WO2014062819A1 - Compositions and methods for improving diagnosis - Google Patents

Compositions and methods for improving diagnosis Download PDF

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WO2014062819A1
WO2014062819A1 PCT/US2013/065268 US2013065268W WO2014062819A1 WO 2014062819 A1 WO2014062819 A1 WO 2014062819A1 US 2013065268 W US2013065268 W US 2013065268W WO 2014062819 A1 WO2014062819 A1 WO 2014062819A1
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urine sample
stabilizing
method
omega
stabilizing agents
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PCT/US2013/065268
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French (fr)
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Richard H. Selinfreund
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Companion Diagnostics Inc.
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/70Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • G01N33/487Physical analysis of biological material of liquid biological material
    • G01N33/493Physical analysis of biological material of liquid biological material urine

Abstract

The invention provides compositions and methods for improving diagnosis. Specifically, the invention provides compositions and methods for enhancing the sensitivity or accuracy of a diagnostic test that uses a urine sample. Hospitals, clinics and physician offices often collect urine samples to be tested later at regional testing laboratories like Lab Core and Quest. The patient samples are transported in most cases without refrigeration to the labs and often sit for days before being tested.

Description

COMPOSITIONS AND METHODS FOR IMPROVING DIAGNOSIS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Applications 61/714,650 and 61/783,959, filed October 16, 2012 and March 14, 2013, respectively, both of which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

[0002] The invention relates to compositions and methods for improving diagnosis. Specifically, the invention relates to compositions and methods for enhancing the sensitivity or accuracy of a diagnostic test that uses a urine sample. BACKGROUND OF THE INVENTION

[0003] Hospitals, clinics and physician offices often collect urine samples to be tested later at regional testing laboratories like Lab Core and Quest. The patient samples are transported in most cases without refrigeration to the labs and often sit for days before being tested. Microbiological contaminants (e.g., bacteria) in the collected samples immediately start to multiply. This growth is associated with changes in pH, and the production of oxygen species, nitric oxide and hydrogen peroxide (H202). Changes in pH and oxidation of the content can alter the molecules to be tested in urine sample. A change in pH can alter the endpoint measurement in diabetes testing. A change in oxidation can significantly change the level of creatinine levels (i.e, a measurement of kidney function). The target of the clinical test is adversely impacted by the contaminants. Accordingly, there is a need to reduce the problems associated with the unavoidable microbiological contamination of urine sample without significantly impacting the process, accuracy or the current cost of testing.

[0004] Furthermore, there is a requirement today for the large testing laboratories that process thousands of urine specimens to purchase calibration controls for each specific tests. Regardless of the test or equipment used, the FDA requires each test to have a calibration control. There is also a requirement by the FDA that these controls be made up in a blank or collected urine sample. For example, one large urine control supplier to the industry processes more than 20 lots of human urine a year. Each lot can be larger than 1000 liters from just one company. Multiple companies provide urine controls with the diagnostic industry which represents 1000's of liters of urine. These companies use processes for preparing the urines for production into controls. The problem develops when these control products are tested for the subsequent contaminant that will cause interference in the control accuracy over time, for example, during the labeled use of the control. In such control samples, oxidation or rancidity can be detected. The oxidation or rancidity can have significant impact on the accuracy of the clinical control being used to calibrate the results for each urine sample being tested. Accordingly, there is a need for a rapid non-interfering method and additives to reduce oxidative damage and pH changes in urine clinical controls.

[0005] Additionally, detection of controlled substances and drugs of abuse are often made with a patient's urine specimen. These samples are collected at one place and then transported to a laboratory that specializes in measuring drugs of addiction and/or drugs of abuse. It is important requirement that these small organic molecules be measured accurately. Problems with sample contaminants often reduce the accuracy of the tests.

[0006] The major problem being a "false-negative," the findings can have a poor outcome when a physician is deciding if another compound is required to treat a patient. In addition, a hiring decision can be made incorrectly if a false-negative is not detected.

[0007] Accordingly, there exists a need for compositions and methods for enhancing the sensitivity or accuracy of a diagnostic test.

