Integrated device for collection, processing, stabilization and storage of samples and methods of use thereof

Abstract

A device for collection, filtration and stable storage of cell-free DNA, extracellular RNA and protein analytes from a saliva sample, and methods of use thereof, as well as methods of use of guanidine thiocyanate as a stabilization agent for stabilizing nucleic acid species. A saliva testing kit is also described.

Description

INTEGRATED DEVICE FOR COLLECTION, PROCESSING, STABILIZATION AND STORAGE OF SAMPLES AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 552,965 filed on February 13, 2024, incorporated herein by reference in its entirety.REFERENCE TO A "SEQUENCE LISTING," SUBMITTED AS AN XML FILE

[0002] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name: “206030-0298- 00WO Sequence Listing.xml”; created on February 12, 2025, and 3,946 bytes in size. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Saliva is the most accessible biofluid in the field of liquid biopsy. In today ’ s date, due to time limitations, manpower consuming procedures and proper storage facilities, most of the clinicians refrain from going through the hassle of collecting a saliva sample. Moreover, the stability of the various nucleic analytes in saliva from the phase of collection till point of analysis other than in central facilities has been a concerning issue. Therefore, saliva, a readily accessible and non-invasively acquired biofluid is still not sought after as it lacks rigorous and standardized collection protocols, or preservatives that stabilize the salivary analytes at ambient temperature.

[0004] Thus, there is a need in the art for a novel collector device for saliva that provides proper stabilization to the salivary analytes. The present invention meets this need.SUMMARY OF THE INVENTION

[0005] In one embodiment, the invention relates to a collector device, comprising: a syringe fluidly connected to a splitter, the splitter fluidly connected to one or more receptacles, the assembly of which defines a proximal end and distal end for the collector device; wherein the syringe comprises a tube forming a lumen with an absorbent pad disposed within the lumen of the tube, and a plunger with a handle at least partially disposed with the lumen of the tube; thesplitter comprises a housing forming an interior volume and having a proximal inlet and one or more distal outlets in fluid connection with the interior volume; and the one or more receptacles each comprise a proximal opening and one or more walls defining an interior volume therein.

[0006] In one embodiment, each receptacle of one or more receptacles comprises a stabilizing compound.

[0007] In one embodiment, the one or more distal outlets comprise at least a first, second and third outlet, and the one or more receptacles comprise at least a first, second and third receptacle, each receptacle correlated with a respective outlet.

[0008] In one embodiment, the first receptacle comprises a stabilizing compound for stabilization of cell-free DNA (cfDNA), the second receptacle comprises a stabilizing compound for stabilization of extracellular RNA (exRNA), and the third container comprises a stabilizing compound for stabilization of protein.

[0009] In one embodiment, the stabilizing compound for stabilizing of cfDNA and the compound for stabilizing of extracellular RNA comprises guanidine thiocyanate (GTC) or guanidinium isothiocyanate (GITC), and further wherein the stabilizing compound for stabilizing of protein comprises ethanol.

[0010] In one embodiment, the device further comprises a filter disposed within the interior volume of the splitter housing.

[0011] In one embodiment, each outlet of the one or more distal outlets comprises a circular flange extending out in a distal direction from the housing, wherein each receptacle is configured to sealingly and releasably attach to the flange of the opening with a compression fit.

[0012] In one embodiment, the plunger comprises a sample volume adequacy indicator positioned at the distal end of the plunger fluidly connected to the lumen of the syringe tube.

[0013] In one embodiment, the plunger comprises a distal compression seal surrounding at least a portion of the plunger.

[0014] In one embodiment, the seal comprises one or more gaskets or O-rings.

[0015] In one embodiment, the plunger comprises a proximal thumb pad having a rounded and textured portion at the proximal end of the plunger.

[0016] In one embodiment, the plunger comprises one or more ergonomic features in the handle of the plunger.

[0017] In some embodiments, one or more ergonomic features comprise any of ridges, knurling, indents, detents, recesses, cutouts, narrowed-regions, finger-recesses, curved portions, or combinations thereof.

[0018] In some embodiments, the tube comprises a transparent plastic compression tube.

[0019] In some embodiments, the housing comprises a removable portion defining at least a portion of housing and the third outlet, configured to be removably and sealingly attached to the housing with a seal. In some embodiments, the seal comprises any of flanges, recesses, O- rings, gaskets, or combinations thereof.

[0020] In some embodiments, the housing has a length ranging between 1 cm and 10 cm, a width ranging between 1 cm and 10 cm, and a height ranging between 1 cm and 10 cm.

[0021] In some embodiments, each flange has a height ranging between 1 mm and 2 cm, and a diameter ranging between 1 mm and 2 cm.

[0022] In some embodiments, interior volume of the housing has a volume ranging between 10 mm3and 1000 mm3.

[0023] In one embodiment, the invention relates to a method of using a collector device, comprising the steps of: providing a collector device, comprising: a syringe fluidly connected to a splitter, the splitter fluidly connected to one or more receptacles, the assembly of which defines a proximal end and distal end for the collector device; wherein the syringe comprises a tube forming a lumen with an absorbent pad disposed within the lumen of the tube, and a plunger with a handle at least partially disposed with the lumen of the tube; the splitter comprises a housing forming an interior volume and having a proximal inlet and one or more distal outlets in fluid connection with the interior volume; and the one or more receptacles each comprise a proximal opening and one or more walls defining an interior volume therein; loading biological fluid from a subject captured on the absorbent pad into the lumen of the syringe tube; depressing the plunger of the syringe to push the liquid from the tube of the syringe into the interior volume of the splitter housing; and collecting one or more analytes in the one or more receptacles.

[0024] In one embodiment, the sample is a saliva sample.

[0025] In one embodiment, the invention relates to a method of detecting a biomarker in a biological fluid sample of a subject, the method comprising, providing a collector device, comprising: a syringe fluidly connected to a splitter, the splitter fluidly connected to one or more receptacles, the assembly of which defines a proximal end and distal end for the collector device; wherein the syringe comprises a tube forming a lumen with an absorbent pad disposed within the lumen of the tube, and a plunger with a handle at least partially disposed with the lumen of the tube; the splitter comprises a housing forming an interior volume and having a proximal inlet and one or more distal outlets in fluid connection with the interior volume; and the one or more receptacles each comprise a proximal opening and one or more walls defining an interior volume therein; loading biological fluid from a subject captured on the absorbent pad into the lumen of the syringe tube; filling the lumen of the syringe tube with one or more liquids; depressing the plunger of the syringe to push the liquid from the tube of the syringe into the interior volume of the splitter housing; collecting one or more analytes in the one or more receptacles, and analyzing the one or more analytes to detect a biomarker of interest in the sample.

[0026] In one embodiment, the sample is a saliva sample.

[0027] In one embodiment, the method comprises collecting cell-free DNA in the first receptacle, extracellular RNA in the second receptacle and protein in the third receptacle.

[0028] In one embodiment, the method further comprises the step of applying a lysis buffer to the lumen of the device following the collecting step, and collecting the lysed cellular products from the compression tube containing genomic DNA (gDNA) and intracellular RNA for analysis in a separate tube.

[0029] In one embodiment, the method comprises analyzing cfDNA, exRNA or proteins or any combination thereof.

[0030] In one embodiment, the method comprises analyzing cfDNA, exRNA, proteins, gDNA or intracellular RNA, or any combination thereof.

[0031] In one embodiment, the invention relates to a method of diagnosing a disease or disorder in a subject, the method comprising, providing a collector device, comprising: a syringe fluidly connected to a splitter,the splitter fluidly connected to one or more receptacles, the assembly of which defines a proximal end and distal end for the collector device; wherein the syringe comprises a tube forming a lumen with an absorbent pad disposed within the lumen of the tube, and a plunger with a handle at least partially disposed with the lumen of the tube; the splitter comprises a housing forming an interior volume and having a proximal inlet and one or more distal outlets in fluid connection with the interior volume; and the one or more receptacles each comprise a proximal opening and one or more walls defining an interior volume therein; loading biological fluid from a subject captured on the absorbent pad into the lumen of the syringe tube; filling the lumen of the syringe tube with one or more liquids; depressing the plunger of the syringe to push the liquid from the tube of the syringe into the interior volume of the splitter housing; collecting one or more analytes in the one or more receptacles, analyzing the one or more analytes to detect a biomarker of interest in the sample, wherein the biomarker is a disease-associated biomarker, and diagnosing the subject as having or at risk of developing the disease associated with the biomarker upon detection of the presence or level of the biomarker as compared to a comparator control.

[0032] In one embodiment, the sample is a saliva sample.

[0033] In one embodiment, the method comprises collecting cell-free DNA in the first receptacle, extracellular RNA in the second receptacle and protein in the third receptacle.

[0034] In one embodiment, the method further comprises the step of applying a lysis buffer to the compression tube of the device following the collecting step, and collecting the lysed cellular products from the compression tube containing genomic DNA (gDNA) and intracellular RNA for analysis in a separate tube.

[0035] In one embodiment, the method comprises analyzing cfDNA, exRNA or proteins or any combination thereof.

[0036] In one embodiment, the method comprises analyzing cfDNA, exRNA, proteins, gDNA or intracellular RNA, or any combination thereof.

[0037] In one embodiment, the invention relates to a composition for stabilization of nucleic acid molecules, the composition comprising guanidine thiocyanate or guanidinium thiocyanate.

[0038] In one embodiment, the invention relates to a method of stabilizing nucleic acid molecules, the method comprising contacting a sample comprising one or more nucleic acid molecule with guanidine thiocyanate. In one embodiment, the one or more nucleic acid molecule comprises cfDNA or exRNA.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0040] Fig. 1 A depicts an exemplary collector device, in some examples referred to as an integrated Saliva Collection, Processing, Stabilization, and Storage (iSCPSS) device, according to aspects of the present invention.

[0041] Fig. IB depicts an exemplary collector device comprising a splitter with filter and collection tubes with stabilizing compounds according to aspects of the present invention.

[0042] Fig. 2 depicts an exemplary collector device with one or more receptacles according to aspects of the present invention.

[0043] Fig. 3 depicts an illustrative computer architecture for an exemplary computer for practicing the various embodiments of the invention.

[0044] Fig. 4A is a diagram depicting an exemplary working principle or method for use of the device for collecting cell-free DNA in a first receptacle, extracellular RNA in a second receptacle and protein in a third receptacle.

[0045] Fig. 4B is a diagram depicting an exemplary method of using the device comprising the step of applying a lysis buffer to the lumen of the device following the collecting step as depicted in Fig. 4A.

[0046] Fig. 5A is a flow diagram depicting an exemplary method collecting one or more analytes from a biological fluid sample from a subject.

[0047] Fig. 5B is a flow diagram depicting an exemplary method of detecting a biomarker in a biological fluid sample of a subject.

[0048] Fig. 5C is a flow diagram depicting an exemplary method of diagnosing a disease or disorder from detection of a biomarker in a biological fluid sample of a subject.

[0049] Fig. 6A depicts data demonstrating the quantification of the DNA concentration and purity using a Nanodrop (spectrophotometer).

[0050] Fig. 6B depicts data demonstrating the quantification of the DNA concentration and purity using a Qubit (Fluorometer).

[0051] Fig. 6C depicts data demonstrating the cfDNA stability in saliva as evaluated using target specific sequence primers from a gene rich enriched area using Droplet Digital PCR (ddPCR).

[0052] Fig. 7A depicts data demonstrating the results of the DNA concentration measured by NanoDrop over 0 & 1 day at ambient temperature with and without GIT stabilizer.

[0053] Fig. 7B depicts data demonstrating the results of the DNA concentration measured by Qubit over 0 & 1 day at ambient temperature without and without GIT stabilizer.

[0054] Fig. 7C depicts data demonstrating the results of a comparison of absolute SCFT / Chr 6 target copy numbers determined by ddPCR Evagreen mix for saliva sample filtered using the standard operating protocol for saliva (SOP) versus the iSCPSS device with GIT over 0 & 1 day.

[0055] Fig. 8A depicts data demonstrating the results of tape station analysis of RNA concentration collected through the disclosed device with GIT stabilizer (1: 1 ratio) over 0, 1, 3, 7 days at ambient temperature.

[0056] Fig. 8B depicts data demonstrating the results of tape station analysis of the percentage of saliva sample collected through the disclosed device with GIT stabilizer (1 : 1 ratio) over 0, 1, 3, 7 days at ambient temperature.

[0057] Fig. 9 depicts data demonstrating the results for a total protein estimation using Bradford Assay over 0, 1, 3, 7 days collected in the proposed integrated device with 20% w / v ethanol stabilizer and stored at ambient temperature.

[0058] Fig. 10A depicts data demonstrating the genomic DNA data integrity (DIN) values over 0 and 1 day using SDS lysis buffer.

[0059] Fig. 1 OB depicts data demonstrating the genomic DNA data integrity (DIN) values over 0 / 1 / 3 / 7 days using a GIT lysis buffer (e.g., GITC).

[0060] Fig. IOC depicts data demonstrating the genomic DNA data integrity (DIN) values over 0 / 1 / 3 / 7 days using a GIT lysis buffer (e.g., GITC) using the integrated device.DETAILED DESCRIPTION

[0061] It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity many other elements found in related systems and methods. Those of ordinary skill in the art may recognize that other elements and / or steps are desirable and / or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art.Definitions

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0063] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0064] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0065] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate.

[0066] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal amenable to the systems, devices, and methods described herein. The patient, subject or individual may be a mammal, and in some instances, a human.

[0067] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Collector Device

[0068] Aspects of the present invention relate to a collector device, in some examples referred to as an integrated Saliva Collection, Processing, Stabilization, and Storage (iSCPSS) device. The disclosed device is an easy to use and cost-effective tool for the split sample collection of rich sources of nucleic acids (e.g., RNA, miRNA, mRNA) and proteins found in saliva. The disclosed device provides a plurality of equivalent samples of saliva (e.g., 2, 3, 4 equivalent samples of saliva) in a short period of time (1 min, 2 min, 3 min) which may then be analyzed for various components (e.g., RNA or protein components). The key importance of the iSCPSS device is an ambient temperature compatible standardized saliva collection technology that allows the concurrent harnessing of saliva RNA, protein, gDNA and cfDNA that is of superb quality that can be preserved (-80C) for downstream molecular analysis. Applications of device 100 in the life sciences and research fields include, but are not limited to, isolation of exosomes, liquid biopsy, and the use of cell free DNA or cell free RNA.

