Device, solution, and method for sample collection related application, analysis, and diagnosis

Abstract

To provide a solution for preserving a cell and / or an extracellular component in a naturally expressed body fluid (e.g., saliva, sputum, urine) for further downstream analysis and / or for diagnosis of a disease state.SOLUTION: A solution may be hypertonic relative to blood. Technique is described for concentrating cells from a sample of naturally expressed body fluid and / or for performing analysis so as to diagnose disease conditions such as cancer, obesity, infection, autism, Alzheimer's disease, blood disorders, cardiovascular disease or disorder, diabetes, vulnerable plaque, LTBI, HIV infection, COPD, ACOS.SELECTED DRAWING: None

Description

Technical Field

[0001] (Field of Disclosure) The present disclosure relates to devices, solutions, and methods for collecting samples of harmful and / or toxic substances, particularly body fluids including naturally occurring body fluids (e.g., oral fluid, urine) or other substances. Additionally, or alternatively, the present disclosure generally relates to the preservation of cells and / or extracellular components from such liquids. Additionally, or alternatively, the present disclosure generally relates to functional genomics. Additionally, or alternatively, the present disclosure relates to the isolation and / or preservation of cells and / or extracellular components from such body fluids for research in, for example, any of diagnosis, genetics, functional genomics, epigenetic research, and biomarker discovery. Additionally, or alternatively, the present disclosure generally relates to the detection of diseases or infections by using naturally occurring body fluids.

Background Art

[0002] Personalized medicine is the customization of treatment to an individual as opposed to one treatment model for all. Personalized medicine involves categorizing patients based on their condition and designing an optimal medical solution only for that category. The advancement of personalized medicine depends on the discovery, validation, and commercialization of biomarkers for stratifying populations for treatment and for the development of diagnostics for screening and early disease detection.

[0003] Epigenetic research is at the forefront of medical research and is involved in the etiology of several physical and mental disorders, including cancer, obesity, diabetes, schizophrenia, and Alzheimer's disease (Alika et al., 2010; Grant et al., 2010; McGowen et al., 2009; McGowen and Szyf, 2010; Plazas-Mayorca and Vrana, 2011; and Portela and Esteller, 2010). In addition, epigenetics may be particularly promising in many scientific and medical fields, including but not limited to cancer, diabetes, drug integration, drug efficacy, childhood aggression, suicidal behavior, aging, inflammation, pain, obesity, schizophrenia, and other mental disorders (Abdolmaleky et al., 2005; Costa et al., 2003; Iwamoto & Kato, 2009; Kuratomi et al., 2007; McGowen & Kato, 2007; McGowen and Szyf, 2010; Peedicayil, 2007; Petronis et al., 1999; McGowen and Szyf, 2010; Plazas-Mayorca and Vrana, 2011; and Zawia et al., 2009).

[0004] The main challenges in the field include the identification of appropriate source samples for home sample collection that are sufficient for large-scale epigenetics research, including whole genome analysis studies. Epigenetics may be a key to understanding the mechanisms of gene-environment interactions, as evidence is increasingly suggesting that epigenetic mechanisms may provide a molecular memory of environmental experiences (Ho, 2010; Kappeler and Meaney, 2010; McGowen et al., 2009; McGowen and Szyf, 2010; Portela and Esteller, 2010; Richards, 2008; Russo et al., 2010; Tsai et al., 2010; and Vlaanderen et al., 2010). Preliminary data from some humans suggest that distinct methylation patterns in peripheral blood cells are associated with social behaviors, including childhood aggression, suicidal behavior, and aging (Kappeler and Meaney, 2010; McGowen et al., 2009; McGowen and Szyf, 2010; Portela and Esteller, 2010; Russo et al., 2010; Tierling et al., 2010; Tsai et al., 2010; and Zhang et al., 2011).

[0005] Due to the heterogeneous nature of human diseases, particularly mental diseases, and the complex interactions of contributing etiological factors, research requires large sample sizes to be reliable and provide significant effects. However, current research options for sample collection for epigenetic studies do not meet this essential requirement of "large sample size." The need for large sample sizes in research also applies to studying these interactions because human-environment interactions are also of a very complex nature involving many contributing factors, and significant effects are required to occur in order to study these interactions. The ability to conduct large-scale "population-scale" (with at least hundreds to thousands of subject samples) epigenetic studies can facilitate the completion of longitudinal studies that introduce new understandings of human-environment interactions and promote the development of epigenetic-based screening diagnoses essential for the advancement of modern medicine. This epigenetic research can lead to an understanding of how the environment affects our epigenome and how this can relate to an individual's health outcomes, and further lead to the development of diagnostic methods for these health disparities, including but not limited to preventive interventions and diagnoses for individuals considered to be at high risk.

[0006] Some epigenetic studies that attempt to quantify environmental and other complex interactions within human populations use blood as the original sample for experiments. Blood can limit the ability of researchers to conduct large-population-scale studies because of the following. 1. Generally requires medical monitoring 2. Involves invasive techniques for collection 3. Has a negative impression that restricts participation 4. Is expensive to collect and transport

[0007] Naturally occurring body fluids, such as saliva and urine, can be suitable additional or alternative original samples for in-home sample collection because of the following. 1. Do not require invasive techniques 2. Do not have the same negative impression as blood 3. Do not require expert monitoring 4. Can be inexpensively collected

[0008] In addition, at least saliva has been shown to contain white blood cells (Dos-Santos et al., 2009). The use of body fluids such as oral fluid and urine can enable large-scale "population-scale" epigenetic studies. In addition, home sample collection of oral fluid or urine can greatly increase the number of participants and make samples more easily shipped / transported by subjects from anywhere in the world, thus enabling a much wider range of research options. For example, the ability to more easily ship samples from anywhere in the world can be particularly useful when the samples are from countries without laboratory infrastructure.

[0009] An organism's genome is a fixed sequence that contains its genetic information and is identical in all cells of the organism. In contrast, an organism's epigenome varies between cell types and changes over the lifetime of the organism. Thus, epigenetic studies may include a single cell type as a sample material source to control for these differences (Johnson and Tricker, 2010; Lister et al., 2009; and Rangwala et al., 2006). For example, human saliva contains multiple cell types, including epithelial cells, cells normally found in the blood (i.e., T cells and B cells), bacteria, and debris (Dos-Santos et al., 2009 and Viet and Schmidt, 2008). The cells in saliva that are most important for epigenetic profiling are those derived from the bloodstream because these cells convey epigenetic information from all over the body (Kappeler and Meaney, 2010; McGowen and Szyf, 2010; McGowen and Szyf, 2010; Righini et al, 2007; Rosas et al., 2011; Vlaanderen et al., 2010 and Zhang et al., 2011).

[0010] In addition, cells in saliva that make up the overwhelming majority of cells (Dos-Santos et al., 2009), such as epithelial cells which are not found in blood, have the ability to "hide" the epigenetic effects seen in T cells (cells that originated in blood) by weakening the influence of the minority of cells (Dos Santos et al., 2009, Lister et al., 2009; and Tierling et al., 2010), so it may not be practical to use the entire saliva DNA. To address these concerns, AboGen has developed a method for separating and extracting different cell types found in body fluids such as saliva by utilizing cell-specific markers and (e.g., magnetic) isolation techniques. This method uses a practical amount of body fluid such as saliva to yield concentrated cells and can be used for downstream biological applications, including large-scale functional genomic studies (e.g., epigenomic studies). For example, saliva sample processing technology enables the collected samples to be processed into a single cell type and have their epigenomes profiled.

[0011] Furthermore, saliva (and other body fluids) can present challenges for cell isolation as a source sample for blood cells in downstream experiments for reasons such as the following. 1. Blood is a transport fluid, while saliva is a digestive fluid that can be rich in proteases, enzymes, and secreted substances, and urine is an excretory fluid consisting of unwanted waste products. 2. Cells do not survive intact in saliva over long periods. While cells can survive in blood for weeks (or even years for some cell types), cell survival in saliva is typically less than 1 hour. The percentages of cells in saliva that survive after 15, 45, and 90 minutes have been reported to be approximately 66%, 33%, and 27% respectively. Saliva presents an environment that is not suitable for whole-cell preservation for analysis or diagnosis due to its physiological properties. Some studies have demonstrated that 95% of cells cannot survive up to 60 minutes (Baron et al., 1999). 3. Some fluids can have a wide pH range, and some of the reported pH values, such as saliva, would be lethal if the blood reached that pH (saliva is 6.2 - 7.4, urine is 4.5 - 8, and blood is 7.35 - 7.45). 4. Some fluids contain more bacteria than blood. 5. Some fluids vary among individuals and contain non - cellular substances that interfere with cell isolation. 6. Some fluids may be abundant in blood but scarce in other naturally occurring body fluids such as saliva or urine. Unlike in blood, other cell types such as epithelial cells greatly outnumber and include blood cells such as T cells. 7. Subsets of lymphocytes in some body fluids such as saliva are very different from the populations of these cell types in blood. For example, only CD4 + CD8 - T cells have been reported to be found in saliva. 8. Some fluids such as saliva are produced daily at a rate of about 0.5 - 1.5 liters per individual per day.

[0012] Therefore, there is a need for new methods to isolate rare cells (i.e., T cells) from saliva, oral fluid, and other naturally occurring body fluids.

[0013] For collecting saliva samples from large groups of people widely geographically dispersed (e.g., for functional genomics research or medical diagnostic methods), several requirements may need to be met for an optimal sample collection device. For example, it may be beneficial to have a sample collection device that securely houses a toxic preservation solution within a closed chamber. Additionally, the sample collection device may be capable of being shipped to the donor along with the securely enclosed toxic solution. The sample collection device may also enable easy and safe collection of donor specimens such as human saliva or urine without the risk of exposing the donor to the toxic solution. Further, the sample collection device may enable the donor to safely mix the toxic solution and the specimen (for specimen preservation) without the risk of exposing the donor to the toxic solution or any other hazard. The sample collection device may also enable the donor to ship the sample to the laboratory in addition to the sample collection device for generally "as is" processing after securely closing the sample collection device. Finally, the sample collection device may further enable a laboratory technician to receive the sample collection device and safely open it for processing without generally any risk of exposure to any hazard.

[0014] In addition, the purification process requires that cells maintain their antigen profiles, and epigenomic profiling requires that their epigenomes be maintained. For this purpose, cells need to be treated in a way that generally allows them to maintain these characteristics. Currently available treatments generally do not meet this need. For example, U.S. Patent Nos. 7,267,980 and 7,749,757 are understood to disclose solutions containing lysine, glycine, and formaldehyde for stabilizing cells from blood. U.S. Patent No. 6,912,932 is understood to disclose reactants that generate multiple species of formaldehyde ammonium complexes for stabilizing blood samples containing platelets when mixed together. However, such solutions mainly address the physiologically non - burdensome environment of blood and cannot address the different properties of saliva. The difficulty of preserving undamaged cells and extracellular components in saliva should not be underestimated. The solutions referenced above do not protect cells from the physiologically harsh environment of saliva and, in particular, cannot preserve a sufficient number of the relatively few rare cells already in saliva to be an effective sample medium for analysis or diagnosis in the currently proposed manner. Thus, there is a need for new solutions and methods that can preserve the antigenicity as well as the epigenome of cells in body fluids such as saliva, oral fluid, and other naturally expressed body fluids, particularly to enable at - home sample collection.

[0015] Personalized medicine is the customization of treatment to an individual as opposed to one overall treatment model. Personalized medicine involves categorizing patients based on their physical condition and exclusively designing the optimal medical solution for that category. The advancement of personalized medicine depends on the discovery, validation, and commercialization of biomarkers for stratifying populations for treatment as well as for the development of diagnostics for screening and early detection.

[0016] Epigenetic research is at the forefront of medical research and is involved in the etiology of several physical and mental diseases, including cancer, obesity, diabetes, schizophrenia, and Alzheimer's disease. In addition, epigenetics may be particularly promising in many scientific and medical fields, including, but not limited to, cancer, diabetes, drug integration, drug efficacy, childhood aggression, suicidal behavior, aging, inflammation, pain, obesity, schizophrenia, and other mental diseases.

[0017] Similar considerations to those discussed above also apply to extracellular components carried in saliva, such as proteins, viruses, and floating DNA / RNA. Such components also degrade rapidly due to the harsh environment of saliva. General blood-based preservation techniques will not be effective for saliva in practice, and for this reason, saliva has not been used as a practical alternative medium for studying such extracellular components, especially in remote in-home sample collection. For example, using current techniques, saliva must be frozen immediately to study its extracellular components. If freezing is not immediate, this can cause changes in the concentration of these factors due to the degradation of factors such as cytokines. Examples of studying extracellular factors in saliva only after freezing include the interleukin family of proteins as well as additional cytokines. International Publication Nos. WO2012 / 177656 and WO2015 / 112496 propose devices, solutions, and methods for in-home collection and preservation of saliva samples suitable for epigenetic research. The entire contents of these documents are incorporated herein by reference as if fully reproduced herein.

Prior Art Documents

Patent Documents

[0018]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Summary of the Invention

Means for Solving the Problems

[0019] (Summary of the Disclosure) The following presents a simplified summary of the present disclosure in order to provide a basic non-limiting understanding of some aspects of the present disclosure.

[0020] Some embodiments of the present disclosure are based on the principles within International Publication No. WO2012 / 177656 and / or International Publication No. WO2015 / 112496.

[0021] Some embodiments of the present disclosure are defined in the appended claims.

[0022] Some embodiments of the present disclosure provide a solution for preserving cells and / or extracellular components in a sample of a naturally occurring body fluid such as oral fluid (e.g., saliva or sputum or fluid from a lung aspirate) or urine. Optionally, the solution may be defined in combination with a device for collecting the fluid sample from a donor.

[0023] As used herein, the term "extracellular" can refer to any and / or all components that are not whole cells and that do not exist within whole cells. For example, "extracellular" can refer to the components that remain when the cells are removed. The extracellular components may include, for example, any of proteins, viruses, cell-free DNA, and / or cell-free RNA.

[0024] Some embodiments of the present disclosure provide a preservation solution that is hypertonic with respect to blood (e.g., prior to mixing with the body fluid).