SUMMARY OF THE INVENTION

[0008] In one embodiment, the invention provides a method for enhancing the sensitivity or accuracy of a diagnostic test that uses a urine sample, the method comprising: pasteurizing said urine sample; and adding one or more stabilizing agents to said urine sample, thereby enhancing the sensitivity or accuracy of said diagnostic test that uses said urine sample. In an exemplary embodiment, the stabilizing agent is capable of reducing the molecules that cause oxidative damage, pH change, or a combination thereof. In some embodiments, the methods of the invention described herein comprise an additional step of measuring against a control sample to confirm accuracy.

[0009] In another embodiment, the invention provides a method for reducing contaminants (e.g., microbial contaminants) in a urine sample for a medical diagnosis, the method comprising: pasteurizing said urine sample; and adding one or more stabilizing agents to said urine sample, thereby reducing said contaminants in said urine sample for said medical diagnosis.

[0010] In another embodiment, the invention provides a method for improving stability or shelf life of a urine sample for a medical diagnosis, the method comprising: pasteurizing said urine sample; and adding one or more stabilizing agents to said urine sample, thereby improving stability or shelf life of said urine sample for said medical diagnosis.

[0011] In another embodiment, the invention provides a diagnostic composition comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample.

[0012] In another embodiment, the invention provides a diagnostic kit comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample.

[0013] In another embodiment, the invention provides a urine sample for a medical diagnosis, said sample comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample.

[0014] In another embodiment, the invention provides a method for enhancing the sensitivity or accuracy of a diagnostic test that uses a urine sample, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides present in said urine sample, thereby enhancing the sensitivity or accuracy of said diagnostic test that uses said urine sample.

[0015] In another embodiment, the invention provides a method for improving stability or shelf life of a urine sample for a medical diagnosis, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides present in said urine sample, thereby improving stability or shelf life of said urine sample for said medical diagnosis.

[0016] In another embodiment, the invention provides a diagnostic composition comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides present in said urine sample, and wherein said agent capable of preventing or inhibiting oxidative damage to nucleosides is a catalase.

[0017] In another embodiment, the invention provides a diagnostic kit comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides present in said urine sample, and wherein said agent capable of preventing or inhibiting oxidative damage to nucleosides is a catalase.

[0018] In another embodiment, the invention provides a urine sample for a medical diagnosis, said sample comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides present in said urine sample, and wherein said agent capable of preventing or inhibiting oxidative damage to nucleosides is a catalase.

[0019] In another embodiment, the invention provides a method for enhancing the sensitivity or accuracy of a diagnostic test that uses a urine sample, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, thereby enhancing the sensitivity or accuracy of said diagnostic test that uses said urine sample. In an exemplary embodiment, said breakdown product is creatinine.

[0020] In another embodiment, the invention provides a method for improving stability or shelf life of a urine sample for a medical diagnosis, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, thereby improving stability or shelf life of said urine sample for said medical diagnosis.

[0021] In another embodiment, the invention provides a method for stabilizing creatinine for the accurate measurement of omega-3 or omega-6 fatty acid in urine samples, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is catalase.

[0022] In another embodiment, the invention provides a diagnostic composition comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, wherein said agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid is a catalase, and wherein said breakdown product is creatinine.

[0023] In another embodiment, the invention provides a diagnostic kit comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, wherein said agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid is a catalase, and wherein said breakdown product is creatinine.

[0024] In another embodiment, the invention provides a urine sample for a medical diagnosis, said sample comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, wherein said agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid is a catalase, and wherein said breakdown product is creatinine.

[0025] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows a peroxide standard curve: Ex 530 nm, Em 590 nm, and cut-off 570 nm.

[0027] Figure 2 shows the comparison of initial peroxide content versus synthetic urine. Emission intensity was normalized and scaled to untreated urine.

[0028] Figure 3 shows the comparison of peroxide content after 3 min exposure at T1C versus untreated urine. Emission intensity was normalized and scaled to untreated urine.

[0029] Figure 4 shows the comparison peroxide content after 0, 3 & 30 min exposure at T1C.