[0069] Referring now to Fig. 1 A and Fig. IB, shown is an exemplary universal collector device 100 according to aspects of the present invention. In some embodiments, device 100 comprises at least one syringe 110 fluidly connected to at least one splitter 140, the splitter 140 fluidly connected to one or more receptacles 170, the assembly of which defines a proximal end 102 and distal end 104 for device 100. In some embodiments, syringe 110 comprises a tube 112 forming a lumen with a plunger 114 at least partially disposed within the lumen of the tube, the syringe 110 configured to fluidly dispense one or more fluids out of an outlet 116. In some embodiments, tube 112 further comprises at least one absorbent pad 118 positioned inside the lumen of the tube 112. In some embodiments, tube 112 is formed from a transparent plastic compression tube. In some embodiments, plunger 114 comprises a sample volume adequacy indicator 120 positioned at the distal end of the plunger configured to fluidly connect to the one or more fluids contained within tube 112. The sample volume adequacy indicator 120 provides a visual indication that an adequate quantity of sample has been collected for downstream analysis. In some embodiments, plunger 114 comprises a distal compression seal 122 and / or a proximal thumb pad 124, and / or one or more ergonomic features 126. In some embodiments, compression seal 122 comprises one or more gaskets or O-rings. In some embodiments, proximal thumb pad 124 comprises a rounded portion on the proximal end of plunger 114. In some embodiments, the one or more ergonomic features 126 comprise any of ridges, knurling, indents, detents, recesses, cutouts, narrowed-regions, finger-recesses, curved portions, or combinations thereof in or on plunger 114. Syringe 110 may fixedly and removably connect or attach to splitter 140 by any attachment means known in the art including but not limited to compression fits, threads, Luer lock, camlocks, or the like.

[0070] In some embodiments, splitter 140 comprises a housing 142 defining an interior volume 144, wherein the housing comprises at least one proximal inlet 146 and one or more distal outlets, each inlet and outlet fluidly connected to interior volume 144. In some embodiments, each of the one or more distal outlets comprise a circular flange 150 extending out in a distal direction from housing 142. In some embodiments, the one or more distal outlets comprise a first outlet 148a, a second outlet 148b, and a third outlet 148c. It should be appreciated herein that splitter 140 may be formed or manufactured as one or more housing 142 comprising one or more components or units, and be modular in nature allowing a user to configure the splitter 140 to have any number of outlets and receptacles. For example, in someembodiments, splitter 140 is formed with one housing 142 that comprises any number of outlets (e g., 2, 3, 4, 5, 6 outlets) and one proximal inlet 146. In a preferred embodiment, splitter 140 comprises a single housing 142 with 3 outlets. In some embodiments, splitter 140 may be formed by one or more removable portions that connect or attach together to create a splitter 140 having any number of outlets. Splitter 140 may include any number of individual components or units that may fixedly and removably connect or attach together to form a splitter 140 having any desired number of outlets. The individual units or components may each have lateral sides forming openings configured to attach to another unit or component of the splitter 140 and form a fluid connection. In some embodiments, each unit or component may comprise only a single lateral opening or more than one lateral opening. In some embodiments, each unit or component of housing 142 may comprise an inlet that may be sealed or capped as required to form an interior volume with only one inlet and multiple outlets. The caps or seals may be used on one or more of the inlets, or the lateral openings to sealingly close an inlet or one of the lateral sides of the individual component or unit once the desired numbers of outlets is achieved.. In one example, splitter 140 is formed of a housing 142 comprising a first outlet 148a, a second outlet 148b, and any number of outlets (e g., a third outlet 148c) may be attached to increase the number of outlets. In some examples, the individual components or units may be referred to herein as removable portions. The removable portions may be added on either lateral side of the portion comprising the inlet for connecting to the syringe. In some embodiments, housing 142 comprises a removable portion 152 defining at least a portion of housing 142 and comprises a third outlet 148c, wherein the removable portion 152 is configured to be removably and sealingly attached to housing 142 with a seal 154. In some embodiments, seal 154 comprises any of flanges, recesses, O-rings, gaskets, or combinations thereof. In some embodiments, a modular housing 142 comprises one central portion with lateral openings, one outlet, and one proximal inlet 146, and comprises lateral removable portions comprising lateral openings each with an outlet, and without proximal inlets 146. In some embodiments, splitter 140 may be referred to as a splitting unit with one or more filters or variable filters. In some embodiments, splitter 140 may be referred to as a universal filtration and splitting unit. In some embodiments, splitter 140 may comprise one or more filters positioned inside interior volume 144.

[0071] In some embodiments, splitter 140 comprises one or more filters positioned before each of the one or mor outlets inside of housing 142. In some embodiments, the one ormore filters comprises one filter positioned at the neck of proximal inlet 146. The one or more filters positioned in splitter 140 are configured to remove cells and unwanted components from the saliva. The clean sample is then promptly delivered to the one or more receptacles 170 for immediate stabilization and use or for long term storage.

[0072] Aspects of the present invention relate to the structure and configuration of interior volume 144 for splitter 140. In some embodiments, the interior volume 144 comprises one open chamber that allows even (3-way) distribution of filtered saliva to emerge into the one or more receptacles 170. Flow from syringe 110 is directed into inlet 146 and proceeds into and through interior volume 144. In some embodiments, interior volume 144 comprises one or more manifolds or channels configured to direct and distribute flow evenly through the interior volume 144, through the one or more filters in splitter 140, and out the one or more outlets. In some embodiments, the volume of interior volume 144 is minimized to reduce dead-space or areas where turbulent flow may form, and comprises substantially curved or round surfaces configured to direct and form a laminar flow of fluid through the splitter 140. In some embodiments, interior volume 144 has a volume ranging between about 50 pL and about 250 pL, or about 80 pL and 140 pL. In some embodiments, interior volume has a volume of about 50 pL, 60 pL, 70 pL, 80 pL, 90 pL, 100 pL, 110 pL, 120 pL, 130 pL, 140 pL, 150 pL, 160 pL, 170 pL, 180 pL, 190 pL, 200 pL, 210 pL, 220 pL, 230 pL, 240 pL, or about 250 pL.

[0073] Now referring in detail to Fig. IB, in some embodiments, device 100 comprises one or more receptacles 170, each receptacle comprising a proximal opening 172 and defining an interior volume 174. In some embodiments, one or more receptacles 170 comprise at least a first receptacle 170a, a second receptacle 170b, and a third receptacle 170c. In some embodiments, one or more receptacles 170 comprises a fourth receptacle 170d.

[0074] In some embodiments, each receptacle of one or more receptacles 170 comprises at least one of a stabilizing compound, a compound for stabilization of cfDNA, guanidine thiocyanate (GIT), guanidinium isothiocyanate (GITC), a compound for stabilization of extracellular RNA, ethanol, and / or 20% ethanol w / v for protein stabilization. In some embodiments, device 100 comprises a splitter 140 removably attached to one or more receptacles 170 that are pre-loaded with GIT or GITC, or a buffer solution comprising GIT or GITC. In one embodiment, device 100 comprises a first collection tube (e.g., first receptacle 170a) pre-loaded with GIT or GITC for collection and stabilization of cfDNA and a second collection tube (e.g.,second receptacle 170b) pre-loaded with GIT or GITC for collection and stabilization of extracellular RNA.

[0075] Now referring to Fig. 2, in some embodiments, the system comprises the disclosed device 100 comprising a sample collection device and detachable separation, filtration component 140 and one or more receptacles 170 which serve as collection tubes for the analytes that are filtered and separated by the device. In some embodiments the system comprises a stabilizing compound 180 (e.g., GITC) for stabilization of cfDNA in collection tube 1 (e.g., first receptacle 170a), a stabilizing compound 182 (e.g., GITC) for stabilization of extracellular RNA in collection tube 2 (e.g., second receptacle 170b), and a stabilizing compound 184 (e.g., 20% ethanol w / v) for protein stabilization in collection tube 3 (e.g., third receptacle 170c), with a genomic lysis buffer 186 (e.g., SDS or GITC lysis buffer) for extraction and stabilization of gDNA and intracellular RNA into compression tube from tube 4 (e.g., fourth receptacle 170d).

[0076] The stabilized nucleic acids of the present invention can be stored at room temperature for longer than 1 week, 2 weeks, 5 weeks, 10 weeks, or 25 weeks or more.

[0077] Aspects of the present invention relate to physical dimensions for device 100. In some embodiments, housing 142 has a length, width, or depth ranging between 1 cm and 10 cm. In some embodiments, inlet 146 has a diameter ranging between 1 mm and 10 mm. In some embodiments, flange 150 comprises a height or diameter ranging between about 1 mm and 2 cm. In some embodiments, interior volume of housing 152 ranges between 10 mm3and 1000 mm3.Computing Device

[0078] In some embodiments, the data derived from the collection and analysis of a sample using the device 100 and / or any disclosed kit may be processed or analyzed using a computing device, such as computer 900 described herein. In some aspects of the present invention, software for executing instructions related to the processing or analysis of a sample, or data derived therefrom, as provided herein may be stored on a non-transitory computer-readable medium, wherein the software performs some or all of the steps related to the processing or analysis of a sample, or data derived therefrom when executed on a processor.

[0079] Aspects of the invention relate to algorithms executed in computer software. Though certain embodiments may be described as written in particular programming languages,or executed on particular operating systems or computing platforms, it is understood that the system and method of the present invention is not limited to any particular computing language, platform, or combination thereof. Software executing the algorithms described herein may be written in any programming language known in the art, compiled, or interpreted, including but not limited to C, C++, C#, Objective-C, Java, JavaScript, MATLAB, Python, PHP, Perl, Ruby, or Visual Basic. It is further understood that elements of the present invention may be executed on any acceptable computing platform, including but not limited to a server, a cloud instance, a workstation, a thin client, a mobile device, an embedded microcontroller, a television, or any other suitable computing device known in the art.

[0080] Parts of this invention are described as software running on a computing device. Though software described herein may be disclosed as operating on one particular computing device (e.g. a dedicated server or a workstation), it is understood in the art that software is intrinsically portable and that most software running on a dedicated server may also be run, for the purposes of the present invention, on any of a wide range of devices including desktop or mobile devices, laptops, tablets, smartphones, watches, wearable electronics or other wireless digital / cellular phones, televisions, cloud instances, embedded microcontrollers, thin client devices, or any other suitable computing device known in the art.

[0081] Similarly, parts of this invention are described as communicating over a variety of wireless or wired computer networks. For the purposes of this invention, the words “network”, “networked”, and “networking” are understood to encompass wired Ethernet, fiber optic connections, wireless connections including any of the various 802.11 standards, cellular WAN infrastructures such as 3G, 4G / LTE, or 5G networks, Bluetooth®, Bluetooth® Low Energy (BLE) or Zigbee® communication links, or any other method by which one electronic device is capable of communicating with another. In some embodiments, elements of the networked portion of the invention may be implemented over a Virtual Private Network (VPN).

[0082] Fig. 3 and the following discussion are intended to provide a brief, general description of a suitable computing environment in which the invention may be implemented. While the invention is described above in the general context of program modules that execute in conjunction with an application program that runs on an operating system on a computer, those skilled in the art will recognize that the invention may also be implemented in combination with other program modules.

[0083] Generally, program modules include routines, programs, components, data structures, and other types of structures that perform particular tasks or implement particular abstract data types. Moreover, those skilled in the art will appreciate that the invention may be practiced with other computer system configurations, including hand-held devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, and the like. The invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

[0084] Fig. 3 depicts an illustrative computer architecture for a computer 900 for practicing the various embodiments of the invention. The computer architecture shown in Fig. 3 illustrates a conventional personal computer, including a central processing unit 950 (“CPU”), a system memory 905, including a random access memory 910 (“RAM”) and a read-only memory (“ROM”) 915, and a system bus 935 that couples the system memory 905 to the CPU 950. A basic input / output system containing the basic routines that help to transfer information between elements within the computer, such as during startup, is stored in the ROM 915. The computer 900 further includes a storage device 920 for storing an operating system 925, application / program 930, and data.

[0085] The storage device 920 is connected to the CPU 950 through a storage controller (not shown) connected to the bus 935. The storage device 920 and its associated computer- readable media provide non-volatile storage for the computer 900. Although the description of computer-readable media contained herein refers to a storage device, such as a hard disk or CD- ROM drive, it should be appreciated by those skilled in the art that computer-readable media can be any available media that can be accessed by the computer 900.

[0086] By way of example, and not to be limiting, computer-readable media may comprise computer storage media. Computer storage media includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid state memory technology, CD-ROM, DVD, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, orany other medium which can be used to store the desired information and which can be accessed by the computer.

[0087] According to various embodiments of the invention, the computer 900 may operate in a networked environment using logical connections to remote computers through a network 940, such as TCP / IP network such as the Internet or an intranet. The computer 900 may connect to the network 940 through a network interface unit 945 connected to the bus 935. It should be appreciated that the network interface unit 945 may also be utilized to connect to other types of networks and remote computer systems.

[0088] The computer 900 may also include an input / output controller 955 for receiving and processing input from a number of input / output devices 960, including a keyboard, a mouse, a touchscreen, a camera, a microphone, a controller, a joystick, or other type of input device. Similarly, the input / output controller 955 may provide output to a display screen, a printer, a speaker, or other type of output device. The computer 900 can connect to the input / output device 960 via a wired connection including, but not limited to, fiber optic, Ethernet, or copper wire or wireless means including, but not limited to, Wi-Fi, Bluetooth, Near-Field Communication (NFC), infrared, or other suitable wired or wireless connections.

[0089] As mentioned briefly above, a number of program modules and data files may be stored in the storage device 920 and / or RAM 910 of the computer 900, including an operating system 925 suitable for controlling the operation of a networked computer. The storage device 920 and RAM 910 may also store one or more applications / programs 930. In particular, the storage device 920 and RAM 910 may store an application / program 930 for providing a variety of functionalities to a user. For instance, the application / program 930 may comprise many types of programs such as a word processing application, a spreadsheet application, a desktop publishing application, a database application, a gaming application, internet browsing application, electronic mail application, messaging application, and the like. According to an embodiment of the present invention, the application / program 930 comprises a multiple functionality software application for providing word processing functionality, slide presentation functionality, spreadsheet functionality, database functionality and the like.

