of obtaining samples from the subject and/or determining or obtaining an amount of total cf-DNA. In any one of such methods, the amount of total cf- DNA can be determined by measuring the amount of Alu fragments in a sample.

In some embodiments, the total cf-DNA can be plotted over time. In some embodiments, threshold values for the points in time may also be plotted. For example, the threshold values can represent desirable or healthy values for the state or condition of a subject. Such plotting can be helpful to determine risk and/or to monitor a subject’s progress. Such threshold values can be determined using data from a sufficient number of subjects. A comparison with a subject’s total cf-DNA levels to such threshold values over a period of time can be used to predict risk. Alternatively, whether or not total cf-DNA amounts increase or decrease over time in a subject can alone be used to predict risk and/or assess the state or condition of the subject.

Increasing levels of total cf-DNA can correlate with increased risk, thus, a clinician may determine that a subject should undergo more frequent sampling if the subject’s total cf- DNA levels is found to increase between time points. If a subject is found to have decreasing levels of total cf-DNA between time points, a clinician may determine that less frequent sampling is sufficient. Additionally, if a subject does not show a decrease, the clinician may determine that additional testing and/or treatment and/or another type of treatment may be necessary. Steps of performing any one or more of the foregoing may be included in any one of the methods provided herein.

Timing and/or frequency of monitoring may also be determined by a comparison to threshold values or other amount(s), such as those determined at other point(s) in time. For example, if the amount of total cf-DNA is equal to or greater a threshold provided herein and/or is increasing, more frequent sampling may be needed, whereas, if the amount of total cf-DNA is less than one of the thresholds provided herein, and/or is not increasing, less frequent sampling may be required. Generally, subjects with higher or increasing amounts of total cf-DNA require closer monitoring and more frequent sampling.

In some embodiments of any one of the methods provided herein, each amount and time point may be recorded in a report or in a database. Threshold values may also be recorded in a report or in a database. Reports with any one or more of the values as provided herein are also provided in an aspect. Reports may be in oral, written (or hard copy) or electronic form, such as in a form that can be visualized or displayed. Preferably, the report provides the amount of total cf-DNA as determined by an amount of Alu fragments in a sample. In some embodiments, the report provides amounts of total cf-DNA as determined by amounts of Alu fragments in samples from a subject over time.

In some embodiments, the amounts are in or entered into a database. In one aspect, a database with such values is provided. From the amount(s), a clinician may assess the need for a treatment or monitoring of a subject. Accordingly, in any one of the methods provided herein, the method can include assessing the amount of total cf-DNA in the subject at more than one point in time. Such assessing can be performed with any one of the methods or compositions provided herein.

As used herein, “amount” refers to any quantitative value for a measurement and can be given in an absolute or relative amount. Further, the amount can be a total amount, frequency, ratio, percentage, etc. As used herein, the term “level” can be used instead of “amount” but is intended to refer to the same types of values. Generally, unless otherwise provided, the amounts provided herein represent the total cf-DNA levels in a sample as determined by the measurement of Alu fragments (e.g., concentrations of Alu fragments).

In some embodiments, any one of the methods provided herein can comprise comparing an amount of total cf-DNA to a threshold value, or to one or more prior amounts, to identify a subject at increased or decreased risk. In some embodiments of any one of the methods provided herein, a subject having an increased amount of total cf-DNA compared to a threshold value, or to one or more prior amounts, is identified as being at increased risk. In some embodiments of any one of the methods provided herein, a subject having a decreased or similar amount of total cf-DNA compared to a threshold value, or to one or more prior amounts, is identified as being at decreased or not increased risk.

“Threshold” or “threshold value” or “cutpoint”, as used herein, refers to any predetermined level or range of levels that is indicative of the presence or absence of a condition or the presence or absence of a risk. The threshold value can take a variety of forms. It can be single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as where the risk in one defined group is double the risk in another defined group. It can be a range, for example, where the tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high- risk group, or into quadrants, the lowest quadrant being subjects with the lowest risk and the highest quadrant being subjects with the highest risk. The threshold value can depend upon the particular population selected. For example, an apparently healthy population will have a different ‘normal’ range. As another example, a threshold value can be determined from baseline values before the presence of a condition or risk or after a course of treatment. Such a baseline can be indicative of a normal or other state in the subject not correlated with the risk or condition that is being tested for. In some embodiments, the threshold value can be a baseline value of the subject being tested. Accordingly, the predetermined values selected may take into account the category in which the subject falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. The threshold value of any one of the methods provided herein, can be any one of the threshold values provided herein, such as in the Examples or Figures. The threshold values can be used for comparisons to make treatment and/or monitoring decisions. The determination can be done based on any one of the comparisons as provided herein with or without other indicators of risk or the state or condition of the subject.

As described above, the level of Alu fragments may be used as a marker for total cf- DNA and it follows risk. In some embodiments, the level of Alu fragments is used as a trend monitor, for example, to determine if a subject is improving (e.g., if the subject’s risk is lessening). In some embodiments, the level of Alu fragments is used as an indicator of absolute risk; that is, near-term risk of poor clinical outcome, condition or state.