[0025] While not desiring to be bound by any specific theory, the inventors understand that the tonicity (or osmolality) of saliva can be a significant factor in making saliva a harsh environment for cell survival. Saliva, and in particular saliva collected in a given sample, has been found to be hypotonic with respect to blood (and thus cells). Cells from blood that enter an individual's saliva are subject to destructive osmosis that draws fluid into the cells, causing the cells to swell, become damaged, and ultimately rupture. This can explain the small number of cells that have been reported to survive intact in saliva. In fact, saliva is a natural lysis solution that lyses cells, making this an unintuitive medium for obtaining a sample of viable whole cells.

[0026] By providing a hypertonic preservation solution that is hypertonic with respect to blood, this solution can neutralize the tonicity towards an isotonic environment (with respect to blood and / or cells) when mixed with a saliva sample. Such an environment can avoid or at least significantly reduce the osmotic pressure on the cells in the collected sample, thereby allowing for a significantly improved yield of preserved intact cells.

[0027] For example, it may be noted that a hypertonic non-lytic preservation solution for preserving cells in saliva represents a distinct difference from preservation solutions intended to preserve or stabilize whole cells in a blood sample. This reflects the significant difference in tonicity between saliva and blood, even if not confirmed by the teachings of the prior art that focus on preserving blood. A non-lytic blood preservative should be isotonic so as not to change the neutral isotonic environment of the blood and to avoid increasing the osmotic pressure on the cells. If a hypertonic preservation solution is mixed with blood, this will change the tonicity of the blood and pose a risk of damage to the cells. Similarly, conventional isotonic blood preservatives used to preserve cells in a saliva sample are not sufficient to protect the cells against the hypotonic environment of saliva and will result in an insufficient yield of preserved cells, as previously described as a deficiency in the teachings of the prior art.

[0028] In some embodiments, the naturally occurring body fluid may be of a type that is naturally hypotonic to cells, for example, but is not limited to oral fluids (including, for example, saliva), and in particular, is not limited to saliva itself.

[0029] As used herein, a fluid can be considered isotonic to blood when the fluid generally has an osmolality within the range including 240 to 320 milliosmoles per liter (mOsm / L). The actual osmolality of blood is generally within the range including 275 to 295 mOsm / L, optionally about 290 mOsm / L. Thus, the term "isotonic" as used herein includes the limit where the difference in osmolality, and thus osmotic pressure, is not significant. A fluid can be considered hypotonic to blood when it has an osmolality less than 240 mOsm / L. A fluid can be considered hypertonic to blood when it has an osmolality greater than 320 mOsm / L.

[0030] In some embodiments disclosed herein, the osmolality of a solution (e.g., prior to mixing with a body fluid) can optionally be defined as being about or at least (either case) "n" times 240 Osm / L, where "n" is a natural number including 2 to 15. For example, "n" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13, 14, or 15.

[0031] In some embodiments disclosed herein, the osmolality of a solution (e.g., prior to mixing with a body fluid) can optionally be defined as being about or at least (either case) "n" times 275 Osm / L, where "n" is a natural number including 2 to 15. For example, "n" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13, 14, or 15.

[0032] In some embodiments disclosed herein, the osmolarity of a solution (e.g., prior to mixing with a body fluid) can optionally be defined as being about or at least (in either case) "n" times that of 290 Osm / L, where "n" is a natural number including 2 to 15. For example, "n" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13, 14, or 15.

[0033] In addition or alternatively, the aggregate salt ion osmolarity in a solution (e.g., prior to mixing with a body fluid) can optionally be about or at least (in either case) "n" times that of 290 Osm / L, where "n" is a natural number including 2 to 15. For example, "n" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0034] In some embodiments disclosed herein, the osmolarity of a solution compared to blood (e.g., prior to mixing with a body fluid) can optionally be defined as being about or at least (in either case) "n" times that of blood, where "n" is a natural number including 2 to 15. For example, "n" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0035] In addition or alternatively, the concentration of sodium chloride (NaCl) in a solution (e.g., prior to mixing with a body fluid) can optionally be about or at least (in either case) "n" times that of concentration "m", where "m" is a value in the range including 8.0 to 9.0 grams per liter (g / L), and "n" is a natural number including 2 to 15. For example, "n" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0036] In addition, or alternatively, the solution may optionally contain phosphate buffered saline (PBS) at a concentration that is about or at least (in either case) “n” times the isotonic PBS concentration of blood (e.g., prior to mixing with body fluid), where “n” is a natural number including 2 to 15. For example, “n” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0037] In some embodiments, the technique for measuring the tonicity / osmolality of a fluid is to measure its electrical conductivity. Conductivity indicates the presence of ions and can be correlated with osmolality. In some embodiments, the solution has a conductivity of at least 15 millisiemens / cm (mS / cm), optionally a value selected from the group of about or at least (in either case) 15.5 mS / cm, 16 mS / cm, 16.5 mS / cm, 17 mS / cm, 17.5 mS / cm, 18 mS / cm, 18.5 mS / cm.

[0038] Electrical conductivity may be defined alone or optionally in combination with one or more other experimental parameters for quantifying the properties of the solution, such as one or both of the following. (i) pH - for example, values within a range including about 6.4 to about 8.4, in some embodiments including about 7.2 to about 7.6, optionally including about 7.3 to 7.6, and / or (ii) Density - for example, values within a range including 1 to 1.015 Kg / liter, in some embodiments including 1.0090 to 1.0112 kg / liter.

[0039] In some embodiments, the solution (prior to mixing with a sample of hypo-osmotic naturally occurring body fluid) has a hypertonicity such that when mixed with a collected sample of body fluid, the combined mixture of the solution and the body fluid is isotonic, and the tonicity is defined with respect to blood.

[0040] In some embodiments, the solution (prior to mixing with the saliva sample) has a tonicity such that when mixed with the collected saliva sample, the combined mixture of the solution and saliva is isotonic, and the tonicity is defined relative to blood.

[0041] Obtaining an isotonic solution can be advantageous not only during the storage (e.g., fixation) of the saliva sample, but also during the period when the stored cells are held in storage awaiting processing or analysis. For example, stored and / or fixed cells can still be vulnerable to osmotic effects that can cause the cells to expand and burst, and / or to be crushed and disrupted internally, depending on the osmotic conditions that affect fluid movement through the cell wall.

[0042] In addition to or alternatively to any of the above, in some embodiments, the solution can preserve cells and / or extracellular components in a naturally occurring body fluid sample (e.g., saliva, sputum, fluid from a lung aspirate, or urine) for at least a period of at least one week, optionally at least two weeks, optionally at least three weeks, optionally at least one month, optionally at least two months, optionally at least three months, to at least a predetermined effectiveness.

[0043] For the purposes of the present disclosure, "preserving" refers to, for example, preventing cells from degrading their antigens such that the cells can be purified or concentrated based on those antigens, and preventing changes to the cell epigenome. "Epigenome" refers to the state or pattern of changes to genomic DNA by covalent modification of DNA or proteins bound to DNA. Examples of such changes include methylation of the 5th position of cytosine in CpG dinucleotides, acetylation of histone lysine residues, and other genetic or non-genetic changes not resulting from changes in the underlying DNA sequence.

[0044] As used herein, the term "efficacy" may mean that at least a predetermined percentage of the cells in the original body fluid sample are preserved. The predetermined percentage may optionally be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%. (Since the mixing of the original sample with the preservation solution increases the net volume of the mixture, thereby diluting the cell concentration, the cell concentration per unit volume can be reduced compared to the original body fluid sample.)

[0045] In addition, or alternatively, efficacy may refer to the number of cells (e.g., a certain type, e.g., T cells) per unit volume. For example, the number of such cells can be at least about 5000 per ml, optionally at least about 10000 per ml, optionally at least about 12000 per ml.

[0046] In addition, or alternatively, in some embodiments, the solution may have a storage period of at least 1 month, optionally at least 2 months, optionally at least 3 months, optionally at least 4 months at room temperature.

[0047] In addition, or alternatively, in some embodiments, a solution for preserving cells and / or extracellular components (e.g., any of proteins, viruses, cell-free DNA, and / or cell-free RNA) in body fluids such as oral fluid (e.g., saliva and / or sputum) and urine is provided for further separation into cell types and extracellular components, and for downstream analysis. Optionally, with respect to the cells, the solution may enable the cells in saliva to retain their antigenicity and cell structure during storage. In addition, or alternatively, optionally, with respect to the extracellular components, the solution may enable the extracellular components (e.g., proteins) to be preserved without degradation and / or without preserving the biological reactivity of extracellular proteins. In some embodiments, the solution may preserve both the cells and extracellular components in a saliva sample.

[0048] The solution can include, but is not limited to, at least one chemical fixative such as paraformaldehyde and at least one protease inhibitor. In some embodiments, the solution can further include, for example, at least one antibacterial agent and / or one or more of serum proteins from humans and / or other animal species. Additionally or alternatively, the solution can be buffered at a pH of about 6.4 to about 8.4, and in some embodiments, about 7.2 to about 7.6.

[0049] Chemical fixation of cells with a preservative can increase the mechanical strength of the cells, enabling the cells to be preserved intact and better withstand natural degradation and osmotic pressure over time. Specifically, storing cells in a fixative for more than a few minutes (so-called "over-fixation") has been reported to increase the solubility resistance of the cells.

[0050] Chemical fixation of extracellular components such as proteins can bind these proteins by cross-linking the proteins to other substances, such as other proteins or DNA. For example, a protein biologically bound to another substance can be more firmly chemically bound to the same substance by cross-linking. A protein that was merely in proximity to another material may be bound to that material by cross-linking. Cross-linking can preserve the protein by reinforcing it against unwanted degradation into smaller segments (e.g., peptides).

[0051] Cross-linking can be at least partially reversible. Reversing the cross-linking can be, for example, a step in the analysis of extracellular components such as proteins, for example, in subsequent downstream processing.

[0052] In addition to, or alternatively to, any of the above, in some embodiments, the solution is substantially detergent-free. Detergents are sometimes used in certain treatments of biological samples to facilitate permeation through cell material, but herein this is considered to be at the expense of increased cell damage and cell loss. According to some embodiments of the present disclosure, avoiding the presence of detergents can enhance the number of cells preserved, which can be particularly advantageous given the relatively small amounts of a particular target cell in a saliva sample.

[0053] As described above, the solution can optionally be hypertonic with respect to blood.

[0054] In addition to, or alternatively to, any of the above, in some embodiments, a sample collection device is provided that contains a preserved sample of a naturally occurring body fluid (e.g., saliva, sputum, or other oral fluid, or urine), and the preserved sample is isotonic with respect to blood. Optionally, the preserved sample can be a mixture of (i) the collected sample of the naturally occurring body fluid and (ii) the preservation solution.

[0055] In addition to, or alternatively to, any of the above, some embodiments of the present disclosure provide for the use of a hypertonic preservation solution for mixing with a sample of a hypotonic naturally occurring body fluid so as to make the resulting mixture isotonic in tonicity, which is defined with respect to blood. By way of example only, the body fluid can be or can contain saliva.

[0056] In some embodiments, a method of preserving cells in one or more body fluids includes contacting the collected cells with a solution, according to one and / or another embodiment of the present disclosure, such that the cells are able to retain their antigenicity and epigenome.

[0057] In some embodiments, a method for preserving extracellular components in one or more body fluids includes contacting a collected sample of the body fluid with a solution according to one embodiment and / or another embodiment of the present disclosure that allows certain extracellular components, such as proteins, to retain their structures without arbitrarily retaining their biological reactivity.

[0058] For example, body fluids may be naturally expressed, including one or more of oral fluid, saliva, sputum, fluid from lung aspirates, urine. In some cases, lung aspirates may also be artificially aspirated or encouraged.

[0059] In some embodiments, a method for separating certain extracellular components (such as any of proteins, viruses, free DNA, and / or free RNA) from cells in a chemically fixed body fluid (such as saliva or another oral fluid, or urine) includes centrifuging the sample to separate the sample into the specific extracellular components in liquid form, such as a cell pellet. The pellet and the liquid component may be processed separately. For example, the liquid component may be processed for analysis of the specific extracellular components. Additionally or alternatively, the pellet may be processed for cell analysis. Optionally, the same sample may provide materials for the analysis of both (i) cells and (ii) the extracellular components (such as any of proteins, viruses, free DNA, and / or free RNA).

[0060] In some embodiments, a method for isolating cells from chemically fixed cells collected from a body fluid, such as saliva or urine, includes centrifuging the cells to separate, for example, DNA and / or other soluble substances from the pellet of cells, bacteria, and debris, concentrating white blood cells from the other contents of the pellet, and isolating specific cells (such as white blood cells) using an antibody conjugated to magnetic beads targeted to a cell-specific marker.

[0061] In some embodiments, a method of isolating a specific type of cell, such as a type of white blood cell (e.g., lymphocyte), from one or more body fluids (e.g., saliva and / or urine) includes one or more of the following steps (and, depending on the embodiment, some or all of the following steps): providing a sample of the body fluid containing chemically fixed cells, optionally centrifuging the body fluid sample to obtain a pellet containing the cells, optionally resuspending the pellet in a buffer, subjecting the resuspended pellet to density gradient separation to obtain a layer of a mixture of white blood cell types (including lymphocytes), contacting the mixture of cell types with a solution containing a specific binding agent for an epitope found on the specific type of white blood cell, and separating the specific type of white blood cell (including lymphocytes) from the mixture of white blood cell types.

[0062] In some embodiments, the specific binding agent can be magnetic beads conjugated to an antibody specific for an epitope found on the specific type of white blood cell, and then the separation step can include, for example, magnetically separating the specific type of white blood cell (including lymphocytes) from the mixture of white blood cell types (although other cell separation techniques are within the scope of the present disclosure).

[0063] In some embodiments, a body fluid (e.g., saliva, sputum (or other oral fluid), or urine) can be mixed with a chemical fixation solution, and the mixture can be removed from the pellet. The pellet can then be resuspended in a buffer. The resuspended pellet can optionally be centrifuged and washed one or more times in the buffer. The washed pellet can then be applied to a hydrophilic polysaccharide mixture to form a gradient. Since the density of cells in other body fluids (e.g., saliva, urine) after chemical fixation for storage can vary depending on the different density of the stored cells, which requires changes in the gradient time, temperature, and / or density for the cells being processed through the density gradient, this gradient can be different from that used for blood.