[0090] The computer 900 in some embodiments can include a variety of sensors 965 for monitoring the environment surrounding and the environment internal to the computer 900. These sensors 965 can include a Global Positioning System (GPS) sensor, a photosensitivesensor, a gyroscope, a magnetometer, thermometer, a proximity sensor, an accelerometer, a microphone, biometric sensor, barometer, humidity sensor, radiation sensor, or any other suitable sensor.Sample collection apparatus and kit

[0091] Embodiments of the invention described herein relate to the analysis of extracellular nucleic acids and proteins derived from a cell-free fluid phase portion of a biological sample. In some embodiments, the integrated Saliva Collection, Processing, Stabilization, and Storage (iSCPSS) device (e.g., device 100) may be provided or designed as an all-in-one kit to collect, process, stabilize, and store biological samples for research and clinical applications such as molecular diagnostics based on protein, RNA, and DNA. In some embodiments, the biological samples are saliva samples.

[0092] In some embodiments, the disclosed kit comprises at least one device 100 and further comprises one or more receptacles 170 which serve as collection tubes for the analytes that are fdtered and separated by the device. In some embodiments, the kit comprises a first, second and third receptacle (170a, 170b, 170c) for the separation and storage of cfDNA, extracellular RNA and proteins. In some embodiments, the first, second and third receptacle (170a, 170b, 170c) are pre-loaded with one or more stabilization agents for stabilization of nucleic acid molecules or proteins. In some embodiments, the kit comprises a fourth receptacle 170d, wherein the fourth receptacle 170d comprises a genomic lysis buffer (e.g., GITC buffer) for stabilization and collection of gDNA and intracellular RNA after collection of other analytes (e.g., cfDNA, RNA or protein).

[0093] Therefore, in some embodiments, the kit comprises components for isolation and stabilization of cfDNA, extracellular RNA, genomic DNA, intracellular RNA, protein, and any combination thereof from a biological fluid sample, in some embodiments, the kit comprises components for isolation and stabilization of cfDNA, extracellular RNA, genomic DNA, intracellular RNA, protein, and any combination thereof from a biological fluid sample.

[0094] The quality of the detected nucleic acids and proteins meet the demand for techniques such as PCR, qPCR, microarray assays, ELISA, Western blot, etc.

[0095] In some embodiments, to obtain samples, subjects can be given an absorbent pad to place under their tongue for a period of time long enough to absorb saliva. Any type of absorbent pad that absorbs saliva can be used, and device 100 will work with any animal that produces saliva. Suitable absorbent materials can include, but are not limited to, nitrocellulose, cellulose acetate, polyethersulfur fabric, cellulose fiber such as paper strips or cotton, nylon, gel foam, fiber glass, polycarbonate, polypropylene, acetate, rayon, polyester absorbent pad, or other synthetic materials capable of collecting saliva. Any other method known in the art can be used to collect saliva. For example, the spitting method can also be used as a means of collecting a sample of saliva. An alternative method of obtaining saliva samples is a method whereby saliva is sucked out of the oral cavity by means of an aspirator. Saliva that has collected in the oral cavity can also be simply dripped out into a sample vessel.

[0096] In an embodiment, the absorbent pad can be individually placed into any syringe (e g., syringe 110) having a fdter attached to the end of the syringe. The filter can be any type of filter described herein capable of separating saliva into a cell-free and fluid phase, for example, a 5.0 pm hydrophilic PVDF filter (Millex-SV, Millipore). The syringe plunger can then be used to push the saliva out of the pad and through the filter into a collection tube (e.g., one or more receptacles 170). The one or more receptacles 170 or tubes can be pre-loaded with specific stabilizer for protein, RNA, and DNA. The tubes can also be pre-loaded with an alcohol solution. The collection apparatus can be any type of commercial collection pad. For example, the SUPER*SAL™ or VERSI*SAL® collection devices (Oasis Diagnostics, Vancouver, WA) can be used to collect saliva samples and further configured to be used with a sample filtration apparatus. In other embodiments, the saliva collection device can separate samples into two or more aliquots following filtration. An exemplary device for dual separation is the ULTRA*SAL-2™ saliva collection device (Oasis Diagnostics, Vancouver, WA). Exemplary saliva collection devices include, but are not limited to, Pure*SAL™ and RNAPro*SAL™ (Chiang, et al., 2015, Biotechniques 58:69-762.)

[0097] An embodiment describes the device 100 and / or kit described herein in a method of collecting saliva. The method can include inserting the sample collection pad into the oral cavity for sufficient time to moisten the sample collection pad, inserting the collection pad into the receiving tube, applying sufficient force to cause the materials collected in the collection padto pass through the filter thereby forming a filtered sample, and collecting the filtered sample into one or more receiving devices (e.g., one or more receptacles 170).

[0098] It can be appreciated that classes of compounds in addition to nucleic acid and protein can be analyzed, e.g., virus, prions, bacteria (e.g., Mycobacterium tuberculosis), carbohydrates such as sugars, lipids, fatty acids, hormones, cholesterol, metabolites, and small molecule drug compounds.

[0099] It can further be appreciated that device 100 and / or the disclosed kit can be used to diagnose a disease in a subject. The disease can include, but is not limited to, autoimmune disease, cancer, infectious disease, metabolic disease, or any combination thereof. For example, in some embodiments, the device 100 and / or kit can be used to separate and filter analytes in a biological fluid sample from a subject which are subsequently analyzed for the presence of one or more disease-associated biomarker. Exemplary biomarkers include, but are not limited to, markers that are detected in cfDNA, extracellular RNA (exRNA), gDNA, intracellular RNA (inRNA), proteins, or any combination thereof.Methods of Use

[0100] Embodiments described herein include methods for streamlined, ambienttemperature processing, stabilization, and storage of multiple analytes derived from biological fluid. In some embodiments, the filtered, separated and collected analytes may be used in one or more downstream analysis methods. In some embodiments, direct analysis of analytes collected from the biological fluid supernatant filtered and stored using a device as described herein, can be performed at ambient temperature. In some embodiments, stabilization agents can be included. Any disclosed method may utilize one or more device 100 and / or one or more of the kits thereof.

[0101] Profiling biological fluid analytes (e.g., at one or more time points over the course of disease progression) can reveal potential biomarkers indicative of disease or different stages of disease. In some embodiments, profiling of biological fluid cfDNA molecules is useful in diagnosis or early detection of disease. In some embodiments, profiling of biological fluid exRNA molecules is useful in diagnosis or early detection of disease. In some embodiments, profiling of biological fluid protein molecules is useful in diagnosis or early detection of disease.In some embodiments, profiling of a combination of cfDNA and exRNA is useful in diagnosis or early detection of disease. In some embodiments, profiling of a combination of cfDNA and protein is useful in diagnosis or early detection of disease. In some embodiments, profiling of a combination of exRNA and protein is useful in diagnosis or early detection of disease. In some embodiments, profiling of a combination of cfDNA, exRNA and protein is useful in diagnosis or early detection of disease. In some embodiments, profiling of a combination of at least one of cfDNA, exRNA and protein and at least one of gDNA and inRNA is useful in diagnosis or early detection of disease.

[0102] In some embodiments, the methods of the invention include filtration, separation and stabilization of cell free DNA and extracellular RNA (exRNA) in separate collection tubes (e.g., one or more receptacles 170) for down stream analysis. In some embodiment, the methods of the invention further include collection of genomic DNA, intracellular RNA (inRNA) or a combination thereof in a separate collection tube following filtration, separation and stabilization of cell free DNA and extracellular RNA. Therefore, in some embodiments, one or more downstream analysis method can be performed for analysis of cfDNA, exRNA, gDNA, inRNA or any combination thereof.

[0103] In some embodiments, the methods include 1) applying a biological fluid sample to collection device as described herein comprising at least a first collection tube for collection of cfDNA, wherein the first collection tube is pre-loaded with a stabilization agent for stabilization of cfDNA, a second collection tube for collection of exRNA, wherein the second collection tube is pre-loaded with a stabilization agent for stabilization of exRNA, and a third collection tube for collection of proteins, wherein the third collection tube is pre-loaded with a stabilization agent for stabilization of proteins, 2) compressing the compression tube of the device to filter and separate the biological fluid sample into the attached collection tubes, and 3) removing the collection tubes from the device for storage and / or downstream analysis of the collected cfDNA, exRNA, proteins, or any combination thereof. In some embodiments, the method further comprises applying a lysis buffer to the compression tube of the device and collecting the lysed cellular products comprising the gDNA and / or inRNA from the compression tube of the device for downstream analysis of the gDNA, inRNA or a combination thereof.

[0104] Nucleic acid stabilization requiring extremely low temperatures or nucleic acid stabilization chemicals can be impractical for field applications or daily clinical operations.Furthermore, nucleic acid stabilizers can affect downstream analyses. Therefore, in some embodiments, the methods described herein include the extraction of extracellular nucleic acids, and the stabilization of the extracted nucleic acids, which can be stored at ambient temperatures until needed for downstream applications. In some embodiments, GIT or GITC can be pre- loaded into one or more collection tube of the device as a stabilization agent for stabilization of nucleic acid molecules.

[0105] The nucleic acids collected using the biological fluid collection apparatus of the present invention can be stored at room temperature for longer than 1 week, 2 weeks, 5 weeks, 10 weeks, or 25 weeks or more.

[0106] Fig. 4A & Fig. 4B show a series of images depicting an exemplary method 200 of using a universal collector (e.g., device 100). In some embodiments, the steps of method 200 comprise: 210 a collector device (e.g., device 100), assembly, or kit is used to collect a saliva sample from a subject, 220 a saliva sample is collected using an absorbent pad in the collector assembly as per instructions, 230 the absorbent pad is removed from the mouth of a subject and inserted into the compression tube of the syringe, 240 the pad is then pushed by the plunger of the syringe to let the sample be collected in the centrifuge or collection tubes — the plunger handle attached to the absorbent pad end is firmly pushed downwards into the syringe or compression tube to transfer saliva from the absorbent pad into collection tubes 1, 2, and 3 with pre-specified stabilizing solutions through the three way splitting unit with filtration, and the device is inverted 2-3 times to mix the sample, 250 after the tubes are detached along with the splitting unit, the compression tube end is sealed as per instructions with paraffin film, 260 the absorbent pad is removed from the assembly, and a genomic lysis buffer is added to the sealed compression tube with paraffin film. In some embodiments, the absorbent pad suspended into the lysis buffer in the tube. In some embodiments, the collection tubes are inverted 2-3 times to let the pad absorb carefully. In some embodiments, the sample is collected after 1 hr by removing the paraffin seal from the rear end of the tube, and the sample is collected by compressing the absorbent pad, and channeling the samples into collection tube for gDNA and intracellular RNA analysis as desired.

[0107] Aspects of the present invention relate to an exemplary method of using a collector device (e.g., device 100, or any disclosed kit comprising one or more device 100). Referring now to Fig. 5A, an exemplary method 300 of using a collector device is shown. Insome embodiments, method 300 comprises the steps of: 301 providing a collector device or kit (e.g., device 100); 302 loading biological fluid from a subject captured on the absorbent pad into the lumen of the syringe tube; 303 depressing the plunger of the syringe to push the liquid from the tube of the syringe into the interior volume of the splitter housing; and 304 collecting one or more analytes in the one or more receptacles. In some embodiments, the sample is a saliva sample.

[0108] Aspects of the present invention relate to a method of detecting a biomarker in a biological fluid sample of a subject. Referring now to Fig. 5B, an exemplary method 400 of detecting a biomarker in a biological fluid sample of a subject is shown. In some embodiments, method 400 comprises the steps of: 401 providing a collector device (e.g., collector device 100); 402 loading biological fluid from a subject captured on the absorbent pad into the lumen of the syringe tube; 403 depressing the plunger of the syringe to push the liquid from the tube of the syringe into the interior volume of the splitter housing; 404 collecting one or more analytes in the one or more receptacles; and 405 analyzing the one or more analytes to detect a biomarker of interest in the sample. In some embodiments, the sample is a saliva sample.

[0109] In some embodiments, any disclosed method further comprises collecting cell- free DNA in the first receptacle, extracellular RNA in the second receptacle and protein in the third receptacle.

[0110] Aspects of the present invention relate to a method of diagnosing a disease or disorder in a subject. Referring now to Fig. 5C, an exemplary method 500 of diagnosing a disease or disorder in a subject is shown. In some embodiments, method 500 comprises the steps of: 501 providing a collector device (e.g., collector device 100); 502 loading biological fluid from a subject captured on an absorbent pad into the lumen of the syringe tube; 503 depressing the plunger of the syringe to push the liquid from the tube of the syringe into the interior volume of the splitter housing; 504 collecting one or more analytes in the one or more receptacles; 505 analyzing the one or more analytes to detect a biomarker of interest in the sample, wherein the biomarker is a disease-associated biomarker; and 506 diagnosing the subject as having or at risk of developing the disease associated with the biomarker upon detection of the presence or level of the biomarker as compared to a comparator control. In some embodiments, the sample is a saliva sample.

[0111] In some embodiments, any disclosed method further comprises collecting cell- free DNA in the first receptacle, extracellular RNA in the second receptacle and protein in the third receptacle.

[0112] In some embodiments, any disclosed method further comprises the step of applying a lysis buffer to the lumen of the device following the collecting step, and collecting the lysed cellular products from the compression tube containing genomic DNA (gDNA) and intracellular RNA for analysis in a separate tube. In some embodiments, any disclosed method further comprises analyzing cfDNA, exRNA or proteins or any combination thereof. In some embodiments, any disclosed method further comprises analyzing cfDNA, exRNA, proteins, gDNA or intracellular RNA, or any combination thereof.

[0113] In some embodiments, any disclosed method further comprises collecting cell- free DNA in the first receptacle, extracellular RNA in the second receptacle and protein in the third receptacle. In some embodiments, any disclosed method further comprises the step of removing the splitter housing from the syringe, applying a temporary seal to the output end of the syringe, applying a lysis buffer to the compression tube of the syringe, removing the temporary seal from the output end of the syringe and depressing the plunger of the syringe to collect the lysed cellular products from the compression tube containing genomic DNA (gDNA) and intracellular RNA for analysis in a separate tube. In some embodiments, any disclosed method further comprises analyzing cfDNA, exRNA or proteins or any combination thereof. In some embodiments, any disclosed method further comprises analyzing cfDNA, exRNA, proteins, gDNA or intracellular RNA, or any combination thereof.