[0030] Figure 5 shows that the stabilizing agent effectively reduces hydrogen peroxide. DETAILED DESCRIPTION OF THE INVENTION

[0031] The invention provides compositions and methods for improving diagnosis. Specifically, the invention provides compositions and methods for enhancing the sensitivity or accuracy of a diagnostic test that uses a urine sample.

[0032] The inventor of the instant application surprisingly and unexpectedly found that urine by pasteurization followed by the addition of a stabilizing agent (e.g., a catalase) reduces peroxide levels by approximately 85%.

[0033] The methods of the invention may include the steps of pasteurizing a urine sample and adding one or more stabilizing agents to the urine sample. In some embodiments, the methods of the invention may include an additional step of measuring against a control sample (e.g., synthetic urine or sterile saline) to confirm accuracy.

[0034] Pasteurization is well known in the art. Any suitable pasteurization method, known to one of skilled in the art, can be used.

[0035] Additionally, the inventor of the instant application surprisingly and unexpectedly found that the addition of a stabilizing agent (e.g., a catalase) prevents or inhibits oxidative damage to nucleosides or nucleic acids in urine samples. The inventor also surprisingly and unexpectedly found that the addition of a stabilizing agent (e.g., a catalase) protects omega 3/6 breakdown products, including creatinine, in clinical urine samples.

[0036] Any suitable stabilizing agent can be used. In one embodiment, the stabilizing agent is capable of reducing the oxidative damage. In another embodiment, the stabilizing agent is capable of reducing the levels of hydrogen peroxide. In yet another embodiment stabilizing agent is capable of reducing pH change. In a further embodiment, the stabilizing agent is capable of reducing the molecules that cause the oxidative damage, pH change, or a combination thereof.

[0037] In one example, the stabilizing agent is a catalase. Other examples of stabilizing agents are fully described in U.S. Patent Application Publications US 20110196085, US 20110195871, US 20110195399, all of which are incorporated by reference herein in their entirety.

[0038] In addition to the stabilizing agent, any suitable carriers or additives, known to one of skilled in the art, can also be added to the sample. Examples of carriers or additives are fully described in U.S. Patent Application Publications US 20110196085, US 20110195871, US 20110195399, all of which are incorporated by reference herein in their entirety.

[0039] The invention can be used to diagnose or detect any biomarkers or metabolites. Also, the invention can be used to diagnose or detect any disease, disorder, or condition.

[0040] Any patent or patent application publication cited herein is incorporated by reference herein in its entirety.

[0041] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

Example 1: Reducing Contamination in Urine Test

[0042] Clinical testing of urine specimens is widely used for detection for multiple disease states. For example, urine tests are routinely used to detect diabetes, kidney disease and as a screen for drugs of addiction and drugs of abuse. The accuracy of these results can have a large impact on early disease detection and as an indication in substance over-use or abuse. In most cases urine is collected at one point and then transported by mail or courier for subsequent laboratory testing. The problem comes from the delay, storage and transport of urine samples. We have found that even under the best collection, storage, processing steps for urine samples being prepared for clinical controls, the samples are showing a significant amount of spoilage or rancidity. Specifically, pH changes, oxidation changes due to the generation of hydrogen peroxide- a general measurement of rancidity are found across samples taken from the industry. [0043] In the present invention, we have developed a rapid method for the identification of contaminants, and a broadly applicable general purpose method to decrease the interfering contaminants. The new process allows the subsequent urine test to be completed accurately.

[0044] A broad spectrum, rapid inexpensive procedure/additive to increase the accuracy of urine tests can include the following steps:

1. a pasteurization process that reduces microorganisms.

2. a group of additives (that reduce molecules that cause oxidative damage and

subsequent pH change to the molecules of interest)

3. a measurement against a control sample (synthetic urine or sterile saline) to confirm accuracy.

[0045] We have identified a broad spectrum approach to significantly reduce interfering contamination in urine test which increase accuracy over 10 fold (in accelerated aging studies).

[0046] We measured multiple lots of urine to test for peroxide and oxide levels that could potentially impact shelf life of urine control products.

[0047] We identified the presence of peroxides and oxide in all urine samples (likely from microorganism contamination).