[0064] In addition, in some embodiments, white blood cells can form a layer on the gradient. The white blood cell layer can be extracted from the gradient and placed in another centrifuge tube where it can be washed and repelleted in a buffer to remove the remaining gradient mixture. The pellet can then be resuspended and cultured in a buffer containing an antibody specific for an antigen that is specific for the cell type to be isolated. In some embodiments, the cell type to be isolated is a T cell and the antigen is a T cell-specific antigen. In some embodiments, the antigen is CD4. The resuspended cells in the buffer are bound by the antibody and can be subjected to a magnetic field that magnetically attracts the cells bound to the antibody-conjugated magnetic beads to the side of the tube. The remaining liquid can then be removed from the tube and the tube can be washed in a buffer. The isolated T cells then remain attracted to the side of the tube and are ready for further processing such as freezing for subsequent downstream experiments (e.g.).

[0065] In some embodiments, a method of preserving cells and / or extracellular components in a naturally occurring body fluid includes contacting the body fluid with a preservation solution according to any of the disclosed embodiments.

[0066] (i) Rare, i.e., a small number of cells, and / or (ii) the ability to obtain a sample of a naturally occurring body fluid that is preserved to provide a yield suitable for use of extracellular components (e.g., any of proteins, viruses, cell-free DNA, and / or cell-free RNA) can provide alternative techniques for blood collection for (i) diagnosing a patient's medical condition and / or (ii) monitoring the effectiveness of treatment. When used as a diagnostic tool for diagnosing the medical condition being treated, this process / product may sometimes be referred to herein as a "companion diagnostic". For example, an MGMT saliva-based assay that measures MGMT promoter methylation may be used as a companion diagnostic. When used as a monitoring tool for monitoring the effectiveness of treatment (e.g., whether a particular treatment is working, or whether an increase or decrease in dosage is effective, or whether the treatment will be fully effective), this process / product may sometimes be referred to herein as a "companion validator".

[0067] Such techniques can also facilitate the ease of obtaining samples from patients or study subjects, enable in-home collection of samples, and thus greatly expand the number of patients or study subjects who can participate in research, diagnostic, or monitoring programs.

[0068] Techniques used to identify specific cell types or other biomarkers in blood samples may also be used (with or without modification) to identify such cells or other biomarkers in naturally occurring body fluids.

[0069] As just one example, the cells or extracellular substances may be analyzed to indicate or identify one or more of the following. Cells with markers (cells or molecules) that identify the regression or aggressiveness of a disease. The disease can be, for example, cancer (optionally, but not limited to, leukemia). In the case of sputum or a fluid containing sputum, the disease can be any one or more of lung cancer, lung carcinoma, non - small cell lung cancer (NSCLC), pneumonia, or tuberculosis. (b) Prenatal cells. (c) Circulating tumor cells (e.g., metastatic or otherwise). (d) Rare forms of normal cells, such as any one or more of the following: - Immature cells such as myelodysplastic syndromes - Epithelial cell subtypes that indicate disease and do not originate from the mouth - Langerhans cells, for example (but not limited to), for the diagnosis of blood diseases (e) One or more biomarkers indicating obesity. (f) One or more biomarkers indicating bacterial vs. viral infections (useful for avoiding unnecessary and inappropriate prescriptions of antibiotics). (g) One or more biomarkers indicating autism. (h) One or more biomarkers indicating Alzheimer's disease. (i) One or more biomarkers indicating hematological diseases. (j) One or more biomarkers indicating cardiovascular diseases or disorders. (k) One or more biomarkers indicating diabetes. (l) One or more biomarkers indicating unstable plaques, and / or immune cell biomarkers, for example, related to immune cell activity. (m) One or more biomarkers and / or one or more released factors indicating the dormant and / or latent and / or occult forms of a disease or infection. For example, such a disease can be LTBI. Additionally, or alternatively, such factors can be peptides and / or cytokines. (n) The presence of HIV infection in a cell by detecting intracellular HIV virus randomly. (o) One or more biomarkers indicating cancer. (p) One or more biomarkers indicating chronic obstructive pulmonary disease (COPD). (q) One or more biomarkers indicating drug-resistant tuberculosis. (r) One or more biomarkers suitable for replicating and / or substituting one or more tests from the categories of blood tests, electrolyte level tests, lipid profile tests, vitamin level tests, hepatitis tests, iron deficiency tests, liver function tests, kidney profile tests, diabetes screening tests, blood picture tests, thyroid profile tests. (s) One or more biomarkers indicating the type of "asthma-COPD overlap syndrome (ACOS)". Optionally, such biomarkers can provide a companion diagnosis for general diagnosis of COPD to detect that COPD is ACOS. Additionally, or alternatively, such biomarkers can provide a companion validator to monitor the effectiveness of targeting ACOS for treatment. Additionally, or alternatively, the general diagnosis of COPD may be performed using, for example, the biomarkers referred to in (p) above, and / or the general diagnosis of COPD may be performed using other conventional diagnostic tests.

[0070] In some embodiments, although not exclusively limited to the diagnosis of rare cells in naturally occurring body fluids (e.g., saliva, sputum, urine), optionally, the techniques used to diagnose a patient's medical condition may include one or more of the following. (a) Flow cytometry (b) Fluorescence-activated cell sorting (FACS) (c) Immunohistochemistry (d) Molecular analysis (e.g., but not limited to, epigenetic; genetic; translational; post-translational). (e) Cytology (f) Protein level (translated) analysis techniques (g) RNA level (translated) analysis for measuring gene expression (h) DNA, including, for example, epigenetic, and / or DNA methylation and histone modifications.

[0071] Although some embodiments of the sample collection devices disclosed herein are described for uses involving collection of body fluids, it is noted that the devices also have specific uses involving collection of any other substances, including harmful and / or toxic fluids.

[0072] Some of the embodiments relate to using preservatives for preserving whole cells and / or extracellular substances, while other embodiments may relate to extracting and preserving nucleic acids in the collected samples. The nucleic acids may be DNA and / or RNA.

[0073] For example, a solution that lyses cells in a sample collected to extract nucleic acids and preserves the nucleic acids may be used.

[0074] By way of example only, reference is made to Annex 1 attached to this description, the entire content of which is incorporated herein as part of the present disclosure.

[0075] Without limiting the present disclosure, a numbered itemized list of specific features and / or aspects disclosed herein follows.

[0076] Item number: 1. A solution for preserving cells and / or extracellular components in naturally occurring body fluids for further downstream analysis and / or for diagnosis of medical conditions, the solution being hypertonic with respect to blood. 2. The solution according to item 1, wherein the naturally occurring body fluid comprises one or more selected from oral fluid, saliva, sputum, urine, fluids of lung aspirates. 3. The solution according to item 1 or 2, wherein the naturally occurring body fluid is hypotonic by nature. 4. The solution according to item 1, 2 or 3, wherein the osmolarity of the solution is "n" times about or at least 240 mOsm / L, and "n" is a natural number including 2 to 15. 5. The solution according to item 1, 2, 3 or 4, wherein the osmolarity of the solution is "n" times about or at least 275 mOsm / L, and "n" is a natural number including 2 to 15. 6. The solution according to item 1, 2, 3, 4 or 5, wherein the osmolarity of the solution is "n" times about or at least 290 mOsm / L, and "n" is a natural number including 2 to 15. 7. The solution according to any of the preceding items, wherein the tonicity of the solution compared to blood is about or at least "n" times that of blood, and "n" is a natural number including 2 to 15. 8. The solution according to any of the preceding items, wherein the aggregate salt ion osmolarity in the solution is "n" times about or at least 290 mOsm / L, and "n" is a natural number including 2 to 15. 9. The concentration of sodium chloride (NaCl) in the solution is about or at least "n" times the concentration "m", where "m" is in the range including 8.0 to 9.0 grams per liter (g / L) per liter, and "n" is a natural number including 2 to 15. The solution according to any of the preceding items. 10. The solution contains phosphate buffered saline (PBS) at a concentration that is about or at least "n" times the blood isotonic PBS concentration, and "n" is a natural number including 2 to 15. The solution according to any of the preceding items. 11. The solution has a hypertonicity such that when mixed with a collected sample of body fluid, the mixture of the solution and the body fluid is together approximately isotonic, and the tonicity is defined with respect to blood. 12. The solution according to any of the preceding items has an electrical conductivity of at least 15 mS / cm. 13. The solution has at least one of the following further parameters, namely, (i) A pH value within the range including 6.4 to about 8.4, and / or (ii) A density within the range including 1 to 1.015 kg / liter The solution according to item 12, having the above properties. 14. The solution according to any one of the preceding items, which is effective for storing cells over a duration selected from at least 2 weeks, or at least 3 weeks, or at least 1 month, or at least 2 months, or at least 3 months. 15. The solution according to any one of the preceding items, which is effective for storing cells when maintained at at least one of the following temperatures or temperature ranges, namely, 4°C to 40°C, 4°C to 30°C, approximately room temperature, about 4°C, about 30°C, about 40°C. 16. The solution according to any one of the preceding items, having a storage period selected from at least one of at least 1 month, at least 2 months, at least 3 months, at least 4 months. 17. The solution according to item 11, wherein the storage period is the storage period at room temperature. 18. The solution according to any one of the preceding items, which is effective for storing at least a predetermined percentage of cells from an initial sample of body fluid, and the predetermined percentage is selected from at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. 19. The solution according to any one of the preceding items, wherein the number of stored cells or a predetermined cell type per 1 ml is at least about 5,000, or at least about 10,000, or at least about 12,000. 20. The solution according to any one of the preceding items, wherein the cells are T cells. 21. The solution according to any one of the preceding items, comprising at least one chemical fixative buffered at a pH of about 6.4 to about 8.4 and at least one protease inhibitor. 22. A solution according to any of the preceding optional items for preserving at least extracellular components in a naturally occurring body fluid for further downstream analysis and / or for diagnosis of a medical condition, the solution optionally comprising at least one chemical fixative buffered at a pH of from about 6.4 to about 8.4. 23. The solution according to item 21 or 22, wherein the chemical fixative is selected from the group consisting of aldehydes. 24. The solution according to item 23, wherein the chemical fixative is paraformaldehyde. 25. The solution according to item 21, 22, 23, or 24, wherein the chemical fixative is present at a concentration of about 1% (v / v). 26. The solution according to any of items 21 - 25, further comprising at least one antibacterial agent and one or more of serum proteins from human and / or other animal species. 27. The solution according to item 26, wherein the antibacterial agent is selected from the group consisting of antibacterial and antifungal antibiotics. 28. The solution according to any of items 21 - 27, wherein the protease inhibitor(s) is / are selected from the group consisting of aspartic protease inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, serine protease inhibitors, threonine protease inhibitors, trypsin inhibitors, Kunitz STI protease inhibitors, and any combination of the foregoing. 29. The solution according to any of items 21 - 28, wherein the protease inhibitor(s) is / are selected from the group consisting of sodium azide, PMSF, aprotinin, leupeptin, pepstatin, natural or synthetic protease inhibitors, mixtures of natural and synthetic protease inhibitors, and any combination of the foregoing. 30. The solution according to any of items 21 - 29, wherein the protease inhibitor(s) is / are sodium azide. 31. The solution according to any of the preceding items, buffered at a pH of from about 7.2 to about 7.6. 32. The buffer solution is a solution according to any one of items 21 to 31, selected from the group consisting of barbital, trisphosphoric acid, citric acid, cacodylic acid, other non-phosphate buffer solutions, and any combination of the foregoing. 33. The buffer solution is a solution according to any one of items 21 to 31, which is a phosphate buffer solution. 34. A method for preserving cells and / or extracellular components in a naturally occurring body fluid, comprising the step of contacting the body fluid with a preservation solution according to any one of the preceding items. 35. A method for preserving cells and / or extracellular components in a hypotonic naturally occurring body fluid, comprising the step of contacting the body fluid with a hypertonic preservation solution, wherein the tonicity is defined with respect to blood. 36. A sample collection device for collecting a naturally occurring body fluid, comprising a preservation solution according to any one of items 1 to 33. 37. A sample collection kit comprising a solution according to any one of items 1 to 33. 38. A fluid sample for downstream analysis of cells and / or extracellular components in a fluid sample, wherein the fluid sample comprises a naturally occurring hypotonic body fluid to be mixed with a preservation solution, and the sample fluid is substantially isotonic, and the tonicity is defined with respect to blood. 39. The fluid sample according to item 38, wherein the naturally occurring body fluid is saliva. 40. A sample collection device containing the fluid sample according to item 38 or 39. 41. A method for diagnosing and / or monitoring a medical condition, comprising: obtaining a sample of a naturally occurring body fluid; contacting the sample with a preservation solution for stabilizing cells and / or extracellular components in the sample; analyzing the stabilized cells and / or extracellular components from the sample to identify, diagnose, or monitor the medical condition; and comprising. 42. The method according to item 41, wherein the naturally occurring body fluid comprises one or more selected from oral fluid, saliva, sputum, urine, and fluid of lung aspirates. 43. The method according to item 41 or 42, wherein the naturally occurring body fluid is saliva. 44. The method according to item 41, 42, or 43, further comprising contacting the sample with a pretreatment agent prior to contacting the sample with a preservation solution, wherein the pretreatment agent is configured to stimulate the release of one or more factors indicative of a medical condition. 45. The method according to item 44, wherein the pretreatment agent comprises one or more antigens associated with a medical condition. 46. The method according to any one of items 41 - 45, further comprising separating at least specific cells from at least specific extracellular components. 47. The method according to item 46, wherein the at least specific extracellular components comprise one or more selected from protein, virus, floating DNA, and / or floating RNA. 48. The method according to any one of items 41 - 47, further comprising concentrating the stabilized cells prior to the analyzing step. 49. The method according to any one of items 41 - 48, further comprising isolating one or more cell types from the stabilized cells prior to the analyzing step. 50. The method according to any one of items 41 - 49, wherein the analyzing step comprises identifying cells and / or extracellular components using cell markers or molecular markers that identify disease regression or aggressiveness. 51. The method according to item 50, wherein the disease is one or more selected from the group consisting of cancer, leukemia, lung cancer, pneumonia, and / or tuberculosis. 52. The method according to any one of items 41 - 51, wherein the analyzing step comprises identifying circulating tumor cells. 53. The method according to any one of items 41 - 52, wherein the analyzing step comprises identifying prenatal cells. 54. The method according to any one of items 41 - 53, further comprising The method described in 54. The method according to any one of items 41 to 53, wherein the analyzing step includes identifying rare forms of normal cells. 55. The method according to item 54, wherein the rare forms of normal cells include one or more selected from the group consisting of immature cells, immature cells indicating myelodysplastic syndrome, epithelial cell subtypes that indicate a disease and do not originate from the mouth, and / or Langerhans cells. 56. The method according to any one of items 41 to 55, wherein the analyzing step includes flow cytometry. The method of 57. The method according to any one of items 41 to 56, wherein the analyzing step includes fluorescence-activated cell sorting (FACS). The method described in any of 58. The method according to any one of items 41 to 57, wherein the analyzing step includes immunohistochemistry. 59. The method according to any one of items 41 to 58, wherein the analyzing step includes molecular analysis. 60. The method according to item 59, wherein the molecular analysis includes any one or more selected from the group consisting of epigenetic analysis, gene analysis, translational analysis, and / or post-translational analysis. 61. The method according to any one of items 41 to 60, wherein the analyzing step includes cytology. 62. The method according to any one of items 41 to 61, wherein the analyzing step includes identifying biomarkers indicating obesity. The method described in any of 63. The method according to any one of items 41 to 62, wherein the analyzing step includes identifying biomarkers indicating obesity. The method described in any of 64. The method according to any one of items 41 to 63, wherein the analyzing step includes identifying biomarkers indicating bacterial infections. The method described in any of 65. The method according to any one of items 41 to 64, wherein the analyzing step includes identifying biomarkers indicating autism. The method described in any of ​66. The step of analyzing includes identifying biomarkers indicative of Alzheimer's disease, The method according to any one of items 41 to 65. 67. The step of analyzing includes identifying biomarkers indicative of a blood disease, item 41 ~ The method according to any one of 66. 68. The step of analyzing includes identifying biomarkers indicative of a cardiovascular disease or disorder The method according to any one of items 41 to 67. 69. The step of analyzing includes identifying biomarkers indicative of diabetes, items 41~ The method according to any one of 68. 70. The step of analyzing includes identifying biomarkers indicative of unstable plaques, item The method according to any one of 41 to 69. 71. The step of analyzing includes identifying biomarkers indicative of latent tuberculosis infection, item The method according to any one of 41 to 70. 72. The step of analyzing includes identifying factors released by treatment of a sample with an antigen before the step of contacting with a preservation solution, The method according to any one of items 41 to 70. 73. The antigen includes a peptide of Mycobacterium tuberculosis, and the factor includes interferon gamma, the method according to item 72. 74. The step of analyzing includes identifying biomarkers indicative of HIV infection, item 4 The method according to any one of 1 to 73. 75. The step of analyzing includes identifying HIV virus infection prior to the presence of detectable antibodies in the sample, The method according to any one of items 41 to 74. 76. The step of analyzing includes identifying intracellular HIV virus in the sample, item The method according to any one of 41 to 75. 77. The step of analyzing includes identifying the HIV virus in T cells, optionally CD4+ T cells The method according to any one of items 41 to 76, including the step of 78. The method according to any one of items 41 to 77, wherein the analyzing step optionally includes assaying the amount of CD4+ T cells to identify an immunodeficiency disorder indicating HIV infection and / or AIDS. 79. The method according to any one of items 41 to 78, wherein the analyzing step includes (i) indicating COPD and / or (ii) indicating ACOS, and / or (iii) identifying a biomarker for distinguishing between the ACOS type and the non-ACOS type of COPD. 80. A method for diagnosing HIV infection, comprising: providing a naturally occurring body fluid sample, optionally a stored sample of saliva, from an individual; and identifying intracellular HIV virus in the sample to diagnose HIV infection in the individual. 81. The method according to item 80, wherein the step of identifying intracellular HIV virus includes identifying the HIV virus in T cells of the sample, optionally CD4+ T cells. 82. The method according to item 81, wherein the providing step includes providing a fixed sample of the naturally occurring body fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Non-limiting embodiments will be described below by way of example only with reference to the accompanying drawings.