[0114] Aspects of the present invention relate to a method of stabilizing nucleic acid molecules. In some embodiments, an exemplary method of stabilizing nucleic acid molecules comprises the steps of contacting a sample comprising one or more nucleic acid molecule with guanidine thiocyanate. In some embodiments, the one or more nucleic acid molecule comprises cfDNA or exRNA.Nucleic Acid Analysis

[0115] Following filtration and collection, the collected nucleic acid molecules can be used in any number of downstream applications known to those of skill in the art.Methodologies for use for analysis of nucleic acids (e.g., cfDNA, exRNA, gDNA, inRNA or any combination thereof) are well known in the art. Such methods can include, but are not limited to, PCR, reverse transcriptase-PCR (RT-PCR), real-time PCR, reverse transcription quantitative real-time PCR (RT-qPCR), ligase chain reaction, strand displacement amplification (SDA), selfsustained sequence replication (3 SR), in situ PCR, DNA sequencing, RNA sequencing, or any combination thereof. Any suitable qualitative or quantitative methods known in the art for detecting a specific nucleic acid (e.g., RNA or DNA) or for assaying the sequence or amount of a nucleic acid (e.g., RNA or DNA) can be used.

[0116] Applicable PCR amplification techniques are described in, e.g., Ausubel et al. and Innis et al., supra. General nucleic acid hybridization methods are described in Anderson, “Nucleic Acid Hybridization,” BIOS Scientific Publishers, 1999. For PCR, a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length. For high stringency PCR amplification, a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y. (1990).

[0117] Real time, quantitative reverse transcriptase PCR (RT-PCR), or reverse transcription quantitative real-time PCR (RT-qPCR) can be used to determine the presence of mutations. RNA extraction can be performed by any method know to those of skill in the art, e.g., methods involving proteinase K tissue digestion and alcohol-based nucleic acid precipitation, treatment with DNase to digest contaminating DNA, RNA purification using silica-gel-membrane technology, methods utilizing commercially available kits such as Trizol and RNeasy, or any combination thereof. Real time RT-PCR can be performed by any method known to those of skill in the art, e.g., Taqman real time PCR using Applied Biosystem assays.

[0118] Nucleic acid primers, or probes can be generated and used in the methods described herein. In some embodiments, the probes comprise at least about 12, 15, 16, 18, 20, 22, 24, or 25 nt fragments of a contiguous sequence of nucleic acid or polypeptide. The probes canbe produced by, for example, chemical synthesis, PCR amplification, generation from longer polynucleotides using restriction enzymes, or other methods well known in the art.

[0119] Nucleic acid probes can be used as diagnostics wherein a biological sample to be analyzed, such as saliva, can be treated, if desired, to extract the nucleic acids contained therein. The resulting nucleic acid from the sample can be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample can be dot blotted without size separation. The nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable stringencies. The probes can be made completely complementary to the target nucleic acid or portion thereof (e.g., to all or a portion of a sequence encoding a target). Therefore, usually high stringency conditions are desirable in order to prevent or at least minimize false positives. However, conditions of high stringency should only be used if the probes are complementary to regions of the target which lack heterogeneity. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time, and concentration of formamide (Sambrook et al. (1989), “Molecular Cloning; A Laboratory Manual,” Second Edition (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.)).

[0120] Nucleic acid probes, or alternatively nucleic acid from the samples, can be provided in solution for such assays, or can be affixed to a support (e.g., solid or semi-solid support). Examples of supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidene fluoride, diazotized paper, nylon membranes, activated beads, and Protein A beads.

[0121] Non-PCR-based, sequence specific DNA amplification techniques can also be used with the invention to detect nucleic acids. An example of such techniques include, but is not necessarily limited to, the Invader assay (see, e.g., Kwiatkowski et al. Mol Diagn. 1999, 4:353-64. See also U.S. Pat. No. 5,846,717).

[0122] Nucleic acids can be detectably labeled. Exemplary detectable labels include, but are not limited to, radiolabels, fluorochromes, (e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2’, 7’ -dimethoxy - 4’,5’-dichloro-6-carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6- carboxy-2’,4’,T,4,7- hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N’,N’-tetramethyl-6-carboxyrho- damine (TAMRA)), radioactive labels, (e.g. ,sup.32p, ,sup.35S, and sup.3H), and the like. The detectable label can involve two stage systems (e.g., biotin-avidin, hapten-anti- hapten antibody, and the like).

[0123] Analysis of nucleic acid mutations derived from biological fluid samples can be performed using techniques known in the art including, without limitation, electrophoretic analysis or sequence analysis. Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis (DGGE). Other methods of nucleic acid analysis include, but is limited to, restriction analyses such as restriction-fragment-length- polymorphism detection based on allele-specific restriction-endonuclease cleavage (Kan and Dozy, Lancet ii :910- 12 (1978)), hybridization with allele-specific oligonucleotide probes (Wallace et al., Nucl. Acids Res. 6:3543-3557 (1978)), including immobilized oligonucleotides (Saiki et al., PNAS 86:6230-6234 (1989)), oligonucleotide arrays (Maskos and Southern, Nucl. Acids Res. 21 :2269-2270 (1993)), oligonucleotide-ligation assay (OLA) (Landegren et al., Science 241 : 1077 (1988)), allele-specific ligation chain reaction (LCR) (Barrany, PNAS 88: 189- 193 (1991)), gap-LCR (Abavaya et al. Nuc.l Acids Res. 23:675-682 (1995)), single-strand- conformation-polymorphism detection (Orita et al., Genomics 5:874-879 (1983)), RNAase cleavage at mis-matched base-pairs (Myers et al., Science 230: 1242 (1985)), cleavage of heteroduplex DNA, methods based on allele specific primer extension, genetic bit analysis (GBA) (Nikiforov et al., Nucl. Acids Res. 22:4167-4175 (1994)), in situ hybridization, Southern blot, Northern blot analysis, denaturing high performance liquid chromatography (DHPLC) (Kim et al., Genetic Testing 12:295-298 (2008)). Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDLTOF / MS; Fu et al., Nat. Biotechnol., 16:381-384 (1998)), and sequencing by hybridization (Chee et al., Science, 274:610-614 (1996); Drmanac et al., Science, 260: 1649- 1652 (1993); Drmanac et al., Nat. Biotechnol., 16:54-58 (1998)), NGS (next-generation sequencing) (Chen et al., Genome Res. 18: 1143-1149 (2008); Srivatsan et al. PloS Genet. 4:el000139 (2008)), Polony sequencing (Porreca et al., Curr. Protoc. Mol. Biol. Chp. 7;Unit7.8(2006), ion semiconductor sequencing (Elliott et al., J.Biomol Tech. 1 :24-30 (2010), DNA nanoball sequencing (Kaji et al., Chem Soc Rev 39:948-56 (2010), single molecule real-time sequencing (Flusberg et al., Nat. Methods 6:461-5 (2010), or nanopore DNA sequencing (Wanunu, Phys Life Rev 9: 125-58 (2012).

[0124] Probes (or sample nucleic acid) can be provided on an array for detection following nucleic acid extraction. Arrays can be created by, for example, spotting polynucleotide probes onto a substrate (e.g., glass, nitrocellulose, and the like) in a two-dimensional matrix or array. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions. Samples of polynucleotides can be detectably labeled (e.g., using radioactive or fluorescent labels) and then hybridized to the probes. Double stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away. Techniques for constructing arrays and methods of using these arrays are described in EP 799 897; WO 97 / 29212; WO 97 / 27317; EP 785 280; WO 97 / 02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP 728 520; U.S. Pat. No. 5,599,695; EP 721 016; U.S. Pat. No. 5,556,752; WO 95 / 22058; and U.S. Pat. No. 5,631,734. Arrays are particularly useful where, for example a single sample is to be analyzed for the presence of two or more nucleic acid target regions, as the probes for each of the target regions, as well as controls (both positive and negative) can be provided on a single array. Arrays thus facilitate rapid and convenience analysis.Protein analysis

[0125] Following filtration and collection, the collected proteins can be used in any number of downstream applications known to those of skill in the art. Methodologies for use for analysis of proteins are well known in the art.

[0126] The methodology for preparing protein in a form that is suitable for detection is well known in the art. Extracellular protein can be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al, supra; and Sambrook et al., supra).

[0127] The protein can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands or substrates using column chromatography. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).

[0128] Antibody reagents can be used in assays to detect proteins in biological fluid samples using any of a number of immunoassays known to those skilled in the art. Immunoassay techniques and protocols are generally described in Price and Newman, "Principles and Practice of Immunoassay," 2nd Edition, Grove's Dictionaries, 1997; and Gosling, "Immunoassays: A Practical Approach," Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used. (See, e.g., Self et al., Curr. Opin. Biotechnol 7:60-65 (1996)). The term immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); immunohistochemical assay, capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence. (See, e.g., Schmalzing et al., Electrophoresis, 25 18:2184-93 (1997); Bao, J Chromatogr. B. Biomed. Sci., 699:463-80 (1997)). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. (See, e.g., Rongen et al.., J. Immunol. Methods, 204: 105-133 (1997)). In addition, nephelometry assays, in which the formation of protein / antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention. Nephelometry assays are commercially available from Beckman Coulter (Brea, CA; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J Clin. Chem. Clin. Biochem., 27:261-276 (1989)).

[0129] Specific immunological binding of an antibody can be detected directly or indirectly. A detectable moiety can be used (direct or indirect detection). A variety of detectablemoieties are well known to those skilled in the art, and can be any material detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions. Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, metals, and the like. Direct labels include fluorescent or luminescent tags, metals, dyes, radionucleodies, and the like, attached to the antibody. An antibody labeled with iodine- 125 (1251) can be used. A chemiluminescence assay using a chemiluminescent antibody specific for nucleic acids or proteins is suitable for sensitive, non-radioactive detection of nucleic acids or protein levels. An antibody labeled with fluorochrome is also suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), P-galactosidase, urease, and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a P-galactosidase detection system can be used with the chromogenic substrate o- nitrophenyl-P-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm. An urease detection system can be used with a substrate such as urebromocresol purple (Sigma Immunochemicals; St. Louis, MO). Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G can also be used as a label agent. These proteins exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al., J. Immunol. 111 :1401-1406 (1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985).

[0130] Western blot (immunoblot) analysis can be used to detect and quantify the presence of an antigen in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind the antigen. The anti-antigen antibodies specifically bind to the antigen on the solid support. These antibodies can be directly labeled or alternatively can be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-antigen antibodies.

[0131] An ELISA method can be used as follows: (1) bind an antibody or antigen to a substrate; (2) contact the bound receptor with a fluid or tissue sample containing the virus, a viral antigen, or antibodies to the virus; (3) contact the above with an antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe color change. The above method can be readily modified to detect presence of an antibody in the sample or a specific protein as well as a virus.

[0132] An antigen and / or a subject’s antibodies to the virus can be detected utilizing a capture assay. Briefly, to detect antibodies in a sample, antibodies to an immunoglobulin, e.g., anti-IgG (or IgM) are bound to a solid phase substrate and used to capture the patient’s immunoglobulin from serum. The antigen, or reactive fragments of the antigen, are then contacted with the solid phase followed by addition of a labeled antibody. The amount of specific antibody can then be quantitated by the amount of labeled antibody binding. A microagglutination test can also be used to detect the presence of an antigen in test samples. Briefly, latex beads are coated with an antibody and mixed with a test sample, such that the antigen in the tissue or body fluids that is specifically reactive with the antibody crosslink with the receptor, causing agglutination. The agglutinated antibody-virus complexes within a precipitate, visible with the naked eye or by spectrophotometer.

[0133] Competitive assays can also be adapted to provide for an indirect measurement of the amount of an antigen present in the sample. Briefly, serum or other body fluids from the subject is reacted with an antibody bound to a substrate (e.g. an ELISA 96-well plate). Excess serum is thoroughly washed away. A labeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonal antibody is then reacted with the previously reacted antibody complex. The amountof inhibition of monoclonal antibody binding is measured relative to a control. Monoclonal antibodies (MABs) can also be used for detection directly in samples by IFA for MABs specifically reactive for the antibody-antigen complex.

[0134] Immunoassays in the competitive binding format can also be used for crossreactivity determinations. For example, an antigen can be immobilized to a solid support. Proteins can be added to the assay that competes for binding of the antisera to the immobilized antigen. The ability of the added proteins to compete for binding of the antisera to the immobilized protein is compared to the ability of the antigen to compete with itself. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the added proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the added considered proteins, e g., distantly related homologs. The immunoabsorbed and pooled antisera can then be used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps an allele or polymorphic variant of an antigen, to the immunogen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of binding is less than 10 times the amount of the antigen that is required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to antigen.

[0135] A signal from a direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 1251; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. Where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Colorimetric or chemiluminescent labels can be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.

[0136] The antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

[0137] One of skill in the art will appreciate that it is often desirable to minimize nonspecific binding in immunoassays. Particularly, where the assay involves an antigen or antibody immobilized on a solid substrate it is desirable to minimize the amount of non-specific binding to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this technique involves coating the substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used.Samples

[0138] In some embodiments, the sample is derived from a biological fluid, comprising a nucleic acid or a mixture of nucleic acids. Such samples include, but are not limited to plasma, serum, blood, a blood fraction, urine, sweat, sputum / oral fluid, saliva, amniotic fluid, or fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.), peritoneal fluid, pleural fluid, and the like. In some embodiments, the sample is a saliva sample. Although the sample is often taken from a human subject (e.g., patient), the assays can be from any mammal, including, but not limited to, dogs, cats, horses, goats, sheep, cattle, pigs, etc.

[0139] The sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. For example, such pretreatment may include preparing plasma from blood, diluting viscous fluids and so forth. Methods ofpretreatment may also involve, but are not limited to, filtration, precipitation, dilution, distillation, mixing, centrifugation, freezing, lyophilization, concentration, amplification, nucleic acid fragmentation, inactivation of interfering components, the addition of reagents, lysing, etc. If such methods of pretreatment are employed with respect to the sample, such pretreatment methods are typically such that the analyte(s) of interest remain in the test sample. Such "treated" or "processed" samples are still considered to be biological samples with respect to the methods described herein.