The threshold values provided herein can be used to determine a risk level to a subject, in an embodiment of any one of the methods provided herein. In some embodiments, the peak amount of Alu fragments is measured. As used herein “peak amount” refers to the highest value of serial samples. Accordingly, if a peak amount of Alu fragments is less than a certain threshold, the subject is at low risk. “Low risk” as used herein, includes a subject that has a shorter length of hospital stay or a shorter ventilation time and/or no cardiac arrest, death or infection. If a peak amount of Alu fragments is greater than a certain threshold, then the subject is at a high risk. “High risk” as used herein, includes a subject that is likely to experience near-term cardiac arrest, death, infection, and/or the need for mechanical circulatory support. Subjects at high risk are also likely to require a longer length of stay in the hospital and/or longer time on a ventilator in some embodiments. In an embodiment of any one of such methods, a subject with a value greater than a threshold may then be selected for treatment and/or further monitoring as provided herein. In an embodiment of any one of such methods provided herein, the method includes a step of further monitoring or treatment of the subject.

The threshold values can also be used for comparisons to make treatment and/or monitoring decisions. For example, an amount equal to or greater than a threshold may be indicative of cardiac arrest. As another example, an amount equal to or greater than a threshold may be indicative of infection. The determination can be done based on any one of the comparisons as provided herein with or without other indicators of such a complication. As another example, if the amount of Alu fragments is equal to or greater than a threshold value and/or increasing over time in any one of the methods provided herein, further monitoring and/or treatment may be indicated.

The amount of Alu fragments may be determined by a number of methods. In some embodiments such a method is a sequencing-based method. The DNA may be analyzed using any suitable next generation or high-throughput sequencing and/or genotyping technique. Examples of next generation and high-throughput sequencing and/or genotyping techniques include, but are not limited to, massively parallel signature sequencing, polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, ion semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, Mass ARRAY®, and Digital Analysis of Selected Regions (DANSR™) (see, e.g., Stein RA (1 September 2008). "Next- Generation Sequencing Update". Genetic Engineering & Biotechnology News 28 (15); Quail, Michael; Smith, Miriam E; Coupland, Paul; Otto, Thomas D; Harris, Simon R; Connor, Thomas R; Bertoni, Anna; Swerdlow, Harold P; Gu, Yong (1 January 2012). "A tale of three next generation sequencing platforms: comparison of Ion torrent, pacific biosciences and illumina MiSeq sequencers". BMC Genomics 13 (1): 341; Liu, Lin; Li, Yinhu; Li, Siliang; Hu, Ni; He, Yimin; Pong, Ray; Lin, Danni; Lu, Lihua; Law, Maggie (1 January 2012). "Comparison of Next-Generation Sequencing Systems". Journal of Biomedicine and Biotechnology 2012: 1- 11; Qualitative and quantitative genotyping using single base primer extension coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MassARRAY®). Methods Mol Biol. 2009;578:307-43; Chu T, Bunce K, Hogge WA, Peters DG. A novel approach toward the challenge of accurately quantifying fetal DNA in maternal plasma. Prenat Diagn 2010;30:1226-9; and Suzuki N, Kamataki A, Yamaki J, Homma Y. Characterization of circulating DNA in healthy human plasma. Clinica chimica acta; International Journal of Clinical Chemistry 2008;387:55-8).

In some embodiments of any one of the methods provided herein, an amplification- based quantitative assay can be used. An amplification-based quantitative assay is any quantitative assay, such as whereby nucleic acids are amplified and the amounts of the nucleic acids (e.g., Alu fragments) can be determined. Such assays also include simple amplification and detection, hybridization techniques, separation technologies, such as electrophoresis, next generation sequencing and the like. In some embodiments, any one of the methods for determining Alu fragment levels may be quantitative PCR, such as real-time quantitative PCR (e.g., Alu-qPCR). Quantitative PCR includes real-time PCR, digital PCR, TAQMAN™, etc.

The use of multiple probes can expand the capability of single-probe real-time PCR. Multiplex real-time PCR uses multiple probe-based assays, in which each assay can have a specific probe labeled with a unique fluorescent dye, resulting in different observed colors for each assay. Real-time PCR instruments can discriminate between the fluorescence generated from different dyes. Different probes can be labeled with different dyes that each have unique emission spectra. Spectral signals are collected with discrete optics, passed through a series of filter sets, and collected by an array of detectors. Spectral overlap between dyes may be corrected by using pure dye spectra to deconvolute the experimental data by matrix algebra.

A probe may be useful for methods of the present disclosure, particularly for those methods that include a quantification step. Any one of the methods provided herein can include the use of a probe in the performance of the PCR assay(s), while any one of the compositions or kits provided herein can include one or more probes. In some embodiments of any one or more of the methods provided herein, the probe in one or more or all of the PCR quantification assays is on the same strand as the mismatch primer and not on the opposite strand. It has been found that in so incorporating the probe in a PCR reaction, additional allele specific discrimination can be provided.