[0078]

Figure 1

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[0086] Devices, solutions, and methods for sample collection, preservation, isolation, and analysis will be further understood in light of the following drawings, the detailed description of the embodiments, and the claims. Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0087] (Detailed description of the embodiments) In some embodiments, reference is made to the disclosures of International Publication No. WO2012 / 177656 and / or International Publication No. WO2015 / 112496, which the present disclosure may incorporate.

[0088] In some embodiments, solutions are disclosed for preserving cells and / or extracellular components (e.g., any of proteins, viruses, cell-free DNA, and / or cell-free RNA) in one or more naturally occurring bodily fluids such as saliva, sputum, fluid from lung aspirates, oral fluid, and / or urine. The solutions may be useful for further separation into cell types and / or extracellular components and for downstream analysis that enables storage of cells and / or extracellular components in the bodily fluid. The cells may be preserved to retain their antigenicity and cellular structure. The extracellular components may be preserved to avoid degradation and breakdown. The proteins may be preserved without preserving their biological reactivity.

[0089] In some embodiments, the solution is optionally disclosed in combination with a sample collection device that is suitable for home use or at least does not require a visit to a medical laboratory. In some embodiments, the solution is referred to in a “as-supplied” state prior to mixing with the collected bodily fluid sample. In some embodiments, the solution is referred to in that state after mixing with the collected bodily fluid sample.

[0090] Referring to FIG. 1, techniques for collecting and processing naturally occurring bodily fluids are illustrated. The process is described with respect to oral fluid containing saliva as the most difficult example. However, it can be understood that the same principles can be applied to the collection and processing of other naturally occurring bodily fluids. The reference to saliva may be replaced by any other such naturally occurring bodily fluid, specifically, oral fluid in general, and / or sputum, and / or fluid from lung aspirates, and / or urine.

[0091] Referring to FIG. 1, the first step 30 may be for the donor to provide a sample of a fluid, such as saliva. The providing may be accomplished by spitting saliva into a collection device, or by collecting saliva by any other technique such as using a swab.

[0092] The second step 32 may be to contact and / or mix a preservation solution for preserving all cells in the collected saliva sample and / or for preserving extracellular components in the collected saliva sample with the collected saliva sample. The solution for preserving cells and / or extracellular components may be beneficial for further separation into cell types and / or extracellular components and for downstream analysis. As previously discussed herein, saliva presents a physiologically harsh environment for the cells of interest and for extracellular components. The cells of interest can begin to be damaged and destroyed within minutes of the sample being collected. In some embodiments, step 32 may be performed relatively soon after step 30 to avoid or reduce the adverse effects of saliva on the yield of intact cells and preserved extracellular components in the collected sample.

[0093] As used herein, "preserving cells" refers to preventing the cells' antigens from being degraded and preventing changes in the cell epigenome so that the cells can be purified or concentrated based on those antigens. "Epigenome" refers to the state or pattern of changes in genomic DNA by covalent modification of DNA or proteins bound to DNA. Examples of such modifications include methylation of cytosine at the 5th position in CpG dinucleotides, acetylation of histone lysine residues, and other genetic or non-genetic changes not resulting from changes in the underlying DNA sequence. Additionally, or alternatively, "preserving cells" may mean that the cells in saliva retain their antigenicity and cell structure during storage.

[0094] As used herein, the term "validity" of preservation may mean that at least a predetermined percentage of the cells in the original body fluid sample are preserved. The predetermined percentage may optionally be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%. (Since the mixing of the original sample with the preservation solution increases the net volume of the mixture, thereby diluting the cell concentration, the cell concentration per unit volume can be reduced compared to the original body fluid sample.)

[0095] In addition, or alternatively, validity may refer to the number of cells (e.g., a certain type, e.g., T cells) per unit volume. For example, the number of such cells may be at least about 5000 per ml, optionally at least about 10000 per ml, optionally at least about 12000 per ml.

[0096] In addition, or alternatively, preserving extracellular components such as proteins means preventing the proteins from being degraded or cleaved into smaller fragments (e.g., peptides). Preserving the proteins may also include immobilizing the proteins by binding them to any material to which the proteins could have been biologically bound prior to preservation and / or to other materials in close proximity to which they bind after preservation. The biological reactivity of the proteins may be neutralized, for example, by mass spectrometry, to enable the proteins to be analyzed in subsequent downstream steps.

[0097] In some embodiments, an optional pretreatment step 42 may be performed so as to mix saliva with a pretreatment agent, e.g., a solution, powder, or solid agent, prior to the second step 32. The pretreatment may be, in some embodiments, a biological agent or a non-biological agent, or a mixture of a biological agent and a non-biological agent. The pretreatment agent may be used, in some embodiments, to react with and / or stimulate the release of and / or otherwise cause the production of one or more specific factors in saliva. Additionally or alternatively, the pretreatment agent may stimulate the biological reactional response of saliva.

[0098] Such a biological reactional response may be less effective (or not effective at all) after saliva is preserved, e.g., by fixation.

[0099] An example of a pretreatment agent may be an antigen to stimulate or cause the release of specific factors from cells such as lymphocytes so as to determine whether the saliva is from an individual infected with a related disease and / or infection. For example, the pretreatment agent may include a peptide. In one specific example used in the detection of latent tuberculosis infection (LTBI) described hereinafter, the peptide may be a peptide from Mycobacterium tuberculosis that is recognized by lymphocytes and stimulates a biological response in the form of the release of factors such as interferon gamma when the saliva is from an individual infected with LTBI. The pretreatment agent may optionally include more than one antigen and / or more than one peptide.

[0100] Some embodiments of the present disclosure provide such a preservation solution that is hypertonic with respect to blood (e.g., prior to mixing with a body fluid).

[0101] While not desiring to be bound by any particular theory, as described above herein, it is believed that the tonicity (or osmolality) of saliva can be a significant factor in making saliva a harsh environment for cell survival. Saliva, and particularly saliva collected in a given sample, has been found to be hypotonic with respect to blood. Cells from blood that enter an individual's saliva are subject to a destructive osmotic pressure that draws fluid into the cells, causing the cells to swell, become damaged, and ultimately rupture. This can explain the small number of cells that have been reported to survive undamaged in saliva. This can also explain, for example, why HIV is less transmissible by saliva than by other body fluids, as cells, including HIV-infected cells, that enter saliva from blood cannot survive long due to cell rupture as a result of osmosis. Similar destruction and cell damage is generally imparted to cells that enter saliva from blood. The body compensates for such cell destruction by providing a high rate of blood flow to the salivary glands, which allows for the replenishment of cells that die in the mouth. The blood flow to the salivary glands has been reported to be about 100 times greater than the blood flow to skeletal muscle in the relaxed state. The blood flow to exercising skeletal muscle increases by up to about 10-fold, but even then, the blood flow to the salivary glands still significantly exceeds that to the exercising muscle by about 10-fold. This provides further evidence of the harsh environment of saliva and the rapid cell death in saliva, and thus requires a constant replenishment of cells.

[0102] By providing a preservation solution that is hypertonic with respect to blood, this solution can neutralize the hypotonicity of saliva and balance the tonicity towards an isotonic environment when mixed with a saliva sample. Such an environment can avoid or at least significantly reduce the osmotic pressure on the cells in the collected sample, thereby allowing the cells to survive without damage for preservation and enabling a significantly improved yield of cells preserved for analysis or diagnosis.

[0103] Obtaining an isotonic solution can be advantageous not only during the storage (e.g., fixation) of saliva samples, but also during the period when the stored cells are waiting for processing or analysis in storage. For example, stored and / or fixed cells may still be susceptible to osmotic effects that can cause the cells to expand and rupture, and / or be crushed and burst internally, depending on the osmotic conditions that affect fluid movement through the cell wall.

[0104] In some embodiments, the naturally occurring body fluid may be of a type that is naturally hypotonic, for example, but is not limited to oral fluids (e.g., those containing saliva), and in particular is not limited to saliva itself.

[0105] In some embodiments disclosed herein, the osmolality of the solution (e.g., prior to mixing with the body fluid) can optionally be defined as being "n" times about or at least (in either case) 240 mOsm / L, where "n" is a natural number including 2 to 15. For example, "n" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13, 14, or 15.

[0106] In some embodiments disclosed herein, the osmolality of the solution (e.g., prior to mixing with the body fluid) can optionally be defined as being "n" times about or at least (in either case) 275 mOsm / L, where "n" is a natural number including 2 to 15. For example, "n" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13, 14, or 15.

[0107] In some embodiments disclosed herein, the osmolality of the solution (e.g., prior to mixing with the body fluid) can optionally be defined as being "n" times about or at least (in either case) 290 mOsm / L, where "n" is a natural number including 2 to 15. For example, "n" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13, 14, or 15.

[0108] In addition, or alternatively, the aggregate salt ion osmolarity in the solution (e.g., prior to mixing with a body fluid) can optionally be about or at least (in either case) an “n” fold of 290 mOsm / L, where “n” is a natural number including 2 to 15. For example, “n” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0109] In some embodiments disclosed herein, the osmolarity of the solution (e.g., prior to mixing with a body fluid) compared to blood can optionally be defined as being about or at least (in either case) an “n” fold of blood, where “n” is a natural number including 2 to 15. For example, “n” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0110] In addition, or alternatively, the concentration of sodium chloride (NaCl) in the solution (e.g., prior to mixing with a body fluid) can optionally be about or at least (in either case) an “n” fold of concentration “m”, where “m” is a value in the range including 8.0 to 9.0 grams per liter, and “n” is a natural number including 2 to 15. For example, “n” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0111] In addition, or alternatively, the solution can optionally contain phosphate buffered saline (PBS) at a concentration (e.g., prior to mixing with a body fluid) that is about or at least (in either case) an “n” fold of blood isotonic PBS concentration, where “n” is a natural number including 2 to 15. For example, “n” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0112] In some embodiments, the solution (prior to mixing with a sample of a hypotonic, naturally occurring body fluid) has a hypertonicity such that when mixed with the collected sample of the body fluid, the combined mixture of the solution and the body fluid is isotonic, and the tonicity is defined relative to blood.