[0140] In various embodiments of the invention, methods of measuring the level of a marker include, but are not limited to, PCR, reverse transcriptase-PCR (RT-PCR), real-time PCR, reverse transcription quantitative real-time PCR (RT-qPCR), ligase chain reaction, strand displacement amplification (SDA), self-sustained sequence replication (3 SR), in situ PCR, DNA sequencing, RNA sequencing, next generation sequencing technology (NGS), an immunochromatography assay, an immunodot assay, a luminescence assay, an ELISA assay, an ELISPOT assay, a microarray assay, a ligand-receptor binding assay, displacement of a ligand from a receptor assay, displacement of a ligand from a shared receptor assay, an immunostaining assay, a Western blot assay, a mass spectrophotometry assay, a radioimmunoassay (RIA), a radioimmunodiffusion assay, a liquid chromatography-tandem mass spectrometry assay, an ouchterlony immunodiffusion assay, reverse phase protein microarray, a rocket immunoelectrophoresis assay, an immunohistostaining assay, an immunoprecipitation assay, a complement fixation assay, FACS, an enzyme-substrate binding assay, an enzymatic assay, an enzymatic assay employing a detectable molecule, such as a chromophore, fluorophore, or radioactive substrate, a substrate binding assay employing such a substrate, a substrate displacement assay employing such a substrate, and a protein chip assay (see also, 2007, Van Emon, Immunoassay and Other Bioanalytical Techniques, CRC Press; 2005, Wild, Immunoassay Handbook, Gulf Professional Publishing; 1996, Diamandis and Christopoulos, Immunoassay, Academic Press; 2005, Joos, Microarrays in Clinical Diagnosis, Humana Press; 2005, Hamdan and Righetti, Proteomics Today, John Wiley and Sons; 2007). Appropriate methods include both high-throughput and low-throughput methods.Multiplex sequencing

[0141] In some embodiments, the nucleic acid molecules isolated and collected using the device and methods described herein can be analyzed using multiplexed sequencing methodologies. The large number of sequencing reads that can be obtained per sequencing run permits the analysis of pooled samples i.e. multiplexing, which maximizes sequencing capacity and reduces workflow. For example, the massively parallel sequencing of eight libraries performed using the eight-lane flow cell of the Illumina Genome Analyzer, and Illumina's HiSeq Systems, can be multiplexed to sequence two or more samples in each lane such that 16, 24, 32 etc. or more samples can be sequenced in a single run. Parallelizing sequencing for multiple samples i.e. multiplex sequencing, requires the incorporation of sample-specific index sequences, also known as barcodes, during the preparation of sequencing libraries. Sequencing indexes are distinct base sequences of about 5, about 10, about 15, about 20 about 25, or more bases that are added at the 3' end of the genomic and marker nucleic acid. The multiplexing system enables sequencing of hundreds of biological samples within a single sequencing run. The preparation of indexed sequencing libraries for sequencing clonally amplified sequences can be performed by incorporating an index sequence into a PCR primer used for cluster amplification. Alternatively, the index sequence can be incorporated into the adaptor, which is ligated to the nucleic acid molecules prior to the PCR amplification. Sequencing of the uniquely marked indexed nucleic acids provides index sequence information that identifies samples in the pooled sample libraries, and sequence information of marker molecules correlates sequencing information of the genomic nucleic acids to the sample source. In embodiments wherein the multiple samples are sequenced individually i.e. singleplex sequencing, marker and cell-free DNA of each sample need only be modified to contain the adaptor sequences as required by the sequencing platform and exclude the indexing sequences.Diagnostic Assays

[0142] In some embodiments, use of the device and methods described herein in the diagnosis, and / or monitoring, and / or treating pathologies is contemplated. For example, the methods can be applied to determining the presence or absence of a disease, to monitoring the progression of a disease and / or the efficacy of a treatment regimen.

[0143] In one aspect, the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein, in a biological fluid sample of a patient isused to diagnose the presence or risk of a disease or disorder in a subject. In some embodiments, the level cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein, in a test sample obtained from a subject can be compared to the level from a comparator control, and a subject is diagnosed has having or being at risk of developing a disease or disorder associated with the biomarker based on an alteration in the presence or level of the biomarker as compared to the comparator control. In one embodiment, the comparator control is a positive control, a negative control, a historical control, a historical norm, or the level of a reference molecule in the biological sample.

[0144] In various embodiments of the assays of the invention, the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein is determined to be elevated when the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein is increased by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, by at least 1000%, by at least 1500%, by at least 2000%, by at least 2500%, by at least 3000%, by at least 4000%, or by at least 5000%, when compared with a comparator control.

[0145] In various embodiments of the assays of the invention, the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein is determined to be elevated when the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein is increased by at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 200 fold, at least 250 fold, at least 500 fold, or at least 1000 fold, when compared with a comparator control.

[0146] In various embodiments of the assays of the invention, the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein is determined to be decreased when the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein is decreased by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, by at least 1000%, by at least 1500%, by at least 2000%, by at least 2500%, by at least 3000%, by at least 4000%, or by at least 5000%, when compared with a comparator control.

[0147] In various embodiments of the assays of the invention, the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein is determined to be decreased when the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein is decreased by at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 200 fold, at least 250 fold, at least 500 fold, or at least 1000 fold, when compared with a comparator control.

[0148] In certain embodiments, other factors may be combined in an algorithm to determine diagnosis with greater accuracy. Exemplary additional factors may include one or more factors selected from the group consisting of cytogenetics, performance status, age, gender, ethnicity, family history, and contemporary diagnosis.Data Processing

[0149] After isolating cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, as described herein, the cfDNA, exRNA, protein, gDNA, inRNA or any combinationthereof, may be detected and / or analyzed by any suitable method and any suitable detection device and / or computer (e.g., computer 900). One or more target analytes in the cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, may be detected and / or analyzed. In some embodiments, the cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, may contain markers that can be used to identify a disease or disorder (e.g., disease-associated biomarkers). In some embodiments, the cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, may also be useful for as a global biomarker in which its increase concentration may be diagnostic of aberrations in the patient’s condition. Therefore, in some embodiments, the invention includes methods of diagnosing subjects based on the identification of a biomarker in cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof.

[0150] In some embodiments, a diagnosis or the presence or absence of an outcome can be determined from the detection and / or analysis results. In some embodiments, the term "outcome" as used herein can refer to the presence, absence, or amount of a biomarker in a population of cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, in the sample. In some embodiments, the term "outcome" as used herein can refer to an increase or decrease in the proportion of total biomarker in cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, in the sample. In some embodiments, the term "outcome" as used herein can refer to identification of a disease, disorder or condition associated with a biomarker or total cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, in the sample. A nonlimiting example of an outcome includes presence or absence of a disease associated with the presence or absence of a target biomarker.

[0151] As described herein, algorithms, software, processors and / or machines, for example, can be utilized to (i) process detection data pertaining to cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, and / or (ii) identify the presence or absence of an outcome.

[0152] The presence or absence of an outcome may be determined for all samples tested, or in some embodiments, the presence or absence of an outcome is determined in a subset of the samples (e.g., samples from individual subjects). An outcome may be determined for about 60, 65, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or greater than 99%, of samples analyzed in a set. A set of samples can include any suitable number of samples, and in some embodiments, a set has about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 samples, or more than 1000 samples. The set may be considered with respect to samples tested in a particular period of time, and / or at a particular location. The set may be otherwise defined by, for example, age and / or ethnicity. The set may be comprised of a sample which is subdivided into subsamples or replicates all or some of which may be tested. The set may comprise a sample from the same subject collected at two different times. An outcome may be determined about 60% or more of the time for a given sample analyzed (e.g., about 65, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or more than 99% of the time for a given sample). Analyzing a higher number of characteristics (e.g., sequence variations) that discriminate alleles can increase the percentage of outcomes determined for the samples (e.g., discriminated in a multiplex analysis). One or more fluid samples (e.g., one or more saliva samples) may be provided by a subject.

[0153] Presence or absence of an outcome can be expressed in any suitable form, and in conjunction with any suitable variable, collectively including, without limitation, ratio, deviation in ratio, frequency, distribution, probability (e.g., odds ratio, p-value), likelihood, percentage, value over a threshold, or risk factor, associated with the presence of a outcome for a subject or sample. An outcome may be provided with one or more variables, including, but not limited to, sensitivity, specificity, standard deviation, probability, ratio, coefficient of variation (CV), threshold, score, probability, confidence level, or combination of the foregoing, in certain embodiments.

[0154] One or more of ratio, sensitivity, specificity and / or confidence level may be expressed as a percentage. The percentage, independently for each variable, may be greater than about 90% (e.g., about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, or greater than 99% (e.g., about 99.5%, or greater, about 99.9% or greater, about 99.95% or greater, about 99.99% or greater)). Coefficient of variation (CV) in some embodiments is expressed as a percentage, and sometimes the percentage is about 10% or less (e.g., about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%, or less than 1% (e.g., about 0.5% or less, about 0.1% or less, about 0.05% or less, about 0.01% or less)). A probability (e.g., that a particular outcome determined by an algorithm is not due to chance) in certain embodiments is expressed as a p-value, and sometimes the p-value is about 0.05 or less (e.g., about 0.05, 0.04, 0.03, 0.02 or 0.01, or less than 0.01 (e.g., about 0.001 or less, about 0.0001 or less, about 0.00001 or less, about 0.000001 or less)).

[0155] For example, scoring or a score may refer to calculating the probability that a particular outcome is actually present or absent in a subject / sample. The value of a score may be used to determine for example the variation, difference, or ratio of amplified nucleic detectable product that may correspond to the actual outcome. For example, calculating a positive score from detectable products can lead to an identification of an outcome, which is particularly relevant to analysis of single samples.

[0156] Simulated (or simulation) data can aid data processing for example by training an algorithm or testing an algorithm. Simulated data may for instance involve hypothetical various samples of different concentrations of methylated uscfDNA and / or mncfDNA in serum, plasma, saliva and the like. Simulated data may be based on what might be expected from a real population or may be skewed to test an algorithm and / or to assign a correct classification based on a simulated data set. Simulated data also is referred to herein as "virtual" data. Simulations can be performed in most instances by a computer program. One possible step in using a simulated data set is to evaluate the confidence of the identified results, i.e. how well the selected positives / negatives match the sample and whether there are additional variations. A common approach is to calculate the probability value (p-value) which estimates the probability of a random sample having better score than the selected one. As p-value calculations can be prohibitive in certain circumstances, an empirical model may be assessed, in which it is assumed that at least one sample matches a reference sample (with or without resolved variations). Alternatively other distributions such as Poisson distribution can be used to describe the probability distribution.

[0157] An algorithm can assign a confidence value to the true positives, true negatives, false positives, and false negatives calculated. The assignment of a likelihood of the occurrence of an outcome can also be based on a certain probability model.

[0158] Simulated data often is generated in an in silico process. As used herein, the term "in silico" refers to research and experiments performed using a computer. In silico methods include, but are not limited to, molecular modeling studies, karyotyping, genetic calculations, biomolecular docking experiments, and virtual representations of molecular structures and / or processes, such as molecular interactions.

[0159] As used herein, a "data processing routine" refers to a process that can be embodied in software that determines the biological significance of acquired data (i.e., theultimate results of an assay). For example, a data processing routine can determine the amount of each nucleotide sequence species based upon the data collected. A data processing routine also may control an instrument and / or a data collection routine based upon results determined. A data processing routine and a data collection routine often are integrated and provide feedback to operate data acquisition by the instrument, and hence provide assay -based judging methods provided herein.

[0160] As used herein, software refers to computer readable program instructions that, when executed by a computer, perform computer operations. Typically, software is provided on a program product containing program instructions recorded on a computer readable medium, including, but not limited to, magnetic media including floppy disks, hard disks, and magnetic tape; and optical media including CD-ROM discs, DVD discs, magneto-optical discs, and other such media on which the program instructions can be recorded.

[0161] Different methods of predicting abnormality or normality can produce different types of results. For any given prediction, there are four possible types of outcomes: true positive, true negative, false positive or false negative. The term "true positive" as used herein refers to a subject correctly diagnosed as having a outcome. The term "false positive" as used herein refers to a subject wrongly identified as having a outcome. The term "true negative" as used herein refers to a subject correctly identified as not having a outcome. The term "false negative" as used herein refers to a subject wrongly identified as not having a outcome. Two measures of performance for any given method can be calculated based on the ratios of these occurrences: (i) a sensitivity value, the fraction of predicted positives that are correctly identified as being positives (e.g., the fraction of nucleotide sequence sets correctly identified by level comparison detection / determination as indicative of outcome, relative to all nucleotide sequence sets identified as such, correctly or incorrectly), thereby reflecting the accuracy of the results in detecting the outcome; and (ii) a specificity value, the fraction of predicted negatives correctly identified as being negative (the fraction of nucleotide sequence sets correctly identified by level comparison detection / determination as indicative of chromosomal normality, relative to all nucleotide sequence sets identified as such, correctly or incorrectly), thereby reflecting accuracy of the results in detecting the outcome.

[0162] The term "sensitivity" as used herein refers to the number of true positives divided by the number of true positives plus the number of false negatives, where sensitivity(sens) may be within the range of 0 < sens < 1 . Ideally, method embodiments herein have the number of false negatives equaling zero or close to equaling zero, so that no subject is wrongly identified as not having at least one outcome when they indeed have at least one outcome. Conversely, an assessment often is made of the ability of a prediction algorithm to classify negatives correctly, a complementary measurement to sensitivity. The term "specificity" as used herein refers to the number of true negatives divided by the number of true negatives plus the number of false positives, where sensitivity (spec) may be within the range of 0 < spec < 1. Ideally, methods embodiments herein have the number of false positives equaling zero or close to equaling zero, so that no subject wrongly identified as having at least one outcome when they do not have the outcome being assessed. Hence, a method that has sensitivity and specificity equaling one, or 100%, sometimes is selected.

[0163] One or more prediction algorithms may be used to determine significance or give meaning to the detection data collected under variable conditions that may be weighed independently of or dependently on each other. The term "variable" as used herein refers to a factor, quantity, or function of an algorithm that has a value or set of values. For example, a variable may be the design of a set of amplified nucleic acid species, the number of sets of amplified nucleic acid species, type of outcome assayed, and the like.