As an example, a TAQMAN™ probe is a hydrolysis probe that has a FAM™ or VIC® dye label on the 5' end, and minor groove binder (MGB) non-fluorescent quencher (NFQ) on the 3' end. The TAQMAN™ probe principle generally relies on the 5 3 exonuclease activity of Taq® polymerase to cleave the dual-labeled TAQMAN™ probe during hybridization to a complementary probe-binding region and fluorophore-based detection. TAQMAN™ probes can increase the specificity of detection in quantitative measurements during the exponential stages of a quantitative PCR reaction.

PCR systems generally rely upon the detection and quantitation of fluorescent dyes or reporters, the signal of which increase in direct proportion to the amount of PCR product in a reaction. For example, in the simplest and most economical format, that reporter can be the double-stranded DNA-specific dye SYBR® Green (Molecular Probes). SYBR® Green is a dye that binds the minor groove of double-stranded DNA. When SYBR® Green dye binds to a double- stranded DNA, the fluorescence intensity increases. As more double- stranded amplicons are produced, SYBR® Green dye signal will increase.

In any one of the methods provided herein the PCR may be digital PCR. Digital PCR involves partitioning of diluted amplification products into a plurality of discrete test sites such that most of the discrete test sites comprise either zero or one amplification product. The amplification products are then analyzed to provide a representation of the frequency of the selected genomic regions of interest in a sample. Analysis of one amplification product per discrete test site results in a binary “yes-or-no” result for each discrete test site, allowing the selected genomic regions of interest to be quantified and the relative frequency of the selected genomic regions of interest in relation to one another be determined. In certain aspects, in addition to or as an alternative, multiple analyses may be performed using amplification products corresponding to genomic regions from predetermined regions. Results from the analysis of two or more predetermined regions can be used to quantify and determine the relative frequency of the number of amplification products. Using two or more predetermined regions to determine the frequency in a sample reduces a possibility of bias through, e.g., variations in amplification efficiency, which may not be readily apparent through a single detection assay. Methods for quantifying DNA using digital PCR are known in the art and have been previously described, for example in U.S. Patent Publication number US20140242582. It should be appreciated that the PCR conditions provided herein may be modified or optimized to work in accordance with any one of the methods described herein. Typically, the PCR conditions are based on the enzyme used, the target template, and/or the primers. In some embodiments, one or more components of the PCR reaction is modified or optimized. Non-limiting examples of the components of a PCR reaction that may be optimized include the template DNA, the primers (e.g., forward primers and reverse primers), the deoxynucleotides (dNTPs), the polymerase, the magnesium concentration, the buffer, the probe (e.g., when performing real-time PCR), the buffer, and the reaction volume.

In any of the foregoing embodiments, any DNA polymerase (enzyme that catalyzes polymerization of DNA nucleotides into a DNA strand) may be utilized, including thermostable polymerases. Suitable polymerase enzymes will be known to those skilled in the art, and include E. coli DNA polymerase, Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, Klenow class polymerases, Taq polymerase, Pfu DNA polymerase, Vent polymerase, bacteriophage 29, REDTaq™ Genomic DNA polymerase, or sequenase. Exemplary polymerases include, but are not limited to Bacillus stearothermophilus pol I, Thermus aquaticus (Taq) pol I, Pyrccoccus furiosus (Pfu), Pyrococcus woesei (Pwo), Thermus flavus (Tfl), Thermus thermophilus (Tth), Thermus litoris (Tli) and Thermotoga maritime (Tma). These enzymes, modified versions of these enzymes, and combination of enzymes, are commercially available from vendors including Roche, Invitrogen, Qiagen, Stratagene, and Applied Biosystems. Representative enzymes include PHUSION® (New England Biolabs, Ipswich, MA), Hot MasterTaq™ (Eppendorf), PHUSION® Mpx (Finnzymes), PyroStart® (Fermentas), KOD (EMD Biosciences), Z-Taq (TAKARA), and CS3AC/LA (KlenTaq, University City, MO).

Salts and buffers include those familiar to those skilled in the art, including those comprising MgC12, and Tris-HCl and KC1, respectively. Typically, 1.5-2.0nM of magnesium is optimal for Taq DNA polymerase, however, the optimal magnesium concentration may depend on template, buffer, DNA and dNTPs as each has the potential to chelate magnesium. If the concentration of magnesium [Mg2+] is too low, a PCR product may not form. If the concentration of magnesium [Mg2+] is too high, undesired PCR products may be seen. In some embodiments the magnesium concentration may be optimized by supplementing magnesium concentration in O.lmM or 0.5mM increments up to about 5 mM.

Buffers used in accordance with the disclosure may contain additives such as surfactants, dimethyl sulfoxide (DMSO), glycerol, bovine serum albumin (BSA) and polyethylene glycol (PEG), as well as others familiar to those skilled in the art. Nucleotides are generally deoxyribonucleoside triphosphates, such as deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP), which are also added to a reaction adequate amount for amplification of the target nucleic acid. In some embodiments, the concentration of one or more dNTPs (e.g., dATP, dCTP, dGTP, dTTP) is from about 10 mM to about 500mM which may depend on the length and number of PCR products produced in a PCR reaction.