[0048] We invented customized procedure & product to treat urine before the calibration controls are added: They include:

> Urine Pasteurization Procedure

> PSP-UE1 (proprietary CDI product to reduce H2O2 )

[0049] Creatinine measurement (a measurement of kidney function) is significantly reduced by hydrogen peroxide or a change in pH. Hydrogen peroxide at t=0, (2 to 6 μΜ) which quickly disappeared. After storage for 15 days at 47°C, Hydrogen Peroxide levels declined to nM levels (20 to 70 nM).

[0050] Hydrogen Peroxide generation and stability is dependent upon a variety of environmental factors, i.e. oxidative generation processes, radical sources, pH, thermal, and inactivating reagents (peroxide & radical scavengers). In the presence of peroxide generators, elevated temperatures may cause increase in peroxide levels. Upon exhaustion of peroxide sources, thermal decomposition of hydrogen peroxide may occur. As a result hydrogen peroxide levels may very well follow a bell shaped curve as a function of time and temperature, i.e. increasing initially followed by an eventual decline. Hydrogen peroxide generation is often associated with radical formation which can in turn initiate a cascade of oxidative and decomposition processes. Reducing peroxide level is important for stability for all urine products (due to general oxidation processes).

[0051] Based on the results disclosed herein, we have fully demonstrated that our invention reduces peroxide levels and enhance stability for urine samples.

[0052] Table 1 below describes a urine treatment plan and Table 2 below describes peroxide concentration and pH of treated (TjC, 3min) and untreated urine.

Figure imgf000010_0001
Figure imgf000011_0001

[0053] Initial Peroxide levels for four lots of urine preparations range from 1.5 to 2.6 μΜ (See Table 2 & Figure 1). Upon short term exposure to elevated temperature, for 3 minutes, peroxide levels increase approximately 16%. Urine H2O2 levels increase with heat

[0054] Initial peroxide levels in urine are appreciable & approximately 25 fold larger than for synthetic urine (Fig 2). For all 4 lots of urine, Peroxide levels increase approximately 16%, upon exposure to T1C for 3 minutes (Fig 3).

[0055] Figure 4 shows that pasteurization alone is not sufficient. H2O2 increases with heat.

[0056] As shown in Figure 5, peroxide content increases significantly upon longer term exposure and thermal pasteurization alone is not sufficient to reduce peroxide.

[0057] As shown in Figure 5, two CDI-PSP reagents were compared (PSP-UE1 VS.PSP-UE2) for their effectiveness. Peroxide levels were compared to pasteurization alone. PSP-UEI is catalase at 20 units/ml (Sigma Aldrich). The range is 0.2 units per ml to 200 units/ml. PSP- UE2 is dimethyl sulfoxide (DMSO - Sigma Aldrich - 2%). The range is 0.2% to 20% with 2% being preferred working concentration (DMSO).

[0058] Heat treatment or pasteurization alone is not effective. Peroxide levels increase climb to approximately 165% of the initial control values. [0059] Treatment of urine by pasteurization followed by the addition of PSP-UE1 reduces peroxide levels by approximately 85% and is an effective method for preparing urine lots at Bio Rad Laboratories for subsequent control additions. The optimum temperature was 75°C for 30 minutes. The range is from lmin to 1 hour (optimum time is 30 minutes). Example 2: Preventing Oxidative Damage of Nucleosides in Urine Samples

[0060] A key advantage of measuring clinical disease in urine samples is that the samples are non-invasive and considered non-hazardous materials when measured at point of care or shipped into a central lab for testing. The problem is that urine stored or shipped can develop peroxides due to micro-contamination. These peroxides can generate multiple oxidative species that can damage the proteins and nucleobases being measured in the clinical urine sample. In the present invention we disclose the use of urine stabilizer PSP-UEI for the reduction of peroxide generated species in urine samples. Specifically, the oxidative damage to guanine base is one major component to oxidative damage.

[0061] Oxidative damage of nucleosides can be a major source of damage to DNA/RNA, tRNA, miRNA in vitro. Multiple companies have collected human urine samples in order to measure disease states, and found that there are high levels of peroxide activity present in collected human urine samples.