[0164] Any suitable type of method or prediction algorithm may be utilized to give significance to the data of the present technology within an acceptable sensitivity and / or specificity. For example, prediction algorithms such as Mann-Whitney U Test, binomial test, log odds ratio, Chi-squared test, z-test, t-test, ANOVA (analysis of variance), regression analysis, neural nets, fuzzy logic, Hidden Markov Models, multiple model state estimation, and the like may be used. One or more methods or prediction algorithms may be determined to give significance to the data having different independent and / or dependent variables of the present technology. And one or more methods or prediction algorithms may be determined not to give significance to the data having different independent and / or dependent variables of the present technology. One may design or change parameters of the different variables of methods described herein based on results of one or more prediction algorithms (e.g., number of sets analyzed, types of nucleotide species in each set).

[0165] Several algorithms may be chosen to be tested. These algorithms then can be trained with raw data. For each new raw data sample, the trained algorithms will assign aclassification to that sample (e g., trisomy or normal). Based on the classifications of the new raw data samples, the trained algorithms' performance may be assessed based on sensitivity and specificity. Finally, an algorithm with the highest sensitivity and / or specificity or combination thereof may be identified.

[0166] Provided are methods for identifying the presence or absence of an outcome that comprise: (a) providing a system, wherein the system comprises distinct software modules, and wherein the distinct software modules comprise a signal detection module, a logic processing module, and a data display organization module; (b) detecting signal information indicating the presence, absence or amount of enriched nucleic acid; (c) receiving, by the logic processing module, the signal information; (d) calling the presence or absence of an outcome by the logic processing module; and (e) organizing, by the data display organization model in response to being called by the logic processing module, a data display indicating the presence or absence of the outcome.

[0167] Provided also are methods for identifying the presence or absence of an outcome, which comprise providing signal information indicating the presence, absence or amount of enriched nucleic acid; providing a system, wherein the system comprises distinct software modules, and wherein the distinct software modules comprise a signal detection module, a logic processing module, and a data display organization module; receiving, by the logic processing module, the signal information; calling the presence or absence of an outcome by the logic processing module; and, organizing, by the data display organization model in response to being called by the logic processing module, a data display indicating the presence or absence of the outcome.

[0168] Provided also are methods for identifying the presence or absence of an outcome, which comprise providing a system, wherein the system comprises distinct software modules, and wherein the distinct software modules comprise a signal detection module, a logic processing module, and a data display organization module; receiving, by the logic processing module, signal information indicating the presence, absence or amount of enriched nucleic acid; calling the presence or absence of an outcome by the logic processing module; and, organizing, by the data display organization model in response to being called by the logic processing module, a data display indicating the presence or absence of the outcome.

[0169] By "providing signal information" is meant any manner of providing the information, including, for example, computer communication means from a local, or remote site, human data entry, or any other method of transmitting signal information. The signal information may be generated in one location and provided to another location.

[0170] By "obtaining" or "receiving" signal information is meant receiving the signal information by computer communication means from a local, or remote site, human data entry, or any other method of receiving signal information. The signal information may be generated in the same location at which it is received, or it may be generated in a different location and transmitted to the receiving location.

[0171] By "indicating" or "representing" the amount is meant that the signal information is related to, or correlates with, for example, the amount of enriched nucleic acid or presence or absence of enriched nucleic acid. The information may be, for example, the calculated data associated with the presence or absence of enriched nucleic acid as obtained, for example, after converting raw data obtained by mass spectrometry.

[0172] Also provided are computer program products, such as, for example, a computer program products comprising a computer usable medium having a computer readable program code embodied therein, the computer readable program code adapted to be executed to implement a method for identifying the presence or absence of an outcome, which comprises (a) providing a system, wherein the system comprises distinct software modules, and wherein the distinct software modules comprise a signal detection module, a logic processing module, and a data display organization module; (b) detecting signal information indicating the presence, absence or amount of enriched nucleic acid; (c) receiving, by the logic processing module, the signal information; (d) calling the presence or absence of an outcome by the logic processing module; and, organizing, by the data display organization model in response to being called by the logic processing module, a data display indicating the presence or absence of the outcome.

[0173] Also provided are computer program products, such as, for example, computer program products comprising a computer usable medium having a computer readable program code embodied therein, the computer readable program code adapted to be executed to implement a method for identifying the presence or absence of an outcome, which comprises providing a system, wherein the system comprises distinct software modules, and wherein the distinct software modules comprise a signal detection module, a logic processing module, and adata display organization module; receiving signal information indicating the presence, absence or amount of enriched nucleic acid; calling the presence or absence of an outcome by the logic processing module; and, organizing, by the data display organization model in response to being called by the logic processing module, a data display indicating the presence or absence of the outcome.

[0174] Also provided are methods identifying the presence or absence of an outcome that comprises: (a) detecting signal information, wherein the signal information indicates presence, absence, or amount of cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, or a biomarker therein; (b) transforming the signal information into identification data, wherein the identification data represents the presence or absence of the outcome, whereby the presence or absence of the outcome is identified based on the signal information; and (c) displaying the identification data.

[0175] Also provided are methods for identifying the presence or absence of an outcome that comprises: (a) providing signal information indicating the presence, absence, or amount of cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, or a biomarker therein; (b) transforming the signal information representing into identification data, wherein the identification data represents the presence or absence of the outcome, whereby the presence or absence of the outcome is identified based on the signal information; and (c) displaying the identification data.

[0176] Also provided are methods for identifying the presence or absence of an outcome that comprises: (a) receiving signal information indicating the presence, absence, or amount of a cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, or a biomarker therein; (b) transforming the signal information into identification data, wherein the identification data represents the presence or absence of the outcome, whereby the presence or absence of the outcome is identified based on the signal information; and (c) displaying the identification data.

[0177] For purposes of these, and similar embodiments, the term "signal information" indicates information readable by any electronic media, including, for example, computers that represent data derived using the present methods. For example, "signal information" can represent the amount of a biomarker of cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof. Signal information, such as in these examples, that represents physical substances may be transformed into identification data, such as a visual display that representsother physical substances, such as, for example, a cfDNA and / or exRNA profile. Identification data may be displayed in any appropriate manner, including, but not limited to, in a computer visual display, by encoding the identification data into computer readable media that may, for example, be transferred to another electronic device (e.g., electronic record), or by creating a hard copy of the display, such as a printout or physical record of information. The information may also be displayed by auditory signal or any other means of information communication. In some embodiments, the signal information may be detection data obtained using methods to detect a biomarker in cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof.

[0178] Once the signal information is detected, it may be forwarded to the logicprocessing module. The logic-processing module may "call" or "identify" the presence or absence of an outcome.

[0179] The term "identifying the presence or absence of an outcome" or "an increased risk of an outcome," as used herein refers to any method for obtaining such information, including, without limitation, obtaining the information from a laboratory file. A laboratory file can be generated by a laboratory that carried out an assay to determine the presence or absence of an outcome. The laboratory may be in the same location or different location (e g., in another country) as the personnel identifying the presence or absence of the outcome from the laboratory file. For example, the laboratory file can be generated in one location and transmitted to another location in which the information therein will be transmitted to the subject. The laboratory file may be in tangible form or electronic form (e.g., computer readable form), in certain embodiments.

[0180] The term "transmitting the presence or absence of the outcome to the subject" or any other information transmitted as used herein refers to communicating the information to the subject, or family member, guardian or designee thereof, in a suitable medium, including, without limitation, in verbal, document, or file form.

[0181] Also provided are methods for providing to a subject a medical prescription based on the presence or absence of an outcome, wherein the presence or absence of the outcome has been determined from the presence, absence, of a biomarker in cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, from a sample from the subject; and providing a medical prescription based on the presence or absence of the outcome to the subject.

[0182] The term "providing a medical prescription" refers to communicating the prescription to the subject, or family member, guardian, or designee thereof, in a suitable medium, including, without limitation, in verbal, document or fde form. The medical prescription may be for any course of action determined by, for example, a medical professional upon reviewing information related to the analysis of cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof. For example, the medical prescription may be for the subject to undergo additional testing or confirmatory testing. In yet another example, the medical prescription may be medical advice to not undergo further testing.

[0183] Also provided are files, such as, for example, a file comprising the presence or absence of outcome for a subject, wherein the presence or absence of the outcome has been determined from the presence, absence, or amount of a biomarker detected in cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, in a sample from the subject. The file may be, for example, but not limited to, a computer readable file, a paper file, or a medical record file.

[0184] Computer program products include, for example, any electronic storage medium that may be used to provide instructions to a computer, such as, for example, a removable storage device, CD-ROMS, a hard disk installed in hard disk drive, signals, magnetic tape, DVDs, optical disks, flash drives, RAM or floppy disk, and the like.

[0185] The systems discussed herein may further comprise general components of computer systems, such as, for example, network servers, laptop systems, desktop systems, handheld systems, personal digital assistants, computing kiosks, and the like. The computer system may comprise one or more input means such as a keyboard, touch screen, mouse, voice recognition or other means to allow the user to enter data into the system. The system may further comprise one or more output means such as a CRT or LCD display screen, speaker, FAX machine, impact printer, inkjet printer, black and white or color laser printer or other means of providing visual, auditory or hardcopy output of information.

[0186] The input and output means may be connected to a central processing unit which may comprise among other components, a microprocessor for executing program instructions and memory for storing program code and data. In some embodiments the methods may be implemented as a single user system located in a single geographical site. In other embodiments methods may be implemented as a multi-user system. In the case of a multi-user implementation, multiple central processing units may be connected by means of a network. The network may belocal, encompassing a single department in one portion of a building, an entire building, span multiple buildings, span a region, span an entire country or be worldwide. The network may be private, being owned and controlled by the provider, or it may be implemented as an Internet based service where the user accesses a web page to enter and retrieve information.

[0187] The various software modules associated with the implementation of the present products and methods can be suitably loaded into the computer system as desired, or the software code can be stored on a computer-readable medium such as a floppy disk, magnetic tape, or an optical disk, or the like. In an online implementation, a server and web site maintained by an organization can be configured to provide software downloads to remote users. As used herein, "module," including grammatical variations thereof, means, a self-contained functional unit which is used with a larger system. For example, a software module is a part of a program that performs a particular task. Thus, provided herein is a machine comprising one or more software modules described herein, where the machine can be, but is not limited to, a computer (e.g., server) having a storage device such as floppy disk, magnetic tape, optical disk, random access memory and / or hard disk drive, for example.

[0188] The present methods may be implemented using hardware, software or a combination thereof and may be implemented in a computer system or other processing system. An example computer system may include one or more processors. A processor can be connected to a communication bus. The computer system may include a main memory, sometimes random-access memory (RAM), and can also include a secondary memory. The secondary memory can include, for example, a hard disk drive and / or a removable storage drive, representing a floppy disk drive, a magnetic tape drive, an optical disk drive, memory card etc. The removable storage drive reads from and / or writes to a removable storage unit in a well- known manner. A removable storage unit includes, but is not limited to, a floppy disk, magnetic tape, optical disk, etc. which is read by and written to by, for example, a removable storage drive. As will be appreciated, the removable storage unit includes a computer usable storage medium having stored therein computer software and / or data.

[0189] Alternatively, secondary memory may include other similar means for allowing computer programs or other instructions to be loaded into a computer system. Such means can include, for example, a removable storage unit and an interface device. Examples of such can include a program cartridge and cartridge interface (such as that found in video game devices), aremovable memory chip (such as an EPROM, or PROM) and associated socket, and other removable storage units and interfaces which allow software and data to be transferred from the removable storage unit to a computer system.

[0190] The computer system may also include a communications interface. A communications interface allows software and data to be transferred between the computer system and external devices. Examples of communications interface can include a modem, a network interface (such as an Ethernet card), a communications port, a PCMCIA slot and card, etc. Software and data transferred via communications interface are in the form of signals, which can be electronic, electromagnetic, optical, or other signals capable of being received by communications interface. These signals are provided to communications interface via a channel. This channel carries signals and can be implemented using wire or cable, fiber optics, a phone line, a cellular phone link, an RF link and other communications channels. Thus, in one example, a communications interface may be used to receive signal information to be detected by the signal detection module.

[0191] In a related aspect, the signal information may be input by a variety of means, including but not limited to, manual input devices or direct data entry devices (DDEs). For example, manual devices may include keyboards, concept keyboards, touch sensitive screens, light pens, mouse, tracker balls, joysticks, graphic tablets, scanners, digital cameras, video digitizers and voice recognition devices. DDEs may include, for example, bar code readers, magnetic strip codes, smart cards, magnetic ink character recognition, optical character recognition, optical mark recognition, and turnaround documents. In one embodiment, an output from a gene or chip reader may serve as an input signal.Determining Effectiveness of Therapy or Prognosis

[0192] In one aspect, the level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein, in a biological fluid sample of a patient is used to monitor the effectiveness of treatment or the prognosis of disease. In some embodiments, the level cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein, in a test sample obtained from a treated patient can be compared to the level from a reference sample obtained from that patient before initiation of a treatment. Clinical monitoring of treatment typically entails that each patient serves as his or her own baselinecontrol. Tn some embodiments, test samples are obtained at multiple time points following administration of the treatment. In these embodiments, measurement of the level of one or more of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or one or more biomarker identified therein, in the test samples provides an indication of the extent and duration of in vivo effect of the treatment.

[0193] Measurement of the total level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein, may allow for the course of treatment of a disease to be monitored. The effectiveness of a treatment regimen for a disease can be monitored by detecting the level of, or a biomarker in, cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, from multiple samples obtained from a subject over time and comparing the detected level of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker identified therein to a control (e.g., a sample from the subject at an earlier time point in disease progression or treatment). For example, a first sample can be obtained before the subject receives treatment and one or more subsequent samples are taken after or during treatment of the subject. Changes in cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker therein, across the samples may provide an indication as to the effectiveness of the therapy. For example, a first sample can be obtained at a first time point to monitor a disease state and one or more subsequent samples are taken at time intervals following the initial time point. Changes in cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, or a biomarker therein, across the samples may provide an indication as to the stage or progression of a disease.