In some embodiments, the concentration of primers used in the PCR reaction may be modified or optimized. In some embodiments, the concentration of a primer (e.g., a forward or reverse primer) in a PCR reaction may be, for example, about 0.05 mM to about 1 mM. In particular embodiments, the concentration of each primer is about 1 nM to about 1 mM. It should be appreciated that the primers in accordance with the disclosure may be used at the same or different concentrations in a PCR reaction. For example, the forward primer of a primer pair may be used at a concentration of 0.5 mM and the reverse primer of the primer pair may be used at 0.1 mM. The concentration of the primer may be based on factors including, but not limited to, primer length, GC content, purity, mismatches with the target DNA or likelihood of forming primer dimers.

In some embodiments, the thermal profile of the PCR reaction is modified or optimized. Non-limiting examples of PCR thermal profile modifications include denaturation temperature and duration, annealing temperature and duration and extension time.

The temperature of the PCR reaction solutions may be sequentially cycled between a denaturing state, an annealing state, and an extension state for a predetermined number of cycles. The actual times and temperatures can be enzyme, primer, and target dependent. For any given reaction, denaturing states can range in certain embodiments from about 70 °C to about 100 °C. In addition, the annealing temperature and time can influence the specificity and efficiency of primer binding to a particular locus within a target nucleic acid and may be important for particular PCR reactions. For any given reaction, annealing states can range in certain embodiments from about 20 °C to about 75 °C. In some embodiments, the annealing state can be from about 46 °C to 64°C. In certain embodiments, the annealing state can be performed at room temperature (e.g., from about 20 °C to about 25 °C).

Extension temperature and time may also impact the allele product yield. For a given enzyme, extension states can range in certain embodiments from about 60 °C to about 75 °C.

Quantification of the amounts of the alleles from a PCR assay can be performed as provided herein or as otherwise would be apparent to one of ordinary skill in the art. As an example, amplification traces are analyzed for consistency and robust quantification. Internal standards may be used to translate the cycle threshold to amount of input nucleic acids (e.g., DNA). The amounts of alleles can be computed as the mean of performant assays and can be adjusted for genotype.

Other methods for determining Alu fragments in a sample are known in the art.

As used herein, the sample from a subject can be a biological sample. Examples of such biological samples include whole blood, plasma, serum, urine, saliva, etc. In some embodiments, addition of further nucleic acids, e.g., a standard, to the sample can be performed.

Various aspects of the present invention may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and are therefore not limited in their application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Also, embodiments of the invention may be implemented as one or more methods, of which an example has been provided. The acts performed as part of the method(s) may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different from illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed. Such terms are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term).

The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing”, “involving”, and variations thereof, is meant to encompass the items listed thereafter and additional items.

Having described several embodiments of the invention in detail, various modifications and improvements will readily occur to those skilled in the art. Such modifications and improvements are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description is by way of example only, and is not intended as limiting. The following description provides examples of the methods provided herein.

EXAMPLES

Example 1 - Examples of Computer-Implemented Embodiments

In some embodiments, the diagnostic techniques described above may be implemented via one or more computing devices executing one or more software facilities to analyze samples for a subject over time, measure nucleic acids (such as Alu fragments) in the samples, and produce a diagnostic result based on one or more of the samples. Fig. 1 illustrates an example of a computer system with which some embodiments may operate, though it should be appreciated that embodiments are not limited to operating with a system of the type illustrated in Fig. 1

The computer system of Fig. 1 includes a subject 802 and a clinician 804 that may obtain a sample 806 from the subject 806. As should be appreciated from the foregoing, the sample 806 may be any suitable sample of biological material for the subject 802 that may be used to measure the presence of nucleic acids (such as Alu fragments) in the subject 802, including a blood sample. The sample 806 may be provided to an analysis device 808, which one of ordinary skill will appreciate from the foregoing will analyze the sample 808 so as to determine (including estimate) a total amount of nucleic acids (such as Alu fragments) in the sample 806 and/or the subject 802. For ease of illustration, the analysis device 808 is depicted as single device, but it should be appreciated that analysis device 808 may take any suitable form and may, in some embodiments, be implemented as multiple devices. To determine the amounts of nucleic acids (such as Alu fragments) in the sample 806 and/or subject 802, the analysis device 808 may perform any of the techniques described above, and is not limited to performing any particular analysis. The analysis device 808 may include one or more processors to execute an analysis facility implemented in software, which may drive the processor(s) to operate other hardware and receive the results of tasks performed by the other hardware to determine on overall result of the analysis, which may be the amounts of nucleic acids (such as Alu fragments) in the sample 806 and/or the subject 802. The analysis facility may be stored in one or more computer-readable storage media, such as a memory of the device 808. In other embodiments, techniques described herein for analyzing a sample may be partially or entirely implemented in one or more special-purpose computer components such as Application Specific Integrated Circuits (ASICs), or through any other suitable form of computer component that may take the place of a software implementation.