[0113] In some embodiments, the solution (prior to mixing with the saliva sample) has a tonicity such that when the solution is mixed with the collected saliva sample, the combined mixture of the solution and saliva is isotonic, and the tonicity is defined with respect to blood.

[0114] The preservation solution can include, but is not limited to, at least one chemical fixative such as paraformaldehyde and at least one protease inhibitor. In some embodiments, the solution can further include at least one antibacterial agent and one or more of serum proteins from humans and / or other animal species. The solution can be buffered at a pH of about 6.4 to about 8.4, preferably about 7.2 to about 7.6.

[0115] In some embodiments, the concentration of the agent in the following description can be the concentration of the sample preservation solution itself. Depending on the body fluid, and in the case of saliva, approximately equal volumes of the solution and the body fluid can be mixed together. This preferably allows cells from the body fluid to retain their antigenicity and DNA for at least one week at room temperature.

[0116] In some embodiments of the present disclosure, the volume of the preservation solution retained and deployed within the device can be, for example, about 100 to about 500 ml, which is relevant for the preservation of cells in urine. Thus, the preservation solution for urine can be either a solution approximately 10 times (10x) concentrated with respect to urine to a 1.5 times (1.5x) solution.

[0117] The "chemical fixative" according to some embodiments is a chemical cross-linking compound used to change cell components so that cells are resistant to degradation. The chemical fixative can also serve to cross-link histones and other DNA-binding proteins to DNA. Such agents are known in the art and include, but are not limited to, paraformaldehyde, formaldehyde, formalin, aldehydes, alcohols, oxidizing agents, mercury agents, picrate, hepes glutamate buffer-mediated organic solvent protection effect (HOPE), combinations of fixatives such as Zambonis fixative, combinations of aldehydes, and synthetic cross-linking reagents. In some embodiments, the chemical fixative is paraformaldehyde. In some embodiments, the chemical fixative is present at a concentration of about 1% (v / v).

[0118] Chemical fixation of extracellular components such as proteins can bind these proteins by cross-linking the proteins to other substances, such as other proteins or DNA. For example, a protein biologically bound to another substance can be more firmly chemically bound to the same substance by cross-linking. A protein that was merely in proximity to another material may be bound to that material by cross-linking. Cross-linking can preserve a protein by reinforcing it against unwanted degradation into smaller segments (e.g., avoiding degradation into peptides).

[0119] For example, the aldehyde groups of paraformaldehyde react with the nitrogen and other atoms of proteins. This reaction causes the formation of methylene bridges between adjacent proteins. This "traps" DNA (e.g., fixation immobilizes the proteins (e.g., transcription factors) bound to DNA in place due to the reaction with the fixative). Not only can DNA be trapped between these methylene bridges, but lipids, carbohydrates, DNA, and RNA can also be trapped.

[0120] The cross-linking can be at least partially reversible. Reversing the cross-linking can be, for example, a step in the downstream processing, for example, for the analysis of extracellular components such as proteins. For example, bacterial proteins and human proteins may cross-link with each other or with floating DNA. Reversing the cross-linking downstream enables the analysis.

[0121] To protect cells from degradation by proteases present in body fluids, in some embodiments, the solution can contain at least one protease inhibitor. In some embodiments, the protease inhibitor can be selected from the group consisting of aspartic protease inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, serine protease inhibitors (e.g., serpins), threonine protease inhibitors, trypsin inhibitors, and Kunitz STI protease inhibitors. Some specific non-limiting examples include sodium azide, PMSF, aprotinin, leupeptin, pepstatin, natural or synthetic proteinase inhibitors, and cocktail mixtures of protease inhibitors. Suitable concentrations of these inhibitors can include, but are not limited to, PMSF (phenylmethylsulfonyl fluoride) serine protease at about 0.1 - 1 mM, benzamidine serine protease at about 1 mM, pepstatin A acid protease at about 1 μg / ml, leupeptin thiol protease at about 1 μg / ml, aprotinin serine protease at about 5 μg / ml, and antipain thiol protease at about 1 μg / ml. In one embodiment, the protease inhibitor is sodium azide at a concentration of about 0.01% (w / v).

[0122] To prevent damage to cells from microbial contamination, some embodiments of the solution contain at least one antibacterial agent. Suitable antibacterial agents include, but are not limited to, antibacterial or antifungal antibiotics.

[0123] Preservation of cell structure is enhanced by the presence of serum proteins, which can be optionally added to the solution in some embodiments. In addition, serum proteins can be used to neutralize the osmotic pressure difference between the cells and the solution. These can be derived from human or other animal sources. In some cases, whole serum can be used. For example, fetal bovine serum can be added at about 1% (v / v) in some embodiments.

[0124] In addition to or as an alternative to any of the above, in some embodiments, the solution is substantially free of detergents. Detergents are sometimes used in certain processing of biological samples (e.g., antibodies conjugated with fluorescent dyes) to facilitate penetration through cellular material, but are considered herein to be at the expense of increased cell damage and cell loss. According to some embodiments of the present disclosure, avoiding the presence of detergents can enhance the number of preserved cells, which may be particularly advantageous, bearing in mind the relatively small amount of certain cells of interest in saliva samples.

[0125] A solution according to the present disclosure may include any combination of the foregoing embodiments.

[0126] In some embodiments of the present disclosure, a method of preserving cells and / or extracellular components in one or more body fluids is disclosed. The method of preserving cells and / or extracellular components can include contacting the body fluid with a solution according to the present disclosure. The body fluid can contain various cell types and / or extracellular components, and the cells and / or extracellular components in the body fluid can be preserved by the solution according to the present disclosure. Although not critical to the present disclosure, a ratio of solution to body fluid of about 1 to 1 is typically used.

[0127] The following examples are intended to further illustrate some embodiments of solutions and methods for preserving cells and / or extracellular components in bodily fluids and are not to be construed as limiting the scope of the disclosure.

[0128] For example, a solution of PBS at pH 7.4, 1% paraformaldehyde, 1% FBS, and 0.01% NaN3 can be added in a 1:1 ratio with saliva, and then T cells can be purified and DNA can be extracted. The results of such a process are shown in Figure 2. These results can demonstrate that the antigenicity of T cells and the integrity of DNA were maintained for at least one week.

[0129] As shown in Figure 3, further tests have demonstrated the effectiveness of the solution in preserving T cells in saliva samples over a long duration, even at high temperatures and even after a long storage period.

[0130] In Figure 3, the horizontal axis represents the number of T cells per milliliter of saliva sample (in thousands) mixed with the preservation solution at a 1:1 ratio as demonstrated by the analysis. The cell concentration is effectively halved compared to the original saliva sample as a result of the 1:1 mixing with the preservation solution.

[0131] The first (leftmost) column shows the number of cells in three samples stored at room temperature for several hours after sample collection and mixing with the preservation solution. The first column serves as a reference for assessing the number of cells in other samples.

[0132] The second, third, and fourth columns compare the number of cells in three groups of three samples, each stored at room temperature for 1 day, 2 days, and 1 month respectively, after sample collection and mixing with the preservation solution. The second to third columns show little variation from each other or from the first column.

[0133] The fifth, sixth, seventh, and eighth cylinders each store for a period of three months after mixing with the sample collection and preservation solution, and compare the number of cells in a group of 10 samples kept at 4°C, room temperature, 30°C, and 40°C, respectively. Since the test was focused on long-term storage, 10 samples (instead of 3 samples) were used. The fifth to eighth cylinders show little variation with each other or with the first cylinder.

[0134] The last cylinder repeats the test for the second cylinder using a group of 10 samples and the preservation solution stored for 4 months (pre-use storage period). As described above, since the test was focused on demonstrating a long-term storage period, 10 samples (instead of 3 samples) were used.

[0135] The graph illustrates that the preservation solution is extremely effective in preserving T cells in saliva over a long duration, over a wide range of temperature conditions, and even after a long storage period. The solution may have similar or corresponding preservation capabilities for other types of cells. The deviation between the number of cells in different cylinders can be explained, at least, by the normal differences in the number of cells from different people providing the samples and / or different samples even from the same individual. The number of cells per 1 ml is also significantly sufficient to enable downstream cell analysis.

[0136] Referring again to FIG. 1, steps 30 and 32, and optional step 42 may be performed using any suitable apparatus or device. In a simple technique, steps 32 and / or step 42 may be performed by opening a separate container containing the preservation solution and / or the pretreatment agent, manually adding the preservation solution and / or the pretreatment agent to the collected sample, and / or manually mixing the solution with the collected sample. However, in some embodiments, steps 30 and 32, optionally step 42, may both be performed (at 34) using the dedicated sample collection device 10 illustrated in FIGS. 4 or 5. The device 10 may be suitable for home use or of a type that at least obviates the need to visit a laboratory. The sample collection device 10 may be portable, for example, to enable a user to provide a sample of a bodily fluid that is naturally expressed, and / or may be provided to a user at home (e.g., via postal or courier delivery). Alternatively, the sample collection device 10 may also be convenient for use in a laboratory or hospital.

[0137] Figures 4 and 5 illustrate two alternative embodiments of the collection device 10. The device 10 may generally comprise a first body (e.g., a tube) 16 having an opening 18 and defining a collection area 20 for naturally occurring body fluids introduced into the device through the opening 18. The device 10 may further comprise a second body (e.g., a sealing device) 22 attachable to or over the opening 18 so as to seal the device 10 after the provided sample has been introduced. The preservation solution 12 and / or the pretreatment agent may optionally be stored in at least one internal chamber 14 of the device and released, for example, to mix with the collected body fluid sample when the user seals the device or performs certain other operations of the device 10. In the embodiment of FIG. 4, the chamber 14 containing the preservation solution 12 is provided in the second body (e.g., the sealing device) 22. In the embodiment of FIG. 5, the chamber 14 containing the preservation solution 12 is provided in the first body (e.g., the tube) 16. In either embodiment, the chamber 14 is configured to be opened by a mechanism (schematically shown at 24) to communicate with the collection area 20. The opening mechanism 24 may respond to the fitting of the second body (e.g., the sealing device) 22 to the first body (e.g., the tube) 16 or certain other manual operations of the device 10. Various mechanisms 24 are envisioned including, but not limited to, the release of an internal closure or cap, the opening of a tap, the rupture of a frangible wall, the removal or displacement of an internal cover, the relative rotation of a cap or nut, one or more than one being selected.

[0138] In the illustrated embodiment, for example, only a single chamber 14 for the preservation solution is shown. Optionally, additional chambers (not shown) may be provided for any pretreatment agent, if used. The additional chambers may be configured to be opened by the above-described opening mechanism or a second opening mechanism. The additional chambers may be opened, for example, prior to the chamber containing the preservation solution. In some embodiments, a sequential opening mechanism may be configured to release the preservation solution only after the pretreatment agent has been released first.

[0139] Further details of the exemplary structures of the device 10 and the release mechanism 24 are provided in the aforementioned International Publication No. WO2012 / 177656, which is incorporated herein by reference.

[0140] The device 10 may be configured for collecting a predetermined sample volume “Vs” of a naturally occurring body fluid. The device may include, for example, a visual fill scale or fill line (e.g., indicated at 26) or other indicia to indicate when a predetermined sample volume Vs has been achieved. In some embodiments, the sample collection space may have a size equal to the predetermined volume Vs, or the sample collection space may be larger in volume. By way of example only, in some embodiments, the predetermined volume Vs may be at least about 1 ml, or at least about 2 ml, or at least about 3 ml, or at least about 4 ml, or at least about 5 ml, or more. By way of example only, in some embodiments, the predetermined volume Vs may be about 5 ml or less, or about 4 ml or less, or about 3 ml or less, or about 2 ml or less, or about 1 ml or less. By way of example only, in some embodiments, the predetermined volume Vs may be, in some embodiments, from about 1 ml to about 5 ml, optionally from about 1 ml to about 4 ml, optionally from about 1 ml to about 3 ml, optionally from about 1 ml to about 2 ml. By way of example only, the predetermined volume Vs may be, in some embodiments, about 1 ml, or about 2 ml, or about 3 ml, or about 4 ml, or about 5 ml.

[0141] In some embodiments, the volume “Vc” of the preservation solution 12 may not be substantially larger than the predetermined sample volume Vs, or optionally, may be smaller than it. The preservation solution volume Vc may be the same as the internal space of the chamber 14 for filling the chamber 14, or the preservation solution volume Vc may be smaller and partially fill the chamber 14.

[0142] By making the volume Vc of the preservation solution not substantially larger than the predetermined sample collection volume Vs, or optionally, smaller than it, the volume increase when the preservation solution 12 is mixed with the collected sample can be equally small. The volume increase can be equivalent to the dilution of the sample with respect to the cell concentration per unit volume.

[0143] Referring again to FIG. 1, in step 36, the collected sample may be stored, and / or transported to a laboratory, for processing (optionally, via an intermediary). For example, in the case of a sample provided at home, the collection device 10 may be sent to a laboratory via a courier or postal service, or to an intermediary for subsequent transportation. The time for transportation and / or storage prior to analysis by the laboratory may range from minutes, hours, days, weeks, or even months. The ability of the preservation solution to stabilize and preserve all cells and / or extracellular components in a body fluid sample over long periods and over a wide range of temperatures enables a wide range of analytical and diagnostic tools to be used to process naturally occurring body fluids as a viable alternative to sampling a patient's blood. Also, the yield of preserved cells in the sample, including the ability to preserve rare, i.e., a small number of cells, and the yield of extracellular components, provides an opportunity to use naturally occurring body fluids for new medical analysis, diagnosis, and treatment monitoring applications.

[0144] In optional step 38, the preserved sample may be processed to concentrate the cell concentration and / or isolate selected cell types and / or separate specific extracellular components (e.g., any of proteins, viruses, cell-free DNA, and / or cell-free RNA).

[0145] In some embodiments of the present disclosure, a method is disclosed that provides a sample of one or more body fluids, such as saliva or urine, that contains chemically fixed cells and / or chemically fixed extracellular components, and optionally centrifuges the body fluid sample. Centrifuging may separate certain extracellular components (e.g., any of proteins, viruses, floating DNA, and / or floating RNA) from the cell pellet that contains bacteria and debris.