[0062] It is secondary clinical advantage for the use PSP-UEI in collected urine sample. Distinction between the bacteria from the patient can be excluded from the contamination of the urine sample. Using the PSP-UEI reagents and process the sample is heated for 3-30 minutes either using heat or chemicals to block micro-contamination growth. This allows for the characterization of bacterial proteins and nucleobases from the patient, not from a contaminated urine sample. The heating step could be between 30°C to 100°C with 70°C being the preferred method for 3 minutes. A chemical method could be used in the sample to generate heat instead of using external heat.

Example 3: Protection of Omega 3/6 Fatty Acid Breakdown Products in Urine for

Clinical Testing

[0063] The inventor of the instant application surprisingly and unexpectedly found the methods to protect omega 3/6 and their breakdown products, including creatinine, in clinical urine samples. Additionally, the inventor surprisingly and unexpectedly found that the biomarker beta albumin can be used as the calibrator molecule between blood and urine samples.

[0064] Currently, the standard test for omega 3/6 ratios are measured in blood samples. This method requires an invasive blood draw, clinical sample refrigeration and follow up laboratory testing. It is commercially important if a patient after dietary intake of omega 3/6 could then take a urine sample at home and then mail the sample to clinical testing laboratory for accurate measurement of Omega 3/6 breakdown products. The breakdown pathway of omega 3 is well characterized and has been detected in urine (/ Lipid Res. 2006 Nov.; 47(11):2515-24. Epub 2006 Aug 30. Oxidized derivatives of omega-3 fatty acids: identification of IPF3 alpha- VI in human urine).

[0065] The breakdown products of omega 6 have been identified in urine. The problem is that the concentration of creatinine in urine samples is not stable, specifically during the transport of samples as non refrigerated samples. In the current invention, we describe a significant advance in the treatment of urine samples to specifically stabilize creatinine for the accurate measurement of omega 3/6 in urine samples. Additionally, we disclose the beta albumin can be used at the molecule found in blood and urine to calibrate the accurate levels between blood and urine samples.

I. Autoxidation

[0066] Autoxidation is a common set of processes that occur spontaneously upon exposure of a substance or mixture to atmospheric oxygen and are accelerated at higher temperatures. The processes often occur via a free radical chain reaction which can be described in three general steps, initiation, propagation and termination. For our purposes autoxidation is a problem when it affects the accuracy of a diagnostic test through biological or biochemical damage of target markers (proteins, ribonucleic acids and small organic molecules). II. Antioxidants

[0067] An antioxidant in a general sense is a substance which inhibits reactions promoted by oxygen, including reactive oxygen species and nitric oxide synthase. Depending upon the exact nature of the reaction and the scope of protection targets (including relevant industry practices), the protection agents differ substantially as well as the methods used for assessment. [0068] Disruption or retardation of autoxidation is a common target of antioxidants.

[0069] Disruption or inhibition of any of the three steps can provide for oxidative protection. The initiation step could be inhibited by a number of ways including 1) limited oxygen exposure (oxygen elimination via nitrogen, helium or argon saturation & degassing), 2) creating a reducing environment, and/or 3) inhibiting oxidative enzyme activity. Interruption of the propagation step is a very common target of antioxidants. Both general and specific radical scavengers are very effective antioxidants. Effective radical scavengers and antioxidants include a variety of dietary, natural and synthetic products. Biologically active antioxidants include both enzymatic and non-enzymatic substances. A key function of sacrificial free radical scavengers is to prevent primary ligand oxidation. This requires that the antioxidant reacts quickly with primary oxygen radicals at low concentration (k6 »k3) and the ensuing antioxidant radical is stable relative to the ligand and reacts slowly with the substrate (K « k3).