[0194] In some embodiments, the disclosure provides a method for monitoring the relative levels of cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, or a biomarker identified therein. In some embodiments, the relative level of cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, or a biomarker identified therein, can be measured over time, where the level at one timepoint is compared to the level at a second timepoint. For example, in some embodiments, the first time point is prior to a treatment and the second timepoint is following treatment.

[0195] In some embodiments, cfDNA, exRNA, protein, gDNA, inRNA levels or biomarkers can be used to identify therapeutics or drugs that are appropriate for a specific subject. For example, a test sample from the subject can be exposed to a therapeutic agent or adrug, and the level of cfDNA, exRNA, protein, gDNA, inRNA, or a biomarker identified therein, can be determined. The level of cfDNA, exRNA, protein, gDNA, inRNA, or a biomarker identified therein, can be compared to a sample derived from the subject before and after treatment or exposure to a therapeutic agent or a drug or can be compared to samples derived from one or more subjects who have shown improvements relative to a disease as a result of such treatment or exposure. Thus, in one aspect, the disclosure provides a method of assessing the efficacy of a therapy with respect to a subject comprising taking a first measurement of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, in a first sample from the subject; effecting the therapy with respect to the subject; taking a second measurement of the cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof in a second sample from the subject and comparing the first and second measurements to assess the efficacy of the therapy.

[0196] Accordingly, treatments or therapeutic regimens for use in can be selected based on the amounts of a specific biomarker in cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof or total cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof in samples obtained from the subjects and compared to a reference value. Two or more treatments or therapeutic regimens can be evaluated in parallel to determine which treatment or therapeutic regimen would be the most efficacious for use in a subject to delay onset, or slow progression of a disease. In various embodiments, a recommendation is made on whether to initiate or continue treatment of a disease.

[0197] A prognosis may be expressed as the amount of time a patient can be expected to survive. Alternatively, a prognosis may refer to the likelihood that the disease goes into remission or to the amount of time the disease can be expected to remain in remission. Prognosis can be expressed in various ways; for example, prognosis can be expressed as a percent chance that a patient will survive after one year, five years, ten years, or the like. Alternatively, prognosis may be expressed as the number of years, on average, that a patient can expect to survive as a result of a condition or disease. The prognosis of a patient may be considered as an expression of relativism, with many factors affecting the ultimate outcome. For example, for patients with certain conditions, prognosis can be appropriately expressed as the likelihood that a condition may be treatable or curable, or the likelihood that a disease will go into remission, whereas for patients with more severe conditions, prognosis may be more appropriately expressed as likelihood of survival for a specified period of time. Additionally, a change in aclinical factor from a baseline level may impact a patient's prognosis, and the degree of change in level of the clinical factor may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations and determining a confidence interval and / or a p value.

[0198] Multiple determinations of cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof can be made, and a temporal change in cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, can be used to determine a prognosis. For example, comparative measurements are made of the cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, in a patient at multiple time points, and a comparison of the cfDNA, exRNA, protein, gDNA, inRNA, or any combination thereof, at two or more time points may be indicative of a particular prognosis.

[0199] In certain embodiments, other prognostic factors may be combined in the algorithm to determine prognosis with greater accuracy. Exemplary additional prognostic factors may include one or more prognostic factors selected from the group consisting of cytogenetics, performance status, age, gender, and contemporary diagnosis.Treatments

[0200] In one aspect, the disclosure provides a method of diagnosing, treating, or preventing a disease or disorder associated with a biomarker identified from analysis of cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, or a general increase or decrease of total cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof. In some embodiments, the method comprises administering to the subject an effective amount of a pharmaceutical agent for the treatment of a disease or disorder identified as being associated with a biomarker identified from analysis of cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof, or a general increase or decrease of total cfDNA, exRNA, protein, gDNA, inRNA or any combination thereof.Guanidinium Isothiocyanate

[0201] In some embodiments, the invention includes compositions comprising guanidine thiocyanate or guanidinium isothiocyanate (GIT or GITC), or a solution comprising GIT orGITC, and methods of use thereof to increase the stability of nucleic acid molecules, including but not limited to cell free DNA, extracellular RNA, at ambient temperature. In some embodiments, the invention comprises methods of stabilizing nucleic acid molecules by contacting a solution comprising the nucleic acid molecules with GIT, GITC or a solution comprising GIT or GITC. In some embodiments, the GIT or GITC can be added, for example, at concentrations of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% GIT or GITC. GIT or GITC can be added, for example, at concentrations of 25-35%, 20-40%, 15-45%, 10- 50%, 5-50%. In some embodiments, the GIT or GITC or a buffer solution comprising GIT or GITC is added at a 1 : 1 ratio with the sample comprising one or more nucleic acid molecule. The stabilized nucleic acids of the present invention can be stored at room temperature for at least 1 week, 2 weeks, 5 weeks, 10 weeks, or 25 weeks or more.

[0202] In some embodiments, a solution containing GIT or GITC can be included in a kit or device which includes a step involving nucleic acid molecules. In some embodiments, a solution containing GIT or GITC can be included in a kit or device for isolation of nucleic acid molecules as a buffer for stabilization and / or storage of the isolated nucleic acid molecules. In some embodiments, a solution containing GIT or GITC can be included in a kit or device for purification of nucleic acid molecules as a buffer for stabilization and / or storage of the purified nucleic acid molecules. In some embodiments, a solution containing GIT or GITC can be included in a kit or device for synthesis of nucleic acid molecules as a buffer for stabilization and / or storage of template nucleic acid molecules, synthesized nucleic acid molecules, or a combination thereof.EXPERIMENTAL EXAMPLES

[0203] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0204] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.Development of iSCPSS device with Guanidine thiocyanate as the preservative for various salivary nucleic acid analytes

[0205] Saliva is the most accessible biofluid in the field of Liquid Biopsy. In today’s date, due to time limitations, manpower consuming procedures and proper storage facilities, most of the clinicians refrain from going through the hassle of collecting a saliva sample. Moreover, the stability of the various nucleic analytes in saliva from the phase of collection till point of analysis other than in central facilities has been a concerning issue. Therefore, saliva, a readily accessible and non-invasively acquired biofluid is still not sought after as it lacks rigorous and standardized collection protocols, or preservatives that stabilize the salivary analytes at ambient temperature.

[0206] An all-in-one integrated device that is hassle free and suited for saliva Collection, Filtration, Processing, Stabilization, and Storage (iSCPSS) of salivary analytes for extended time periods at ambient temperature while maintaining the biological stability of the analytes is most crucial. The disclosed device and method, in some embodiments, is related to the design concept of a saliva collecting device that is able to collect and facilitate filtration of salivary components, including debris and remaining important analytes like extracellular proteins (Chiang, et al., 2015, Biotechniques 58:69-762), and nucleic acids, including cell-free DNA (Cheng et al., 2022, iScience, 25: 104554). This device further collects various analytes in a compartmentalized manner for better stabilization, storage and downstream analysis.

[0207] The invention further relates, in part, to the use of Guanidine thiocyanate (GIT or GITC) as a preservative when added to the saliva sample at 1 : 1 ratio. It was discovered that GIT stabilizes salivary analytes including cell free DNA (cfDNA), genomic DNA (gDNA) and RNA over a longer period (approximately 7 days) at ambient temperature without degradation. To date, there has been no specific preservative reported to preserve the salivary nucleic acids at ambient temperature for longer periods.

[0208] Thus, the present invention includes a collector assembly that assists in collecting samples and further compositions and methods that stabilize salivary nucleic acids (cfDNA, gDNA, RNA) with GIT at ambient temperatures. The disclosed invention provides a novel device and method for collecting and preserving saliva — opening new avenues for current liquid biopsy approaches and paving innovative pathways towards biomarker discovery and disease monitoring. In some embodiments, the disclosed iSCPSS (Integrated Saliva device for Collection, Processing, Stabilization and Storage of salivary analytes) device comprises GIT as a preservative, presenting a novel solution for a universal problem.

[0209] A universal collection device is disclosed, and is referred to herein as an iSCPSS device (integrated saliva collection, processing, stabilization and storage) for saliva collection. In some embodiments, the iSCPSS device comprises preservatives that may be added to the collected samples for stability of the salivary nucleic acids (cfDNA, gDNA and RNA) when stored at ambient temperature over extended storage time.

[0210] A method of using the disclosed iSCPSS device is provided herein. In some embodiments, the method includes separation of cell free DNA (cfDNA), extracellular RNA and protein by compressing the sample in the absorbent pad through a three-way splitting unit with filter into three collection tubes.

[0211] Guanidine thiocyanate (GIT) which is known to shelter DNA and RNA with its chaotropic action and various other roles, has never been represented as a salivary nucleic acid preservative. The role of GIT was investigated with the disclosed device, and it was discovered that the addition of GIT to saliva samples preserves the salivary analytes (cfDNA and ex-RNA) for up to 7 days at ambient temperature with no degradation. Therefore, out of the three analytes, cfDNA and extracellular RNA are stabilized independently using this stabilizing solution (which is in some examples a GIT 1 : 1 ratio with Saliva sample). For protein 20% v / v ethanol is still preferred. In the next step, the absorbent pad in the collection tube is suspended with Guanidine thiocyanate lysis buffer (GITC: 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% (w / v) sarcosyl (N-lauroylsarcosine), and 0.1 M 2-mercaptoethanol) for stabilizing genomic DNA (gDNA) and intracellular RNA inside the compression tube. All the stabilized analytes using this collector assembly; cfDNA, gDNA, RNA, and protein may either be used immediately or stored for long periods of time pending analysis.

[0212] Saliva is an ideal component for “OMIC” analysis as its most accessible biofluid for detection, surveillance and monitoring a disease status. The salivary “omics” methodologies currently identified as “Salivaomics”. Saliva being noninvasive, can be collected by a nonprofessional, even by patient at the point of care, making it relatively cost-effective. In the recent era of SARS-CoV-2 outbreak, a simple and non-invasive sampling with a quick and low-cost approach was in demand and saliva emerged as the biofluid of choice. Alas, literature review suggests very few studies which have assessed the impact of sampling devices and procedures on the analytes concentration and recovery. There is a lack of standardized sampling procedures too. The analytes present in saliva can be a powerful alternative to blood for clinical applications, but standardization still remains a far cry. Though multiple commercially available kits for saliva collection like Oragene / Norgen / etc have claimed for salivary nucleic acid stability at room temperature but validation is still awaited.

[0213] The first step was to develop an integrated device platform, iSCPSS for collection, processing, stabilization, and storage of saliva. An existing collector assembly was modified and re-designed to provide a user-friendly integrated point-of-care collection device that can be handled by non-professionals if necessary.

[0214] The Universal Collector device disclosed herein is a novel solution for a universal problem. The disclosed concept for an exemplary universal collector is referred to in some examples as an integrated device for collection, processing, stabilization and storage (iSCPSS). The iSCPSS is particularly suited for Collection, Processing, Stabilization, and Storage of Salivary analytes for extended time periods while maintaining the biological activity of the salivary analytes.

[0215] In some examples, the invention relates to a method of using the disclosed universal collection device. In some embodiments, the method comprises any of the steps of: saliva specimen is collected for cell free DNA (cfDNA), extracellular RNA, protein, or any combination thereof, by compressing the sample in the absorbent pad through a small splitting unit into three standard collection tubes. Each of the samples collected is stabilized independently using specific stabilizing solutions. Later the absorbent pad in the collection tube is suspended in lysis buffer for stabilizing genomic DNA (gDNA) and intracellular RNA and remains inside the compression tube. All the stabilized analytes using this collector assembly,(e g., gDNA, intracellular RNA, extra cellular RNA, cfDNA and protein) may be used immediately or stored for long periods of time pending analysis.

[0216] Exemplary features of the iSCPSS device include: collection of saliva sample from a subject using the assembly of the integrated device; fdtration and channelizing the saliva specimen to produce a fdtered sample that is free of cells; and collection of the fdtered sample in at least three collection receptacles.

[0217] Fig. 1 A & Fig. IB depict an exemplary collector device according to aspects of the present invention. Fig. 2 is a diagram depicting an exemplary working principle for the collector device of the present invention. The disclosed design concept comprises 1) Introduction of a three way splitter with filter; 2) Pre-Addition of a stabilizing compound (e.g., GITC) for stabilization of cfDNA in collection tube 1; 3) Pre-Addition of a stabilizing compound (e.g., GITC) for stabilization of extracellular RNA in collection tube 2; 4) Pre-Addition of 20% ethanol w / v for Protein stabilization in collection tube 3. In some embodiments, the system further comprises 5) an additional tube (tube 4) comprising genomic lysis buffer (e.g., GITC lysis buffer) for stabilization of gDNA and intracellular RNA, wherein the lysis buffer can be added into the compression tube from tube 4 after collection of analytes (cfDNA, RNA and protein) for stabilization and subsequent collection of gDNA, intracellular RNA or the combination thereof.

[0218] Disclosed with the universal collector device is a three-way splitter with filter, each channel connected to one of three collection tubes. In some embodiments, the device comprises a pre-addition of stabilizing compound (e.g., GITC) for stabilization of cfDNA in collection tube 1. In some embodiments, the device comprises the pre-addition of stabilizing compound (e.g. GITC) for stabilization of extracellular RNA in collection tube 2. In some embodiments, the device comprises the pre-addition of 20% ethanol w / v for Protein stabilization in collection tube 3.

[0219] Fig. 4A & Fig. 4B depict an exemplary method of using the universal collector. The collector based on an existing design assembly with modifications is used to collect the saliva sample. Saliva sample is collected using the absorbent pad in the collector assembly as per instructions. Following sample collection, the absorbent pad is inserted into the compression tube. The plunger handle attached to the absorbent pad end is firmly pushed downwards into the compression tube to transfer the collected sample from the absorbent pad through the three-waysplitter with filters into collection tubes containing pre-specified stabilizing solutions. In some embodiments, tubes 1, 2 and 3 are detached from the compression tube, along with the splitting unit. The split sample in tubes 1, 2 and 3 can then be further processed. In some embodiments, the end of the compression tube end is sealed (e.g., using paraffin film), the absorbent pad is removed from the assembly, and a lysis buffer (e.g., SDS or GITC lysis buffer) is added to the sealed compression tube. The absorbent pad is then reinserted into the compression tube so that it is suspended into the lysis buffer in the tube. Following lysis of the sample remaining on the absorbent pad, the sealed end of the compression tube is opened and the plunger handle attached to the absorbent pad end is firmly pushed downwards into the compression tube to transfer the lysed sample products into tube 4 or a new collection tube for further processing. In some embodiments, gDNA and intracellular RNA analysis are performed on the lysed sample products as desired.