[0146] Depending on the particular substance of interest, either or both of the pellet or the liquid extracellular components may be further processed or analyzed.

[0147] For example, the method can further include the step of concentrating white blood cells, including lymphocytes, from other contents of the pellet. Additionally, specific cells may be isolated using antibodies conjugated to magnetic beads that target cell-specific markers.

[0148] In some embodiments, the present disclosure is a method of isolating a specific type of white blood cell (including, but not limited to, lymphocytes in particular) from a body fluid (i.e., saliva, urine, etc.), and includes, for example, one or more of the following (and in some embodiments, some or all of the following steps): providing a sample body fluid containing chemically fixed cells, optionally centrifuging the body fluid sample to obtain a pellet containing cells, optionally resuspending the pellet in a buffer, subjecting the resuspended pellet to density gradient separation to obtain a layer of a mixture of white blood cell types (including lymphocytes), contacting the cell type mixture with a solution containing a specific binding agent for an epitope found on a specific type of white blood cell, separating the specific type of white blood cell (including lymphocytes) from the white blood cell type mixture.

[0149] In some embodiments, the specific binding agent can include magnetic beads that bind to an antibody specific for an epitope found on a particular type of white blood cell, and the separating step may include magnetically separating a particular type of white blood cell (including lymphocytes) from a mixture of white blood cell types, although any method (and corresponding system / device) for separating cell types from each other is within the scope of the present disclosure. Magnetic separation is just one method for doing so.

[0150] Cells can be chemically fixed prior to undergoing the methods according to the present disclosure. Cells can be chemically fixed, for example, by contacting a sample of saliva with a chemical fixation solution. This is done to preserve the cells over time at ambient temperature. This can also enable a complete study of the epigenome, as it allows for the study of histone modifications and other protein-DNA interactions from body fluid samples where they have accumulated. Histones must be chemically fixed to the DNA in order to be studied. Without fixation, histones generally cannot remain bound to the DNA and the proteins will degrade over time.

[0151] In some embodiments, the buffer can include sodium azide, and the buffer can include phosphate buffered saline and sodium azide. In some embodiments, the buffer may further include fetal bovine serum. In some embodiments, the buffer has a pH of from about 7.2 to about 7.6.

[0152] In some embodiments, the cells are washed once in the buffer. This is done in practice to remove soluble substances and, in the case of saliva, to remove what has been classified as the "buccal" layer (Dos-Santos et al, 2009).

[0153] In some embodiments, the mixture of white blood cells is washed one or more times in the buffer prior to separation. This is preferably done to remove any residual density gradient solution from the mixture of cell types.

[0154] In this process, the antibody may bind to a specific type of white blood cell, and thus, a specific type of white blood cell may be bound to magnetic beads. The specific type of white blood cell can then be separated from any other cell type by placing the magnetic beads in a magnetic field and removing any remaining liquid to obtain the isolated cells of the specific type of white blood cell.

[0155] In some embodiments, the specific type of white blood cell can be a lymphocyte, and the lymphocyte can be a T cell. In such embodiments, the antibody used can be specific to an antigen specific to T cells (e.g., an antigen that is CD4). In some embodiments, the isolated blood cells can then be frozen prior to further processing such as prior to epigenetic analysis.

Example

[0156] The following examples are intended to further illustrate exemplary embodiments of the methods of the present disclosure and are not intended to limit the scope of the present disclosure. (Example 1)

[0157] Example 1: Isolate T cells from body fluid (e.g., saliva). Saliva is collected and mixed with a preservation solution. The cells are then pelleted by centrifugation and the processing solution is removed. The cells are then resuspended in approximately 6 ml of buffer (PBS, pH 7.4, 1% FBS, 0.01% NaN3), then washed once in the buffer and pelleted again. The pellet is resuspended in approximately 6 mL of PBS - 15 FBS - 0.01% NaN3 and subjected to density gradient centrifugation using Ficoll® (GE Healthcare) with a density of 1.082 - 1.072 g / ml. The white blood cells are centrifuged at the interface between the polysaccharide and the buffer while bacteria, debris, and any other particulate matter are pelleted at the bottom of the tube. The cells are extracted from the tube and placed in a new tube. The cells are then washed once in Hank's balanced salt solution and then once in PBS - NaN3 - FBS buffer to remove any remaining density gradient solution that may have been taken up during extraction of the white blood cells from the interface.

[0158] Here, the sample contains highly concentrated white blood cells with minimal bacteria and minimal debris. This step can also greatly reduce other cell types such as epithelial cells. The cells can then be incubated in a buffer (PBS-NaN3-FBS) with an antibody that targets CD4 conjugated to magnetic beads (Dynabeads® Invitrogen®). The sample can then be placed in a magnetic field, where the beads are carried to the side of the tube and the liquid is removed. The liquid may contain all that is not bound to the beads through the antibody. T cells can bind to the antibody and cannot be removed due to the magnetic field. The beads and attached cells can be washed in buffer to eliminate any non-specific or weak binding to other cells, bacteria, or other debris found in body fluids such as saliva or urine. The cells can then be frozen for subsequent downstream processing and analysis. Isolation of T cells can be confirmed by light microscopy (T cells are very distinct compared to epithelial cells and bacteria) (see Figure 6). Additionally, flow cytometry and F.A.C.S. analysis using antibodies against CD3, CD4, and CD8 can confirm the visual assessment of the isolated cells. The T cells can then be titrated from the body fluid to determine the number of T cells per unit (ml) of body fluid to determine the amount of body fluid for a sufficient number of cells for downstream experiments (see Figures 6 and 7). The isolated cells can be shown to have intact and suitable DNA for downstream use (see Figure 2).

[0159] Referring again to FIG. 1, at step 40, the sample of interest (optionally, with the cell type concentrated or separated and / or isolated from step 38) is further processed for analysis or diagnosis. As described above, step 40 may be performed on the isolated cells or on the isolated extracellular components. Optionally, the collected sample may provide both a whole cell sample and an extracellular component sample, and step 40 may be performed separately for each. Step 40 may include the same or similar analysis for each, or may include different analyses.

[0160] The ability to obtain samples of naturally occurring body fluids that are rare, i.e., that are preserved to provide a usable yield of only a small number of cells and / or extracellular components, may provide an alternative technique to blood sampling for the diagnosis and / or monitoring of a patient's medical condition. Cellular and extracellular analysis may be used as a companion diagnosis and / or as a companion validator.

[0161] Techniques used to identify specific cell types or other biomarkers in a blood sample may also be used (either with or without modification) to identify such cells or other biomarkers in a naturally occurring body fluid.

[0162] By way of example only, the cells and / or extracellular components can be analyzed to diagnose, indicate, identify, and / or monitor one or more of the following. (a) Cells with markers (cells or molecules) that identify the regression or aggressiveness of a disease. The disease can be, for example, cancer (optionally, but not limited to leukemia). In the case of sputum or a fluid containing sputum, the disease can be any one or more of lung cancer, lung carcinoma, non-small cell lung cancer (NSCLC), pneumonia, or tuberculosis. (b) Fetal cells. (c) Circulating tumor cells (e.g., metastatic or otherwise). (d) Rare forms of normal cells, such as any one or more of the following: - Immature cells such as myelodysplastic syndromes - Epithelial cell subtypes that show disease and do not originate from the mouth - Langerhans cells, for example (but not limited to), for the diagnosis of blood diseases (e) One or more biomarkers indicating obesity. (Discussed below) (f) One or more biomarkers indicating bacterial infection vs. viral infection (useful for avoiding unnecessary and inappropriate prescribing of antibiotics). For example, · The TRAIL protein (TNF-related apoptosis-inducing ligand) can be used to distinguish infections (bacterial vs. viral). This is very useful for combating antibiotic-resistant bacteria. · The TRAIL protein is a cytokine secreted by most cells and induces the process of apoptosis as a ligand. (g) One or more biomarkers indicating autism. For example, · Affects the establishment of oxytocin-affinity binding. As a negative effect, oxytocin in ASD is negatively correlated with 5-HT. Polymorphisms in the OXTR gene encoding the oxytocin receptor are associated with autism, pair-coding, social behavior, emotional impact, and ASD traits. Melatonin has been well established to play a good role in the 24-hour cycle and seasonal rhythm, as well as the modulation of immune response and neuroplasticity. Melatonin is synthesized from 5-HT, which is then converted to N-acetylserotonin and then to melatonin via an enzyme, namely, acetylserotonin methyltransferase (ASMT). The process is inhibited by daylight. Low levels of melatonin in the plasma of ASD patients are thought to be due to the absence of this ASMT enzyme (epigenetic (DNA, protein, and RNA applicable analysis)). ASMT gene variants are associated with ASD. The same gene also harbors disruptive coding mutations in 6 out of 398 (1.51%) ASD individuals, while none were found in any of the 437 controls. Immunological Biomarkers of Autism (Extracellular and Intracellular): - Elevated IL-1 (Interleukin 1) - Elevated IFN-γ (Interferon gamma) - Overproduction of the anti-inflammatory cytokine, IL-10 (Interleukin 10) - Abnormal maturation (post-thymic maturation) of T lymphocytes with increased native and decreased differentiated (i.e., CD4+ and CD8+) T cell numbers (h) One or more biomarkers indicating Alzheimer's disease. (i) One or more biomarkers indicating a blood (hetotological) disease. (j) One or more biomarkers indicating a cardiovascular disease or disorder. (k) One or more biomarkers indicating diabetes. (l) One or more biomarkers indicating unstable plaques (plack), and / or immune cell biomarkers, for example, related to immune cell activity. (m) One or more biomarkers and / or one or more released factors indicative of dormancy and / or latency and / or occult forms of a disease or infection. For example, such a disease can be LTBI. Additionally, or alternatively, such factors can be peptides and / or cytokines.

[0163] In some embodiments, the pretreatment agent may be added to the saliva prior to the storage step (step 42 of FIG. 1 as described above). The pretreatment agent may include, for example, an antigen to the detected disease or infection to cause or stimulate the release of specific factors in the saliva, if the individual has the disease or infection. LTBI may be detected by using a pretreatment agent of an antigen (e.g., a peptide of Mycobacterium tuberculosis) that is recognized by lymphocytes and causes the release of factors such as interferon gamma in the saliva from an infected individual. The released factors such as interferon gamma can then be assayed (e.g., quantitatively or qualitatively) from the stored saliva, and a diagnosis can be made.

[0164] Additionally, or alternatively, in some embodiments, lymphocytes, e.g., T cells, may be analyzed epigenetically for LTBI based on changes that cause the cells to release interferon gamma when a peptide from Mycobacterium tuberculosis is added to the cells (including transcriptional and / or translational analysis). This may be done, for example, with or without the addition step 42 using a pretreatment agent with the antigen.

[0165] (n) The presence of HIV infection in a cell.

[0166] Here, a non-limiting discussion of HIV infection continues. Additional and / or alternative embodiments are also further described below.

[0167] Any of the current saliva-based released antibody / factor assays may be enhanced by the use of the storage techniques of the present disclosure.

[0168] However, current saliva-based techniques generally have the drawback of relying on the human immune system to produce detectable antibodies / factors that are associated with a delay of at least six weeks. Such techniques can be classified based on extracellular factors. During this time, existing saliva-based tests cannot diagnose the presence of HIV infection in an individual, and in certain cases, can lead to specific results that are misleading and / or can lead to an individual unknowingly spreading the infection to others.

[0169] The present disclosure also proposes the use of saliva (or another naturally expressed body fluid) to enable the detection of HIV infection (i) during the period before the body's immune system produces detectable antibodies / factors and / or (ii) without a delay of at least six weeks after an individual is infected.

[0170] In some embodiments, the present disclosure enables the analysis of the HIV virus within preserved infected cells in saliva (or another naturally expressed body fluid). T cells, including blood cells such as CD4+ T cells, may be preserved and / or isolated and / or analyzed to identify the presence of the intracellular HIV virus. Thus, the present disclosure enables intracellular analysis, for example, from saliva, and enables the detection of the HIV virus much earlier than the extracellular tests described above after infection, for example, at least six weeks or more earlier.

[0171] In some embodiments, within the T cells, viral RNA or reverse transcribed DNA, or proteins carried by the virus (e.g., any of reverse transcriptase, protease, ribonuclease, and integrase) may be analyzed.

[0172] In some embodiments, for example, after the preservation of saliva and the isolation of CD4+ T cells, the cells may be lysed, and DNA and / or RNA and / or intracellular proteins may be obtained and / or isolated. Optionally, PCR may be performed using a probe that targets a sequence specific to the HIV virus. Additionally, or alternatively, the diagnostic platform may be able to examine proteins that are used for the integration of HIV into the DNA genome during initial infection. Such an analysis may be essentially proteomics and may include assays such as mass spectrometry, ELISA, or other antibody-mediated analyses. The antibody analysis may be performed with respect to the proteins of the HIV virus (not those released by immune cells as described above).

[0173] Additionally, or alternatively, since HIV actively eliminates CD4+ cells from the body's immune repertoire and causes AIDS, the measurement of the number of CD4+ cells in a sample (e.g., saliva) may itself indicate HIV infection. In some embodiments of the present disclosure, the number of CD4+ cells may be assayed, for example, by flow cytometry and / or FACS, to provide such an indicator from the preserved sample.

[0174] (o) One or more biomarkers indicative of cancer.

[0175] (p) One or more biomarkers indicative of chronic obstructive pulmonary disease (COPD).

[0176] (q) One or more biomarkers indicative of drug-resistant tuberculosis.

[0177] (r) One or more biomarkers suitable for replicating and / or substituting for one or more tests from the categories of blood tests, electrolyte levels, lipid tests, vitamin levels, hepatitis tests, iron deficiency tests, liver function tests, kidney profile tests, diabetes screening tests, blood picture tests, thyroid profile tests.

[0178] (s) One or more biomarkers indicative of the type of "asthma-COPD overlap syndrome (ACOS)". Optionally, such biomarkers can provide a companion diagnosis for the general diagnosis of COPD to detect that the COPD is ACOS. Additionally, or alternatively, such biomarkers can provide a companion validator to monitor the effectiveness of targeting ACOS for treatment. Additionally, or alternatively, the general diagnosis of COPD may be performed using, for example, the biomarkers referenced in (p) above and / or the general diagnosis of COPD may be performed using other conventional diagnostic tests.