In some embodiments, the clinician 804 may directly provide the sample 806 to the analysis device 808 and may operate the device 808 in addition to obtaining the sample 806 from the subject 802, while in other embodiments the device 808 may be located geographically remote from the clinician 804 and subject 802 and the sample 806 may need to be shipped or otherwise transferred to a location of the analysis device 808. The sample 806 may in some embodiments be provided to the analysis device 808 together with (e.g., input via any suitable interface) an identifier for the sample 806 and/or the subject 802, for a date and/or time at which the sample 806 was obtained, or other information describing or identifying the sample 806.

The analysis device 808 may in some embodiments be configured to provide a result of the analysis performed on the sample 806 to a computing device 810, which may include a data store 810A that may be implemented as a database or other suitable data store. The computing device 810 may in some embodiments be implemented as one or more servers, including as one or more physical and/or virtual machines of a distributed computing platform such as a cloud service provider. In other embodiments, the device 810 may be implemented as a desktop or laptop personal computer, a smart mobile phone, a tablet computer, a special-purpose hardware device, or other computing device.

In some embodiments, the analysis device 808 may communicate the result of its analysis to the device 810 via one or more wired and/or wireless, local and/or wide-area computer communication networks, including the Internet. The result of the analysis may be communicated using any suitable protocol and may be communicated together with the information describing or identifying the sample 806, such as an identifier for the sample 806 and/or subject 802 or a date and/or time the sample 806 was obtained.

The computing device 810 may include one or more processors to execute a diagnostic facility implemented in software, which may drive the processor(s) to perform diagnostic techniques described herein. The diagnostic facility may be stored in one or more computer-readable storage media, such as a memory of the device 810. In other embodiments, techniques described herein for analyzing a sample may be partially or entirely implemented in one or more special-purpose computer components such as Application Specific Integrated Circuits (ASICs), or through any other suitable form of computer component that may take the place of a software implementation. The diagnostic facility may receive the result of the analysis and the information describing or identifying the sample 806 and may store that information in the data store 810A. The information may be stored in the data store 810A in association with other information for the subject 802, such as in a case that information regarding prior samples for the subject 802 was previously received and stored by the diagnostic facility. The information regarding multiple samples may be associated using a common identifier, such as an identifier for the subject 802. In some cases, the data store 810A may include information for multiple different subjects.

The diagnostic facility may also be operated to analyze results of the analysis of one or more samples 806 for a particular subject 802, identified by user input, so as to determine a diagnosis for the subject 802. The diagnosis may be a conclusion of a risk that the subject 802 has, may have, or may in the future develop a particular condition. The diagnostic facility may determine the diagnosis using any of the various examples described above, including by comparing the amounts of nucleic acids (such as Alu fragments) determined for a particular sample 806 to one or more thresholds or by comparing a change over time in the amounts of nucleic acids (such as cell-free DNA) determined for samples 806 over time to one or more thresholds. For example, the diagnostic facility may determine a risk to the subject 802 of a condition by comparing a total amount of nucleic acids (such as Alu fragments) for one or more samples 806 to a threshold. Based on the comparisons to the thresholds, the diagnostic facility may produce an output indicative of a risk to the subject 802 of a condition.

As should be appreciated from the foregoing, in some embodiments, the diagnostic facility may be configured with different thresholds to which amounts of nucleic acids (such as Alu fragments) may be compared. The different thresholds may, for example, correspond to different demographic groups (age, gender, race, economic class, presence or absence of a particular procedure/condition/other in medical history, or other demographic categories), different conditions, and/or other parameters or combinations of parameters. In such embodiments, the diagnostic facility may be configured to select thresholds against which amounts of nucleic acids (such as Alu fragments) are to be compared, with different thresholds stored in memory of the computing device 810. The selection may thus be based on demographic information for the subject 802 in embodiments in which thresholds differ based on demographic group, and in these cases demographic information for the subject 802 may be provided to the diagnostic facility or retrieved (from another computing device, or a data store that may be the same or different from the data store 810A, or from any other suitable source) by the diagnostic facility using an identifier for the subject 802. The selection may additionally or alternatively be based on the condition for which a risk is to be determined, and the diagnostic facility may prior to determining the risk receive as input a condition and use the condition to select the thresholds on which to base the determination of risk. It should be appreciated that the diagnostic facility is not limited to selecting thresholds in any particular manner, in embodiments in which multiple thresholds are supported.

In some embodiments, the diagnostic facility may be configured to output for presentation to a user a user interface that includes a diagnosis of a risk and/or a basis for the diagnosis for a subject 802. The basis for the diagnosis may include, for example, amounts of nucleic acids (such as Alu fragments) detected in one or more samples 806 for a subject 802. In some embodiments, user interfaces may include any of the examples of results, values, amounts, graphs, etc. discussed above. They can include results, values, amounts, etc. over time. For example, in some embodiments, a user interface may incorporate a graph similar to that shown in any one of the figures provided herein. In such a case, in some cases the graph may be annotated to indicate to a user how different regions of the graph may correspond to different diagnoses that may be produced from an analysis of data displayed in the graph. For example, thresholds against which the graphed data may be compared to determine the analysis may be imposed on the graph(s).