III. Antioxidant Capacity Methods.

[0070] The means and methods of assessing antioxidant capacity are as varied as the targets. As in the case of rancidity a single unifying assay has not been adopted. A single method was not suitable since the nature of the prevention targets, active ROS agent and the environmental context varies greatly between systems. On the basis of the chemical reactions involved, the major antioxidant capacity assays can be roughly divided into two categories: 1) hydrogen atom transfer (HAT) reaction based assays and (2) single electron transfer (ET) reaction based assays. ET assays are based on the electron transfer reaction: probe (oxidant) + e (form antioxidant) → reduced probe + oxidized antioxidant. The change in ABS or Fl intensity is plotted as a function of antioxidant concentration. This is calibrated versus Trolox or gallic acid and expressed as TE or GAE equivalents, respectively. HAT based assays generally are composed of a synthetic free radical generator, an oxidizable probe, and an antioxidant. HAT & ET based assays measure radical or oxidant scavenging capacity relative to the standard of synthetic urine.

IV. Urine Samples:

[0071] Inventors of the instant application have analyzed urine samples in conjunction with controls. In all clinical urine samples tested (24 hours at room temperature or longer) the presence of hydrogen peroxide was detected. The initial levels of hydrogen peroxide at in clinical urine are very low. However, the levels in clinical samples 24-72 hours after collection ranged from 2μΜ to 6μΜ which quickly decreased to nanomolar levels.

[0072] Hydrogen Peroxide generation and stability is dependent upon a variety of environmental factors, i.e. oxidative generation processes, radical sources, pH, thermal, and inactivating reagents (peroxide & radical scavengers). In the presence of peroxide generators, elevated temperatures may be expected to increase peroxide levels. Upon exhaustion of peroxide sources, thermal decomposition of hydrogen peroxide takes place. As a result hydrogen peroxide levels may very well follow a bell shaped curve as a function of time and temperature, i.e. increasing initially followed by an eventual decline. Hydrogen peroxide generation is often associated with radical formation which can in turn initiate a cascade of oxidative and decomposition processes. Reducing peroxide level is important for stability for all urine products (due to general oxidation processes). This causes significant oxidation to some of the organic molecules that are the target for illegal drugs of abuse testing. The oxidation of targeted molecules can significantly increase the individual sample processing before LC/MS/MS measurements can be made. This market is looking for a broad spectrum urine "method" that allows laboratories to make fewer sample processing steps before LC/MS/MS testing. Ideally, they would like to minimize the processing steps for each sample and go straight to procedures that allow them to dilute urine and shoot them into the detection instruments.

[0073] Preservation of target molecules is not just limited to target molecules for illegal drugs testing market. This is equally important for the rapid processing of samples for prescription pain drug abuse testing markets, and also for the broader urine sample diagnostic markets for diagnostic markets linked to disease and cancer detection. For example, Trovagene requires the tRNA samples to be stabilized in collected clinical urine samples for subsequent testing in their CLEA certified laboratory.

V. Procedure for Clinical Urine Samples.

[0074] Inventors developed a two- step procedure to treat urine before shipping/testing. Add μg/ml quantities of PSP-UE1 to urine samples followed by pasteurization for 30 minutes at 70°C. Heat treatment or pasteurization alone is not effective. Peroxide levels increase to approximately 165% of the initial control values. Treatment of urine by pasteurization followed by the addition of PSP-UEi reduces peroxide levels by approximately 85% and is an effective method for preparing clinical urine samples for all subsequent diagnostic measurements.

[0075] PSP-UE1 can protect omega 3/6 and their breakdown products, including creatinine, in clinical urine samples.