[0179] With respect to (a) above, additional information regarding non-small cell lung cancer (NSCLC) is described here as only one example. Approximately 80% of all lung cancers are NSCLC (5). Early diagnosis and treatment for NSCLC optimize the outcome (12). The main components of conventional diagnosis involve direct visualization of suspicious cancer cells by microscopy (6). This is an established laboratory use involving lung biopsy material.

[0180] In contrast, sample collection of oral fluid using the techniques of the present disclosure may provide easier access to the respiratory cancer cell source. Oral fluid testing is considered to be able to provide a "liquid biopsy" that can at least support and even enhance current clinical practice (3, 4, 7). Oral fluid diagnostic methods are considered to be extensible to other cancers (48) as well as several other diseases suitable for this matrix (49).

[0181] The detection and identification of putative cancer cells, specifically cancer stem cells (CSC), are part of current cancer research and patient management activities (8). Based on recent scientific research data findings, a suitable biomarker panel that enables the identification of NSCLC / CSC (in patients already diagnosed) may provide significant advantages.

[0182] Exemplary biomarkers may include CD133 and / or EpCAM (10,11). Additionally, or alternatively, exemplary biomarkers may include EGFR and / or ALK.

[0183] Some biomarkers can be detected, for example, in the preserved whole cells of a body fluid sample. Additionally, or alternatively, some biomarkers may be detected, for example, in a preserved sample of lysed cells from a body fluid collected (optionally refer to Annex 1 using a preservation solution that accesses nucleic acids in some embodiments of the present disclosure).

[0184] CD133 is a biomarker strongly associated with immortalized cells (stem cells), and EpCAM (epithelial cell adhesion molecule) is a well-known biomarker that is exclusively expressed in epithelia or epithelial-derived neoplasms. EpCAM can be used as a diagnostic marker for various solid tissue cancers. It appears to play a role in tumor development and cancer metastasis. Thus, it can also play the role of a potential prognostic marker and a potential target for immunotherapy strategies. EpCAM capture / detection is an important component of existing circulating tumor cell (CTC) assays in whole blood that are already in clinical practice.

[0185] Used both as a reflective and monitoring clinical assay via the techniques of the present disclosure, physicians can obtain valuable information about the progression and prognosis of patients. The repertoire of such additional targets suitable for testing (gene rearrangements, SNPs, etc.) can be extensive for any tumor and subject to active research (5, 6, 13).

[0186] As an advantage, the techniques of the present disclosure may enable patient sample procurement and stabilization to be achieved more readily than with other approaches. Subsequent assays may be facilitated using the retained patient material via a "liquid biopsy" proposal (14). There are numerous attractive biomarkers that can be evaluated by reflex testing (5, 6, 13). For example, in patients diagnosed with NSCLC, it is highly advantageous to be able to determine early drug treatment and subsequent options, as drug resistance unfortunately occurs via genomic sequence variations and can occur prior to or at any time during drug treatment.

[0187] Two assays that may be relevant to patient management involve testing for mutations in any of several genes known to be involved in treatment drug resistance and monitoring (5, 6, 15, 16). In advanced cases, mutations in the epidermal growth factor receptor gene (EGFR) and chromosomal rearrangements of the ALK gene may be important for drug selection. At a finer level, several other genes are also important for disease treatment such as VEGF, but EGFR / ALK are the main direct focus of treatment regulation (50).

[0188] EGFR-specific drugs, namely gefitinib ((Iressa)-Astra Zeneca), erlotinib ((Tarceva)-Genentech), afatinib ((Gilotrif)-Boehringer Ingelheim), require analysis and monitoring of EGFR sequence changes. Drugs focused on ALK, namely crizotinib ((Xalkori)-Pfizer) and ceritinib ((Zykadia)-Novartis), also require monitoring of patient gene rearrangements.

[0189] Important advantages can be achieved with respect to the management (vigilance) of safety information on the initial and ongoing patient gene sequences. These tests can be facilitated, for example, via tumor cell investigations using preserved lysed body fluid samples (e.g., using the techniques of Appendix 1).

[0190] The assay may be performed more directly by nucleic acid hybridization assays via advanced cytological methods (e.g., IHC, FISH) (9, 18) based on preserved whole cell samples or by using preserved lysed samples (e.g., using the techniques of Annex 1 optimized to isolate nucleic acids) (19).

[0191] Optionally, assays are envisioned that use an investigation of CD133 / EpCAM of cells carried by oral fluid, optionally followed by a reflex assay of drug resistance-related genes such as (but not limited to) EGFR / ALK. Approximately 10% of NSCLC tumors may exhibit EGFR or ALK mutations (50).

[0192] Regarding (e) above, further information is described here as only one example. Global and gene-specific changes in epigenetics (DNA methylation and histone acetylation) are correlated with obesity. Selected genes whose expression is likely to be causally related to obesity (analysis at the protein and RNA levels): · Leptin (LEP) · Leptin receptor (LEPR) · Brain-derived neurotrophic factor (BDNF) · Proopiomelanocortin (POMC) · Single-minded homolog 1 (SIM1) · Neurotrophic tyrosine kinase receptor type 2 (NTRK2) · FTO (fat mass and obesity) Selected epigenetic biomarkers shown to be associated with obesity:

Table A-1

Table A-2

[0193] Referring to (n) above, the following provides additional and / or alternative illustrative information. There have been significant advances in nucleic acid amplification, detection, and sequencing over the past decade (Reviews 27, 37, 38). Reagents, primer sequences, and protocols exist and are readily available. In addition to high throughput and esoteric assays, there continues to be a need for facile sample handling and processing in otherwise routine applications. Saliva and oral fluids generally have seen advances in recent years as a novel matrix for nucleic acid testing (39, 40, 41). There is significant interest in utilizing this relatively "user-friendly" fluid for various gene / sequence detection methods (42).

[0194] A saliva-based test for the BRACA1 gene sequence (breast cancer susceptibility) has been announced as in development (Color Genomics) and has shown initial utility as an alternative to existing cell blood-based tests. Most established providers of gene sequence platforms have adapted to specific protocols for the saliva matrix (43), and thus there is good reason to expect that saliva may be used in the future for the detection of other infectious agents, analytes, and nucleic acid sequence alterations (44, 45).

[0195] For future reference, a related collection of source links for this document (47, 48) is provided with respect to available techniques.

[0196] Exemplary biomarkers for HIV sequence detection can be, for example, CD4 cells for analysis via FISH / IHC methods. Further biomarker detection via PCR will also be discussed later below.

[0197] The techniques disclosed herein provide a saliva sample collection device that enables the preservation and isolation of cell spectra from saliva or induced sputum. These cells may be visualized by several microscopic methods (9). As described above for NSCLC, this existing system also enables the investigation of fixed cells by numerous immunohistochemical methods (IHC) (18). This analysis may include the detection of CD4 cells, which indicate any of several HIV protein biomarkers. Abundant IHC-eligible detection antibodies are commercially available for this purpose (9). The sample collection techniques disclosed herein may be suitable for sequence-specific nucleic acid amplification and detection methods such as gene-specific FISH (fluorescence in situ hybridization) (51). This is an important and widely accepted method similar to cytological analysis that enables the detection of DNA / RNA sequences (both native genomes and viruses) within fixed cells by common microscopy and imaging techniques. There are widely accepted and effective protocols available for this method. The selection of optimized hybridization probes and PCR primers targeting various genomic regions of the HIV virus is widely understood and readily available (52).

[0198] The detection of specific HIV virus sequences as both integrated proviruses and virion particles involves the treatment of microscope slides containing preserved oral fluid cells with "labeled" hybridization probes specific to well-known HIV genomic sequences. Specifically, in CD4 cells, where the viral life cycle is best understood, the "proviral" sequences integrated into the chromosome hybridize to chemically labeled probes and can then be visualized (53). There are high-sensitivity protocols that have been validated for low-copy virus loads (54). These probes are synthetic matches to the viral genomic sequences and may carry any of several available signals that generate a "label" moiety (biotin / streptavidin, ferritin, HRP enzyme, fluorophore, etc.).

[0199] Additional or alternative exemplary biomarkers for HIV sequence detection may involve PCR. Detection of HIV sequences by PCR is not technically complex, and many reagent systems and amplification detection options are available. Sequences, probes, and primers are widely understood and available. Many established protocols exist (Reviews 37, 38, 47).

[0200] Both the HIV virus and anti-HIV antibodies can be detected in saliva, providing an alternative to blood for detecting anti-HIV antibodies to diagnose HIV infection (42, 43). Saliva HIV antibody tests using dedicated devices for appropriate saliva specimen collection have been approved in recent years by the US Food and Drug Administration (FDA) (e.g., OraSure). These tests are used in settings with limited resources, as well as in domestic public clinics where rapid testing and ease of use are preferred.

[0201] The Centers for Disease Control (CDC) includes rapid and frequent HIV testing as part of its policy recommendations for HIV prevention programs (http: / / www.cdc.gov / hiv / testing / clinical / ). In the case of virion particles, limited data indicate that while transmissibility may be low, the viral genomic sequence may still be detectable by sensitive PCR "viral load" assays. This appears to be particularly true during the early stages of infection and perhaps during the "window" period when antibodies first appear (39, 40, 55).

[0202] Initial studies have shown that 42% - 91% of individuals with early HIV infection were positive by sensitive PCR assays using saliva matrix (41, 55). Indeed, a dramatic reduction in HIV RNA was seen in patients who initiated combination drug therapy. Thus, oral fluid / saliva sample collection devices that can be seamlessly integrated with a well-formatted HIV-specific PCR detection system have the potential to provide significant advantages in clinical and surveillance settings.

[0203] Referring to the above (p), further information is described here as only one example. COPD is a complex respiratory inflammatory disease that is extremely difficult to diagnose clearly (20, 21, 22). It presents clinical symptoms similar to other respiratory syndromes and leads to complex serum biomarker changes that are not specific in themselves other than as indicators of "inflammation" throughout the system. However, it is understood to be the fourth leading cause of death worldwide. It is currently not curable, but there are treatments to extend lifespan.

[0204] Using the principles of the present disclosure, one or more of the following biomarkers, namely, VAP-1, CD44, CD11b, eosinophil levels (e.g., oral fluid eosinophil levels and / or sputum eosinophil levels), may be suitable for detecting COPD in a collected body fluid sample.

[0205] There is an important ongoing discussion regarding the most appropriate diagnostic biomarker(s) option(s) for treatment management. Recent research data have identified additional new biomarkers of inflammation and disease progression, and the field is rapidly identifying more specific cellular markers for both infiltration and inflammation that may provide more effective interventions (23, 24). The sample collection and analysis techniques described herein may provide a simple tool that directly evaluates early cellular diagnostic indicators of respiratory failure and contributes to early treatment intervention.

[0206] Many of the current biomarkers for respiratory / inflammatory diseases are soluble analytes that are easily and routinely measured in blood, but there is also new data indicating that invasive neutrophils and eosinophils play important roles in airway failure (25, 26, 27, 28, 29).

[0207] One recent study has identified a novel biomarker, VAP-1, present on airway endothelial cells as a molecular “brake” element at the initial stage of the adhesion cascade, which is responsible for mediating and attenuating infiltrating neutrophils involved in the inflammatory process (30). Experimental drugs that inhibit this ligand have been shown to have dramatic effects in animal models of respiratory diseases, specifically in a COPD model.

[0208] The proper functioning of neutrophils in lung defense is integrated with their ability to exit the microvasculature and migrate through tissue to the target site. In response to organ injury, neutrophils upregulate the expression of specific cell surface receptors (co-expression of CD44 and CD11b, among others), and these biomarkers then bind to adhesion molecules on endothelial cells such as VAP-1 (31, 32, 33). Enabling the movement of neutrophils into the underlying parenchyma through the endothelial cell lining is essential for health, but if this process is not limited, uncontrolled inflammation results. CD44 and CD11b on neutrophil PBMCs have been shown to have such upregulatory behavior and function as early indicators of COPD-related airway neutrophil infiltration and may possibly enable the continuous monitoring of disease progression or treatment. Indeed, the idea that a specific subset of infiltrating neutrophils (such as measured in sputum) can propagate or exacerbate COPD has generated interest in new treatments.

[0209] In addition or alternatively, eosinophil levels may provide additional indicators, particularly as biomarkers for ACOS (see further examples below for (s)).

[0210] Regarding the above (q), further information about tuberculosis is described here as only one example. The diagnosis and treatment of Mycobacterium tuberculosis continue to be a major health concern in both developing and developed countries. In the past decade, it has become clear that the bacterium can develop resistance to the limited armamentarium or treatment drugs currently available. In addition, understanding the status of individual patients, as far as the types and frequencies of resistance-related mutations are concerned, will enable physicians to better treat and even anticipate the threatening shifts and pandemics of new infectious pathogens. This is a technical challenge involving nucleic acid sequence analysis and extensive gene databases on drug resistance alleles. Since the infectious organism is clearly a respiratory agent, the techniques of the present disclosure, including the ease and safety of patient management, can provide important advantages for patient management.

[0211] Some biomarkers may be detected, for example, in the preserved whole cells of a body fluid sample. In addition, or alternatively, some biomarkers may be detected, for example, in a preserved sample of lysed cells from a body fluid collected using, for example, a preservation solution (optionally, see Appendix 1, which accesses nucleic acids in some embodiments of the present disclosure).

[0212] Regarding the above (r), heretofore, it has not been feasible to replace conventional blood tests with saliva-based tests. However, by using the techniques of the present disclosure, most conventional blood tests (almost all whole blood counts and the evaluation of many diagnostic markers) can now be performed using saliva.

[0213] The following is a table (in the first column) listing many (but not all) of the tests commonly ordered by physicians who require blood draws. This first column lists various tests that, together, evaluate most of the important factors in the blood to ensure the patient's health and proper balance. Most of these tests are not specific stand-alone diagnostic methods, but rather factors that contribute to and aid in diagnosis. The middle column indicates the feasibility of implementing the techniques of the present disclosure, as presently contemplated, as well as alternative and / or substitute tests that use saliva samples instead of blood samples. The right column indicates that commercially available saliva kits for genetic analysis can be used to perform these tests, whether currently available or not, in accordance with the best thinking and understanding.