A user interface including a graph, particularly with the lines and/or shading, may provide a user with a far more intuitive and faster-to-review interface to determine a risk of the subject 802 based on amounts of nucleic acids (such as Alu fragments), than may be provided through other user interfaces. It should be appreciated, however, that embodiments are not limited to being implemented with any particular user interface.

In some embodiments, the diagnostic facility may output the diagnosis or a user interface to one or more other computing devices 814 (including devices 814A, 814B) that may be operated by the subject 802 and/or a clinician, which may be the clinician 804 or another clinician. The diagnostic facility may transmit the diagnosis and/or user interface to the device 814 via the network(s) 812.

Techniques operating according to the principles described herein may be implemented in any suitable manner. Included in the discussion above are a series of flow charts showing the steps and acts of various processes that determine a risk of a condition based on an analysis of amounts of nucleic acids (such as Alu fragments). The processing and decision blocks discussed above represent steps and acts that may be included in algorithms that carry out these various processes. Algorithms derived from these processes may be implemented as software integrated with and directing the operation of one or more single- or multi-purpose processors, may be implemented as functionally-equivalent circuits such as a Digital Signal Processing (DSP) circuit or an Application-Specific Integrated Circuit (ASIC), or may be implemented in any other suitable manner. It should be appreciated that embodiments are not limited to any particular syntax or operation of any particular circuit or of any particular programming language or type of programming language. Rather, one skilled in the art may use the description above to fabricate circuits or to implement computer software algorithms to perform the processing of a particular apparatus carrying out the types of techniques described herein. It should also be appreciated that, unless otherwise indicated herein, the particular sequence of steps and/or acts described above is merely illustrative of the algorithms that may be implemented and can be varied in implementations and embodiments of the principles described herein.

Accordingly, in some embodiments, the techniques described herein may be embodied in computer-executable instructions implemented as software, including as application software, system software, firmware, middleware, embedded code, or any other suitable type of computer code. Such computer-executable instructions may be written using any of a number of suitable programming languages and/or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine.

When techniques described herein are embodied as computer-executable instructions, these computer-executable instructions may be implemented in any suitable manner, including as a number of functional facilities, each providing one or more operations to complete execution of algorithms operating according to these techniques. A “functional facility,” however instantiated, is a structural component of a computer system that, when integrated with and executed by one or more computers, causes the one or more computers to perform a specific operational role. A functional facility may be a portion of or an entire software element. For example, a functional facility may be implemented as a function of a process, or as a discrete process, or as any other suitable unit of processing. If techniques described herein are implemented as multiple functional facilities, each functional facility may be implemented in its own way; all need not be implemented the same way. Additionally, these functional facilities may be executed in parallel and/or serially, as appropriate, and may pass information between one another using a shared memory on the computer(s) on which they are executing, using a message passing protocol, or in any other suitable way. Generally, functional facilities include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the functional facilities may be combined or distributed as desired in the systems in which they operate. In some implementations, one or more functional facilities carrying out techniques herein may together form a complete software package. These functional facilities may, in alternative embodiments, be adapted to interact with other, unrelated functional facilities and/or processes, to implement a software program application.

Some exemplary functional facilities have been described herein for carrying out one or more tasks. It should be appreciated, though, that the functional facilities and division of tasks described is merely illustrative of the type of functional facilities that may implement the exemplary techniques described herein, and that embodiments are not limited to being implemented in any specific number, division, or type of functional facilities. In some implementations, all functionality may be implemented in a single functional facility. It should also be appreciated that, in some implementations, some of the functional facilities described herein may be implemented together with or separately from others (i.e., as a single unit or separate units), or some of these functional facilities may not be implemented.

Computer-executable instructions implementing the techniques described herein (when implemented as one or more functional facilities or in any other manner) may, in some embodiments, be encoded on one or more computer-readable media to provide functionality to the media. Computer-readable media include magnetic media such as a hard disk drive, optical media such as a Compact Disk (CD) or a Digital Versatile Disk (DVD), a persistent or non-persistent solid-state memory (e.g., Flash memory, Magnetic RAM, etc.), or any other suitable storage media. Such a computer-readable medium may be implemented in any suitable manner, including as a portion of a computing device or as a stand-alone, separate storage medium. As used herein, “computer-readable media” (also called “computer-readable storage media”) refers to tangible storage media. Tangible storage media are non-transitory and have at least one physical, structural component. In a “computer-readable medium,” as used herein, at least one physical, structural component has at least one physical property that may be altered in some way during a process of creating the medium with embedded information, a process of recording information thereon, or any other process of encoding the medium with information. For example, a magnetization state of a portion of a physical structure of a computer-readable medium may be altered during a recording process. In some, but not all, implementations in which the techniques may be embodied as computer-executable instructions, these instructions may be executed on one or more suitable computing device(s) operating in any suitable computer system, including the exemplary computer system of Fig. 1, or one or more computing devices (or one or more processors of one or more computing devices) may be programmed to execute the computer-executable instructions. A computing device or processor may be programmed to execute instructions when the instructions are stored in a manner accessible to the computing device or processor, such as in a data store (e.g., an on-chip cache or instruction register, a computer-readable storage medium accessible via a bus, etc.). Functional facilities comprising these computer- executable instructions may be integrated with and direct the operation of a single multi purpose programmable digital computing device, a coordinated system of two or more multi purpose computing device sharing processing power and jointly carrying out the techniques described herein, a single computing device or coordinated system of computing device (co located or geographically distributed) dedicated to executing the techniques described herein, one or more Field-Programmable Gate Arrays (FPGAs) for carrying out the techniques described herein, or any other suitable system.