[0220] The function of the Universal Collector Device is described herein. The collector is used to filter, separate and collect multiple types of analytes from a saliva sample. The saliva sample is collected using the absorbent pad in the collector assembly as per the disclosed methods. The absorbent pad is removed from the mouth and inserted into the compression tube. It is then pushed to let the sample be collected in the centrifuge tubes. The plunger handle attached to the absorbent pad end is firmly pushed downwards into the compression tube to transfer saliva from the absorbent pad into collection tubes 1, 2 and 3 with pre-loaded stabilizing solutions through the three-way splitting unit with filtration. In some embodiments, the method includes inverting the collector device 2-3 times to mix the sample.

[0221] After the tubes are detached along with the splitting unit, the compression tube end is sealed as per instructions with a paraffin film. The absorbent pad is removed from the universal collector device. In some embodiments, a genomic lysis buffer is added to the sealed compression tube with paraffin film. The absorbent pad suspended into the lysis buffer in the tube and inverted 2-3 times to let the pad absorb carefully. The sample is collected after 1 hr by removing the paraffin seal from the rear end of the tube. The sample is collected by compressing the absorbent pad, channeling the samples into a fourth collection tube for gDNA and intracellular RNA analysis as desired.

[0222] Described herein are the methods and results revealing guanidine thiocyanate (GIT) as a preservative for stabilizing nucleic acids cfDNA & ex-RNA in saliva. The firstcollection tube 1 was pre-loaded with Guanidine stabilizing solution (GIT) at ratio of 1 : 1 with saliva sample to produce a GIT containing fdtered sample comprising a cell free DNA sample, the second collection tube 2 was pre-loaded with Guanidine stabilizing solution (GIT) at ratio of 1 : 1 with saliva sample to produce a GIT containing fdtered sample comprising an extracellular RNA sample, the third collection tube 3 was pre-loaded 20% w / v ethanol to produce an ethanol- containing fdtered sample comprising a protein sample, wherein the protein sample and the nucleic acids (e.g. cfDNA and ex-RNA samples) are stabilized for at least 7 days when stored at ambient temperature.

[0223] An analysis was performed on the GIT containing fdtered sample collected in the first tube 1 and second tube 2 collecting devices comprising of a cfDNA / ex-RNA and third tube 3 for protein analysis on the 20% w / v ethanol containing fdtered sample.

[0224] The fdtered sample collected for cell free DNA (cfDNA) / extracellular RNA (exRNA) was analyzed using the integrated assembly, fdtered saliva sample was collected over different time points (0, 1, 3, 7 days) and stored at ambient temperature in the preloaded collection tube 1 & 2 with GIT as stabilizer in 1 : 1 ratio with saliva sample.

[0225] Extraction of cfDNA from the saliva sample was done using QIAamp Circulating Nucleic Acid Kit following the Purification of Circulating microRNA protocol following the manufacturer instructions.

[0226] Quantification of the DNA concentration and purity was done by measuring the absorbance at 260 nm and 280 nm using a Nanodrop(spectrophotometer) (shown in Fig. 6A) and Qubit (Fluorometer) (shown in Fig. 6B). For ddPCR analysis, endogenous target sequence was identified (SCFT / Chr6 target) (shown in Table 1). The gene rich regions were evaluated from NGS data available of saliva using IGV software. Primary gene rich areas were located and sequences without any errors within 80-110 bp were finalized. Primers were designed. cfDNA stability in saliva was evaluated by using target specific sequence primers from a gene rich enriched area using ddPCR (shown in Fig. 6C). Further analysis was done in comparison to standard operating protocol for saliva (SOP) with the collector over 0 & 1 day with and without the GIT stabilizer at ambient temperature (shown in Fig. 7A, Fig. 7B & Fig. 7C).

[0227] Fig. 7A & Fig. 7B show the results of the DNA concentration measured by NanoDrop and Qubit with sample collected with SOP versus results from the disclose device, with or without the GIT stabilizer over 0, 1 days at ambient temperature. Fig. 7C shows theresults of the comparison of absolute SCFT / Chr 6 target copy numbers determined by ddPCR Evagreen mix for saliva sample filtered from the SOP versus the results of the disclosed device with or without GIT over 0, 1 days at ambient temperature.Table 1: Identification of endogenous targets for cell free DNA (cfDNA)_

[0228] Extraction of ex-RNA from the saliva sample was done using miRNeasy Kit following the Purification of Circulating microRNA protocol following the manufacturer instructions. Following, an RNA concentration assessment was done.

[0229] Fig. 8A & Fig. 8B show the results of tape station analysis of RNA concentration and percentage of saliva sample collected through the disclosed device with GIT stabilizer (1 : 1 ratio) over 0, 1, 3, 7 days at ambient temperature.

[0230] Another embodiment for analysis of filtered sample collected for protein using the disclosed device is described. Filtered saliva sample was collected over different time points (0, 1, 3, 7 days) and stored at ambient temperature in the 20% w / v ethanol preloaded collection tube 3. Protein analysis on the 20% w / v ethanol containing filtered sample was done for total protein using Bradford Assay for quantitative assessment (shown in Fig. 9). Fig. 9 shows the results for a total protein estimation using Bradford Assay over 0, 1, 3, 7 days collected in the proposed integrated device with 20% w / v ethanol stabilizer and stored at ambient temperature.

[0231] Described herein are the methods and results revealing Guanidine thiocyanate lysis buffer (GITC) as a preservative for gDNA & intracellular RNA in saliva. In some embodiments, the device and method further comprise the re-suspension of the absorbent pad in GITC solution and channelizing the suspended specimen to produce a sample after 1 hr incubation in the compression tube and collection of the suspended GITC sample in a fourth receptacle from the resuspended GITC lysis buffer for gDNA stability assessment. An analysis was performed on the filtered sample collected in a fourth receptacle for gDNA analysis or intracellular RNA analysis on the GITC containing filtered sample.

[0232] In some embodiments, the GITC stabilizing compound comprises Guanidinium thiocyanate (GIT) powder dissolved in 0. IM Tris HCL (pH 7.6) in a 65 degree water bath to form a stabilizing solution. In some embodiments, an amount of 0.1M Tris HCL (pH 7.6) is added to the solution to achieve the desired volume. In some embodiments, 0.5M EDTA and Triton X 100 are mixed and added to the solution make the final stabilizing solution.

[0233] Analysis of filtered sample collected using the disclosed device was performed for genomic DNA (gDNA). The resuspended GITC lysis buffer for gDNA stability assessment was done over different time points (0, 1, 3, 7 days) and at ambient temperature. Extraction of DNA from the saliva samples using QIAamp Circulating Nucleic Acid Kit following the Purification of Circulating microRNA protocol.

[0234] Quantification of the DNA concentration and purity done by measuring the absorbance at 260 nm and 280 nm using a Nanodrop(spectrophotometer). Genomic DNA integrity (DIN) in saliva evaluated using Tapestation.Table 2: gDNA concentration measured using Nanodrop (Spectrophotometer) with GIT buffer collected in the proposed integrated device for immediate processing to assess recovery.

[0235] Fig. 10A, Fig. 10B, & Fig. 10C depict a schematic representation of the experimental design of gDNA integrity (DIN) collected in falcon tube at ambient temperature and 4 °C using SDS buffer showing degradation over 0, 1 day (Fig. 10A), GITC buffer showing stability over 0 / 1 / 2 / 3 / 7 days with no significant difference in ambient versus 4 degree storage conditions (Fig. 10B), and using GITC buffer collected in the disclosed device over 0, 1, 3 days stored at ambient temperature showing no degradation (Fig. 10C).

[0236] Data generated using the saliva samples collected through the integrated device with GIT / GITC added to the samples at different time points clearly suggests that GIT acts as a preservative for the salivary nucleic acids over a period of 7 days at ambient temperature.

[0237] Saliva, a readily accessible and non-invasively acquired biofluid is still not sought after as it lacks rigorous and standardized collection protocols, or preservatives that stabilize thesalivary analytes at ambient temperature. The invention iSCPSS enables saliva collection for all salivary analytes with a stable preservative at ambient temperature over a longer period. This shall change the near future scenario and advocate for non-invasive biofluid collection and facilitate non-invasive modalities for understanding, monitoring disease process and accordingly regulate therapeutic interventions promptly and competently.

[0238] The disclosures of each and every patent, patent application, and publication cited herein are hereby each incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS1. A collector device, comprising: a syringe fluidly connected to a splitter, the splitter fluidly connected to one or more receptacles, the assembly of which defines a proximal end and distal end for the collector device; wherein the syringe comprises a tube forming a lumen with an absorbent pad disposed within the lumen of the tube, and a plunger with a handle at least partially disposed with the lumen of the tube; the splitter comprises a housing forming an interior volume and having a proximal inlet and one or more distal outlets in fluid connection with the interior volume; and the one or more receptacles each comprise a proximal opening and one or more walls defining an interior volume therein.

2. The device of claim 1, wherein each receptacle of one or more receptacles comprises a stabilizing compound.

3. The device of claim 2, wherein the one or more distal outlets comprise at least a first, second and third outlet, and the one or more receptacles comprise at least a first, second and third receptacle, each receptacle correlated with a respective outlet.

4. The device of claim 3, wherein the first receptacle comprises a stabilizing compound for stabilization of cell-free DNA (cfDNA), the second receptacle comprises a stabilizing compound for stabilization of extracellular RNA, and the third container comprises a stabilizing compound for stabilization of protein.

5. The device of claim 4, wherein the stabilizing compound for stabilizing of cfDNA and the compound for stabilizing of extracellular RNA comprises guanidine thiocyanate (GIT) or guanidinium isothiocyanate (GITC), and further wherein the stabilizing compound for stabilizing of protein comprises ethanol.

6. The device of claim 5, wherein the splitter further comprises a filter disposed within the interior volume.

7. The device of claim 6, wherein each outlet of the one or more distal outlets comprises a circular flange extending out in a distal direction from the housing, wherein each receptacle is configured to sealingly and releasably attach to the flange of the opening with a compression fit.

8. The device of claim 7, wherein the plunger comprises a sample volume adequacy indicator positioned at the distal end of the plunger fluidly connected to the lumen of the syringe tube.

9. The device of claim 8, wherein the housing has a length ranging between 1 cm and 10 cm, a width ranging between 1 cm and 10 cm, and a height ranging between 1 cm and 10 cm.

10. The device of claim 9, wherein each flange has a height ranging between 1 mm and 2 cm, and a diameter ranging between 1 mm and 2 cm.

11. The device of claim 10, wherein the interior volume of the housing has a volume ranging between 10 mm3and 1000 mm3.

12. A saliva testing kit comprising a device of any one of claims 1 to 11.

13. The saliva testing kit of claim 12 further comprising at least one additional receptacle comprising a lysis buffer.

14. The saliva testing kit of claim 12, wherein the lysis buffer comprises SDS or GITC lysis buffer.

15. A method of using a collector device, comprising the steps of:providing the device of any one of claims 1 to 11 or the saliva testing kit of any one of claims 12 to 14; loading biological fluid from a subject captured on the fdter into the lumen of the syringe tube; depressing the plunger of the syringe to push the liquid from the tube of the syringe into the interior volume of the splitter housing; and collecting one or more analytes in the one or more receptacles.

16. The method of claim 15, wherein the sample is a saliva sample.

17. A method of detecting a biomarker in a biological fluid sample of a subject, the method comprising, providing the device any one of claims 1 to 11 or the saliva testing kit of any one of claims 12 to 14; loading biological fluid from a subject captured on the filter into the lumen of the syringe tube; depressing the plunger of the syringe to push the liquid from the tube of the syringe into the interior volume of the splitter housing; collecting one or more analytes in the one or more receptacles, and analyzing the one or more analytes to detect a biomarker of interest in the sample.

18. The method of claim 17, wherein the sample is a saliva sample.

19. The method of claim 17, wherein the method comprises collecting cell-free DNA in the first receptacle, extracellular RNA in the second receptacle and protein in the third receptacle.

20. The method of claim 17, further comprising the step of applying a lysis buffer to the lumen of the device following the collecting step, and collecting the lysed cellular products from the compression tube containing genomic DNA (gDNA) and intracellular RNA for analysis in a separate tube.

21. The method of claim 17, wherein the method comprises analyzing cfDNA, exRNA or proteins or any combination thereof.

22. The method of claim 20, wherein the method comprises analyzing cfDNA, exRNA, proteins, gDNA or intracellular RNA, or any combination thereof.

23. A method of diagnosing a disease or disorder in a subject, the method comprising, providing the device of any one of claims 1 to 11 or the saliva testing kit of any one of claims 12 to 14; loading biological fluid from a subject captured on the filter into the lumen of the syringe tube; depressing the plunger of the syringe to push the liquid from the tube of the syringe into the interior volume of the splitter housing; collecting one or more analytes in the one or more receptacles; analyzing the one or more analytes to detect a biomarker of interest in the sample, wherein the biomarker is a disease-associated biomarker; and diagnosing the subject as having or at risk of developing the disease associated with the biomarker upon detection of the presence or level of the biomarker as compared to a comparator control.

24. The method of claim 23, wherein the sample is a saliva sample.

25. The method of claim 23, wherein the method comprises collecting cell-free DNA in the first receptacle, extracellular RNA in the second receptacle and protein in the third receptacle.

26. The method of claim 23, further comprising the step of applying a lysis buffer to the compression tube of the device following the collecting step, and collecting the lysed cellular products from the compression tube containing genomic DNA (gDNA) and intracellular RNA for analysis in a separate tube.

27. The method of claim 23, wherein the method comprises analyzing cfDNA, exRNA or proteins or any combination thereof.

28. The method of claim 26, wherein the method comprises analyzing cfDNA, exRNA, proteins, gDNA or intracellular RNA, or any combination thereof.

29. A composition for stabilization of nucleic acid molecules, the composition comprising guanidine thiocyanate or guanidinium thiocyanate.

30. A method of stabilizing nucleic acid molecules, the method comprising contacting a sample comprising one or more nucleic acid molecule with guanidine thiocyanate.

31. The method of claim 30, wherein the one or more nucleic acid molecule comprises cfDNA or exRNA.

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