[0076] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A method for enhancing the sensitivity or accuracy of a diagnostic test that uses a urine sample, the method comprising: pasteurizing said urine sample; and adding one or more stabilizing agents to said urine sample, thereby enhancing the sensitivity or accuracy of said diagnostic test that uses said urine sample.
2. The method of claim 1 , wherein the method further comprising the step of measuring against a control sample.
3. The method of claim 1, wherein said is stabilizing agent is capable of reducing the oxidative damage.
4. The method of claim 1, wherein said is stabilizing agent is capable of reducing the levels of hydrogen peroxide.
5. The method of claim 1, wherein said is stabilizing agent is capable of reducing pH change.
6. The method of claim 1, wherein said is stabilizing agent is capable of reducing the molecules that cause the oxidative damage, pH change, or a combination thereof.
7. A method for reducing contaminants in a urine sample for a medical diagnosis, the method comprising: pasteurizing said urine sample; and adding one or more stabilizing agents to said urine sample, thereby reducing said contaminants in said urine sample for said medical diagnosis.
8. The method of claim 7, wherein said is stabilizing agent is capable of reducing the oxidative damage.
9. The method of claim 7, wherein said is stabilizing agent is capable of reducing the levels of hydrogen peroxide.
10. The method of claim 7, wherein said is stabilizing agent is capable of reducing pH change.
11. The method of claim 7, wherein said is stabilizing agent is capable of reducing the molecules that cause the oxidative damage, pH change, or a combination thereof.
12. A method for improving stability or shelf life of a urine sample for a medical diagnosis, the method comprising: pasteurizing said urine sample; and adding one or more stabilizing agents to said urine sample, thereby improving stability or shelf life of said urine sample for said medical diagnosis.
13. A diagnostic composition comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample.
14. The composition of claim 13, wherein said is stabilizing agent is capable of reducing the oxidative damage.
15. The composition of claim 13, wherein said is stabilizing agent is capable of reducing the levels of hydrogen peroxide.
16. The composition of claim 13, wherein said is stabilizing agent is capable of reducing pH change.
17. The composition of claim 13, wherein said is stabilizing agent is capable of reducing the molecules that cause the oxidative damage, pH change, or a combination thereof.
18. A diagnostic kit comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample.
19. A urine sample for a medical diagnosis, said sample comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample.
20. A method for enhancing the sensitivity or accuracy of a diagnostic test that uses a urine sample, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides or nucleic acids present in said urine sample, thereby enhancing the sensitivity or accuracy of said diagnostic test that uses said urine sample.
21. The method of claim 20, wherein said agent capable of preventing or inhibiting oxidative damage to nucleosides or nucleic acids is a catalase.
22. A method for improving stability or shelf life of a urine sample for a medical diagnosis, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides or nucleic acids present in said urine sample, thereby improving stability or shelf life of said urine sample for said medical diagnosis.
23. The method of claim 22, wherein said agent capable of preventing or inhibiting oxidative damage to nucleosides or nucleic acids is a catalase.
24. A diagnostic composition comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides or nucleic acids present in said urine sample, and wherein said agent capable of preventing or inhibiting oxidative damage is a catalase.
25. A diagnostic kit comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides or nucleic acids present in said urine sample, and wherein said agent capable of preventing or inhibiting oxidative damage is a catalase.
26. A urine sample for a medical diagnosis, said sample comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of preventing or inhibiting oxidative damage to nucleosides or nucleic acids present in said urine sample, and wherein said agent capable of preventing or inhibiting oxidative damage is a catalase.
27. A method for enhancing the sensitivity or accuracy of a diagnostic test that uses a urine sample, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, thereby enhancing the sensitivity or accuracy of said diagnostic test that uses said urine sample.
28. The method of claim 27, wherein said agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid is a catalase.
29. The method of claim 27, wherein said breakdown product is creatinine.
30. A method for improving stability or shelf life of a urine sample for a medical diagnosis, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, thereby improving stability or shelf life of said urine sample for said medical diagnosis.
31. The method of claim 30, wherein said agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid is a catalase.
32. The method of claim 30, wherein said breakdown product is creatinine.
33. A method for stabilizing creatinine for the accurate measurement of omega-3 or omega-6 fatty acid in urine samples, the method comprising: adding one or more stabilizing agents to said urine sample, wherein at least one of said one or more stabilizing agents is catalase.
34. A diagnostic composition comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, wherein said agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid is a catalase, and wherein said breakdown product is creatinine.
35. A diagnostic kit comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, wherein said agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid is a catalase, and wherein said breakdown product is creatinine.
36. A urine sample for a medical diagnosis, said sample comprising: an effective amount of one or more stabilizing agents that enhance the stability or shelf of a urine sample, wherein at least one of said one or more stabilizing agents is an agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid in said urine sample, wherein said agent capable of stabilizing a breakdown product of omega-3 or omega-6 fatty acid is a catalase, and wherein said breakdown product is creatinine.
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