Embodiments have been described where the techniques are implemented in circuitry and/or computer-executable instructions. It should be appreciated that some embodiments may be in the form of a method, of which at least one example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments. Any one of the aforementioned, including the aforementioned devices, systems, embodiments, methods, techniques, algorithms, media, hardware, software, interfaces, processors, displays, networks, inputs, outputs or any combination thereof are provided herein in other aspects.

Example 2 - Alu Fragment Correlation with Post-Surgical Complications

The myTAIHEART DNA Fragmentation Assay (ALU test) /AKA Cell Lysis Monitoring test is Taqman-based and uses primers specific for two different lengths (115 and 147 bp) of ALU sequence, with a shared probe for both amplicons. ALU 115 bp amplification detects both shorter fragments from apoptosis as well as longer fragments derived from non-apoptotic lysis. Only longer fragments derived from non-apoptotic lysis are detected by ALU 247 bp amplification. The ratio of longer ALU247 fragments to shorter ALU115 fragments (the ALU ratio) increases as ex vivo lysis increases, providing a useful qualitative quality assurance tool to monitor presence of potentially problematic ex vivo leukocyte lysis during collection and processing of plasma samples. An ALU ratio below ~ 0.4 indicates the cf-DNA in the sample is almost entirely the result of cellular apoptosis and acceptably free of leukocyte lysis during sample collection and processing.

The myTAIHEART DNA Fragmentation Assay (ALU test) is performed on cfDNA extract after quantification of extract TCF concentration by RNaseP qPCR. Input was 50 pg, run in triplicate for both Alu fragment length amplifications against a five-point human gDNA standard curve. ALU115 and ALU247 amplifications were performed individually for each primer pair on a Roche Lightcycler 480 (LC280) using a shared TaqMan™ probe. The Lightcycler software was used to calculate a standard curve for the run by plotting the known DNA concentration of each standard dilution on the x-axis and the mean crossing point (Cp) value for those dilutions on the y-axis, also calculating the slope and amplification efficiency for each run. TCF concentrations of patient samples were individually determined by the Lightcycler software for the ALU115 and ALU247 amplifications using the calculated standard curve equation and the mean Cp as input. Results generated by the LC480 Abs Quant/2nd Derivative Max algorithm captured in a report were used to determine Alu ratio by dividing the ALU247 concentration by the ALU115 concentration.

Analytical quality metrics developed to ensure validity of a myTAIHEART DNA Fragmentation Assay run included required ranges for ALU115 and ALU247 amplification efficiency, standard deviations of standard curve points, quantifications in pg/pl of low, medium, and high ALU 115 and ALU247 controls, fragment ratios of specified standards, no template control (NTC) mean Cp, and specified standard Cp.

For determination of LoQ for the short and long fragment components, human gDNA prepared as described in was diluted in 0.1X TE to concentrations of 4, 2, 1, 0.5 and 0.25 pg/pl. Briefly, plasma was spiked with short fragments of DNA obtained by Covaris ME220 (Woodburn, MA) focused ultrasonication (“shearing”) of gDNA from the paired cellular component (buffy coat) to a size distribution primarily in the range of 130-180 bp, approximating that of cfDNA. Resultant fragment lengths were evaluated on an Agilent 2100 Bioanalyzer (Santa Clara, CA) with a high sensitivity DNA chip to confirm production of the targeted range as determined by base pair size of maximum fluorescence values (North et al 2020). LoQ for each assay was determined according guidelines outlined in CLSI EP17- A2 [91]. The LoQ for the short and long fragment assays were determined to be 0.25 pg/pl (North et al 2020). The total cell-free DNA and Alu fragments of subjects who had undergone cardiopulmonary bypass procedures (CBP) (N = 120) were examined (Figs. 2-3). Fig. 2 shows the levels of total cf-DNA (left) and Alu fragments (right) in the combined outcome of death, mechanical circulatory support (MCS) or cardiac arrest (CA). The bold lines show the levels for the poor outcome patients. Fig. 3 shows the correlation between total cf-DNA and death (left) and Alu fragment levels and death (right).

Pre-surgery (left of the “SG” line in Figs. 2-3), the signal from total cf-DNA and Alu fragment levels is mild, but present. The mean total cf-DNA pre-surgery for those with no bad outcomes (e.g., death, mechanical circulatory support (MCS) or cardiac arrest (CA)), was 10^L0.87 ng/mL plasma (N=l 11), while the mean total cf-DNA pre-surgery of the group with bad outcomes (N=9) was 10^L1.21 ng/mL plasma (p=0.03). A difference was also detected in the Alu fragment measurements: the mean Alu levels pre-surgery for those with no bad outcomes (e.g., death, mechanical circulatory support (MCS) or cardiac arrest (CA)), was 10^L2.26 pg/pL plasma (N=l 11), while the mean Alu levels pre-surgery of the group with bad outcomes (N=9) was 10^L2.62 pg/pL plasma (p=0.04).

The distinction is more pronounced following surgery. One hour after CPB, the mean Alu levels were 10^L2.87 pg/uL (no bad outcomes; N=lll) compared to mean Alu levels of 10^L3.59 (bad outcomes; N=9); p O.Ol. Similarly, the total cf-DNA levels were 10^L1.60 ng/mL vs 10^L2.18 n/mL, pcO.Ol.

Claims

What is claimed is: CLAIMS

1. A method of measuring total cell-free DNA (cf-DNA) in a sample from a subject, the method comprising determining an amount of Alu fragments in the sample.

2. The method of claim 1, wherein the method further comprises reporting and/or recording the amount of Alu fragments.

3. The method of claim 1 or 2, wherein the Alu fragments are Alu 115 fragments.

4. The method of any one of the preceding claims, wherein the amount of Alu fragments is determined or obtained using an amplification-based quantification assay.

5. The method of claim 4, wherein the amplification-based quantification assay is quantitative real-time PCR (qRT-PCR) or digital PCR.

6. The method of any one of the preceding claims, wherein the subject is a transplant subject.

7. The method of any claim 6, wherein the transplant subject is a heart transplant subject.

8. The method of any one of claims 1-5, wherein the subject is a surgical subject.

9. The method of claim 8, wherein the subject has undergone heart surgery.

10. The method of any one of claims 1-5, wherein the subject has a pro -inflammatory response.

11. The method of any one of claims 1-5 and 10, wherein the subject has an infection, such as COVID-19.

12. The method of any one of claims 1-5, wherein the subject is on mechanical support.

13. The method of any one of claims 1-5, wherein the subject has sepsis or septic shock.

14. The method of any one of the preceding claims, wherein the sample is a blood, plasma or serum sample.

15. The method of any one of the preceding claims, wherein the Alu fragment amount is provided in a report.

16. The method of any one of the preceding claims, wherein the Alu fragment amount is recorded in a database.

17. A method of assessing a sample from a subject, the method comprising:

(a) obtaining an amount of Alu fragments in a sample from the subject, wherein the subject has, is suspected of having, has had, or is at risk of having any one of the conditions or complications provided herein; and

(b) making an assessment based on the amount of Alu fragments.

18. The method of claim 17, wherein the method further comprises reporting and/or recording the amount of Alu fragments.

19. The method of claim 17 or 18, wherein the making an assessment comprises comparing the amount of Alu fragments to a threshold Alu fragment value or at least one prior Alu fragment amount.

20. The method of any one of claims 17-19, wherein the method further comprises determining that the subject has, or as being at increased risk of having the condition or complication based on the amount of Alu fragments, such as compared to the threshold Alu fragment value and/or at least one prior Alu fragment amount.

21. The method of any one of claims 17-20, wherein the obtaining an amount of Alu fragments is any one of the methods provided herein, such as the method of any one of claims 1-12.

22. The method of any one of claims 17-21, wherein the method further comprises determining a treatment or monitoring regimen for the subject based on the amount of Alu fragments, such as compared to the threshold Alu fragments value and/or at least one prior Alu fragment amount.

23. The method of any one of claims 17-22, wherein the method further comprises classifying the subject as having or as being at increased risk of having the condition or complication based on the amount of Alu fragments, such as compared to the threshold Alu fragment value and/or at least one prior Alu fragment amount.

24. The method of any one claims 17-23, wherein an amount of Alu fragments that is greater than the threshold value and/or is increased relative to the amount from an earlier time point represents an increased or increasing risk.

25. The method of any one of claims 17-23, wherein an amount of Alu fragments that is lower than the threshold value and/or is decreased relative to the amount from an earlier time point represents a decreased or decreasing risk.

26. The method of any one of claims 22-25, wherein the determining a monitoring regimen comprises determining the amount of Alu fragments in the subject over time or at a subsequent point in time, or suggesting such monitoring to the subject.

27. The method of any one of claims 22-26, wherein the time between samples is decreased if the amount of Alu fragments is increased relative to the threshold or an amount from an earlier time point.

28. The method of any one of claims 22-27, wherein the determining a treatment regimen comprises selecting or suggesting a treatment for the subject.

29. The method of any one of claims 22-28, wherein the determining a treatment regimen comprises treating the subject.

30. The method of any one of claims 22-29, wherein the treatment comprises any one of the treatments provided herein.

31. The method of any one of claims 22-29, wherein the determining a treatment regimen comprises providing information about a treatment to the subject.

32. The method of any one of the preceding claims, wherein the subject is receiving or has received treatment for any one of the conditions or complications provided herein.

33. The method of any one of the preceding claims, wherein the threshold is any one of the thresholds provided herein, including any one of the Examples or Figures.