[0214] Categories of blood factors to be evaluated: Electrolytes: Blood electrolyte measurements are routinely ordered for monitoring and evaluating a patient's general health. Electrolyte measurements allow for the detection of any imbalances in the body and the monitoring of acid-base balance abnormalities. Abnormalities in the balance of any individual electrolyte can be a legitimate reason for further investigation and monitoring and / or can indicate a larger health problem.

[0215] Lipids: Lipids contained in the blood are evaluated as part of a cardiac risk assessment screening to determine whether a patient is at risk of developing cardiovascular disease. Thus, these tests can also help determine the need to change a patient's medications and / or the dosage of a medication.

[0216] Vitamins: Vitamin D and vitamin B12 are often measured to determine deficiency or, in the case of deficiency, adequate supplementation. Additionally, vitamin B12 is often tested in the diagnosis of neuropathy and certain anemias.

[0217] Hepatitis: HBsAg is the surface antigen on the hepatitis B virus. Detection of this factor in the blood confirms the diagnosis of hepatitis B.

[0218] Iron deficiency profile: These tests are used to evaluate whether the level of iron in a patient's body is too high or too low and the corresponding need for supplementation.

[0219] Liver function tests: Screening for and detecting hepatitis and liver diseases. Both acute and chronic hepatitis as well as liver disorders are evaluated by the fluctuations of these factors and enzymes.

[0220] Kidney profile: This series of tests is useful for the management and diagnosis of kidney-related symptoms. These tests can simply constitute part of the conventional blood tests to ensure the proper function of the kidneys, or can serve the function of screening and / or tracking patients who have or are at risk of developing kidney diseases.

[0221] Diabetes screening: These are tests that are directed to help monitor and manage a patient's diabetes by diagnosing whether the patient has diabetes and by evaluating the level of blood sugar control.

[0222] Blood picture: Also known as a complete blood count (CBC), it is a broad screening tool that evaluates a patient's general health and detects a number of diseases such as anemia, infections, and certain cancers. CBC includes the assessment of the cellular components of blood (red blood cells, white blood cells, platelets, and their ratios), as well as the measurement of various aspects and factors in the blood.

[0223] Thyroid profile: A series of tests to assess the proper function of the thyroid or to diagnose imbalances indicating thyroid diseases. The tables referred to above follow here below.

Table B-1

Table B-2

Table B-3

Table B-4

[0224] Regarding the above (s), further information on "asthma-COPD overlap syndrome" (ACOS) is provided here as only one example. ACOS is a condition that occurs when a patient has both COPD and asthma. As already explained for the above (p), "chronic obstructive pulmonary disease" (COPD) affects approximately 5% of the population and is associated with both high morbidity and mortality rates. The condition is characterized by chronically poor airflow in the patient, as well as both emphysema and chronic bronchitis. In recent years, a new subtype of COPD, "asthma-COPD overlap syndrome" (ACOS), has been found to afflict 10-40% of patients (Leena George, 2016). ACOS presents symptoms of COPD and asthma, as well as mixed pathology, all leading to a more extreme form of COPD. Typically, the onset of ACOS occurs at an earlier age than in other COPD patients, and the annual healthcare costs for these patients are three times higher ($2,307 for asthma patients, $4,879 for COPD patients, $14,917 for ACOS patients) (Fadia T. Shaya, 2009).

[0225] ACOS is jointly defined by GOLD (Global initiative for Chronic Obstructive Lung Disease) and GINA (Global Initiative for Asthma) as "characterized by persistent airflow limitation, with some features usually associated with asthma and some features usually associated with COPD". Due to the ambiguity of this definition, ACOS is currently not only poorly defined but also difficult to diagnose based on presentations in the clinic. However, since it is understood that patients with ACOS can respond sub-optimally to normal COPD treatments, a correct diagnosis is important. Conversely, ACOS treatments are expensive and should be targeted at patients with symptoms that are likely to be treated successfully without much failure.

[0226] The sample collection, preservation, and analysis techniques described herein may be particularly suitable for enabling the subsequent staining and measurement of preservation and patient eosinophil counts while allowing for the simultaneous detection of other important cells and related biomarkers from the same sample (see above). These techniques may provide clinicians with valuable new seranostic tools for evaluating and monitoring the treatment of COPD patients.

[0227] As further clinical data develop, this application may also play a role, for example, in the differential diagnosis of asthma.

[0228] ACOS has been shown to respond to certain drugs that can help manage patients in a stable state and, more importantly, manage and prevent exacerbations. One such group of drugs is inhaled and oral corticosteroid therapy. Steroids are considered a broad - spectrum approach to managing ACOS due to their ability to reduce eosinophilic bronchial mucosal inflammation (Leena George, 2016). Other narrow - spectrum treatment approaches that would specifically target eosinophilic / Th - 2 - mediated inflammation are currently being studied for use in ACOS (Leena George, 2016). Such treatment approaches not only improve the quality of life of ACOS patients by managing symptoms and exacerbations but also have the potential to reduce healthcare costs and the resulting economic burden.

[0229] Based on this understanding of ACOS and the ongoing treatment approaches, it becomes even more important to develop sufficient and efficient diagnostic tools that can not only aid in the diagnosis of ACOS but also serve as companion diagnostics and / or companion validators to justify the effectiveness of treatment approaches. Conventional options for blood tests and bronchoscopies each have their limitations and drawbacks. Sputum tests have been shown to be an advantage in COPD patients due to the local infiltration of inflammatory cells, suggesting that higher concentrations of inflammatory cells such as eosinophils may be present in the sputum. However, sputum samples are also cumbersome and difficult to obtain. Oral fluid has been shown to contain saliva and sputum, and due to the increased sputum production in COPD / ACOS patients, oral fluid can present a minimally invasive means for the diagnosis and evaluation of many cells and inflammatory biomarkers. The preservation of all contents of oral fluid and the use of the techniques described herein for the isolation and evaluation of eosinophils and major biomarkers can provide significant advantages that have not been achieved or even conceived of before.

[0230] We have discussed biomarkers that can be detected or analyzed for different medical conditions. The following description will now explain different techniques that can be used, for example, to perform many of the above.

[0231] In some embodiments, without being limited exclusively to the analysis of rare cells and / or extracellular components in naturally occurring body fluids (e.g., saliva, sputum, urine), techniques that can optionally be used to diagnose or monitor a patient's medical condition may include one or more of the following. (a) Flow cytometry (b) Fluorescence-activated cell sorting (FACS) (c) Immunohistochemistry (d) Molecular analysis (e.g., limited to any of epigenetic; genetic; translational; post-translational) (e) Cytology (f) Protein-level (post-translational) analysis techniques (g) RNA-level (post-translational) analysis for measuring gene expression (h) DNA, including, for example, epigenetic and / or DNA methylation and histone modifications.

[0232] The following additional examples are intended to further illustrate embodiments of the exemplary methods of the present disclosure and are not intended to limit the scope of the present disclosure. (Example 1)

[0233] Flow cytometry may be used to diagnose various forms of cancer. This is, for example, a test used by, for example, the Cancer Treatment Center of America to diagnose leukemia and lymphoma. Flow cytometry may also be used for the analysis of other types of cells, such as platelet cells in the blood. For example, Quest Diagnostics utilizes flow cytometry to diagnose paroxysmal nocturnal hemoglobinuria (PHN). (Example 2)

[0234] FACS may be used to diagnose various cell markers, including markers for cancer. This is an assay used by Quest, for example, for the diagnosis of cell markers such as CD57, CD8, CD3, etc. FACS analysis can also be useful for monitoring the status of HIV / AIDS treatment by investigating CD4 assays, and BD Biosciences offers, for example, the FACS Count, a system designed for high-confidence CD4 assays. TM FACS analysis is also used to assess the morphology of cell death and mitochondrial damage. For example, EMD Millipore offers various assays for assessing cell health and integrity. (Example 3)

[0235] Immunohistochemistry may be used to diagnose various cell markers, including markers for cancer and other disorders / diseases. This is an assay used by Quest, for example, for the diagnosis of a number of cell markers involved in diseases. As just one example, reference may be made to http: / / www.questdiagnostics.com / home / physicians / testing-services / specialists / hospitals-lab-staff / specimen-handling / immunohistochemistry.html.

[0236] Immunohistochemistry may also be used in the diagnosis of carcinomas and some sarcomas through the detection of cytokeratins. For example, Invitrogen offers immunohistochemistry kits for the detection of cytokeratins. (Example 4)

[0237] Molecular analysis (e.g., but not limited to, epigenetic, genetic, translational, post-translational). Molecular analysis is used by Qiagen and other companies to assess DNA methylation and other epigenetic marks for the diagnosis of various diseases such as cancer by methods including, for example, bisulfite sequencing and methylated DNA immunoprecipitation (MDIP). Molecular analysis is used by the Cancer Treatment Center of America for molecular profiling of various biomarkers found in the DNA of tumor tissue samples. Such tests not only enable the diagnosis of cancer, for example, colorectal cancer, but also facilitate personalized treatment. (Example 5)

[0238] Cytology may be used to diagnose various cell markers, including markers for thyroid cancer and other disorders / diseases. This is, for example, a test used by Quest for the diagnosis of thyroid cancer. As an example only, http: / / www.questdiagnostics.com / testcenter / testguide.action?dc=TG_Thyroid_Cancer may be referenced. (Example 6)

[0239] Protein level (post-transcriptional). Examples of downstream analysis techniques may include analysis of proteomic biomarker panels using mass spectrometry, Western blot, and arrays. Analysis of intracellular protein expression (levels) may be used to analyze the effects of drugs, chemicals, non-chemical inhibitors, or biological inhibitors of a protein or series of proteins.

[0240] This includes measurements regarding the knockdown / knockout / inhibition or activation effects on genes that can modify RNA or proteins or non-coding DNA sequences. This includes proteins / RNA of the epigenome (enzymes such as DNA methyltransferases, DNA demethylases, histone methyltransferases, histone demethylases, histone acetyltransferases, and histone deacetylases (specifically, this includes enzymes such as DNMT1 / 2 / 3 and all subtypes, MBD2, MeCP2, and all histone modifying enzymes such as histone (de)acetylases (de)methylases, etc.)), and can be used in combination with the analysis of epigenetic downstream effects (DNA methylation or histone modification at either the global or gene-specific level, and / or RNA and / or DNA analysis, etc.). (Example 7)

[0241] RNA levels (transcription) for measuring gene expression. There are a number of candidate genes identified in the literature that are used either individually or on a panel to discriminate diseases / disorders, or as companion diagnostics or biomarkers for pharmacological and / or treatment interventions for a number of diseases and disorders, including cancer, neurological diseases, rare diseases, and pediatric diseases / disorders. Furthermore, different types of RNA can be analyzed from isolated cells (this includes microRNA and messenger RNA, etc.). Analytical techniques include (to name a few) PCR, whole genome arrays, and biomarker panels.

[0242] RNA downstream applications may include steps of analyzing cells for RNA or mRNA interference using inhibitors such as siRNA or other chemical and / or non-chemical inhibitors. This includes steps of measuring the levels of RNA by various forms of PCR, expression arrays (examples include affymetirx or agilent human genome expression arrays and custom arrays, which may or may not include known biomarkers for specific diseases or disorders). (Example 8)

[0243] DNA (genetic and epigenetic, including DNA methylation and histone modification). This may include techniques such as its use with biomarker panels, bisulfite sequencing analysis, methylation immunoprecipitation analysis, and whole genome array platforms (including those based on gene panels, agilent, affymetrix, and customized arrays).

[0244] DNA analysis may be used to measure the effects of inhibition of methylation and demethylation both globally and gene-specifically. In the global case, the inhibitors may include biological and chemical ones (e.g., 5azaC). Additionally, this may be used to measure the effects on histones, for example, using arrays, western blots, and mass spectrometry.

[0245] Any mention of publications or other documents, including but not limited to patents, patent applications, papers, web pages, books, etc., presented in this application is hereby incorporated by reference in its entirety into this specification.

[0246] Although several variations have been described in detail above, other modifications are possible. For example, any logical flow depicted in the accompanying drawings and described herein does not require the particular order or sequence shown to achieve the desired result. Other implementations may also be within the scope of at least some of the following exemplary claims.

[0247] Exemplary embodiments of devices, systems, and methods are described herein. As described anywhere, these embodiments are described for illustrative purposes only and not for limitation. Other embodiments will also be contemplated as possible, as will be apparent from the teachings contained herein, and will be covered by the present disclosure. Accordingly, the scope and range of the present disclosure should not be limited by any of the foregoing embodiments, but should be defined only in accordance with the claims supported by the present disclosure and their equivalents. Also, embodiments of the present disclosure may further include any elements from any other disclosed method, system, and device, including any and all elements corresponding to the collection, preservation, separation, and isolation of cells from body fluids (e.g., saliva, urine), as well as the collection of other substances containing toxic and / or harmful substances / fluids (and the preservation, separation, and isolation of their components). In other words, elements from one or another disclosed embodiment may be replaceable with elements from other disclosed embodiments. References (Incorporated herein by reference)

[0248]

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[0249] The present disclosure also relates to devices, solutions, and methods for collecting samples of body fluids or other substances, including harmful and / or toxic substances, specifically samples of naturally occurring body fluids (e.g., saliva, urine). Additionally or alternatively, the present disclosure generally relates to functional genetics and / or the isolation and preservation of DNA from such body fluids for (e.g.) subsequent genetic studies. Additionally or alternatively, the present disclosure generally relates to features that are suitable for home use.

[0250] International Publication No. WO2003 / 104251 (DNA Genotek Inc.) describes compositions and methods for preserving and extracting nucleic acids from collected samples of saliva. The composition includes chelating agents, denaturing agents, and buffers to maintain the pH of the composition within a desirable range for DNA and / or RNA. The composition may also include reducing agents and / or antibacterial agents. The document also describes a saliva sample collection container having a chamber containing the composition, and a technique for releasing the composition upon disassembly of a separation barrier when a cap is fitted to the container.

[0251] The concept of an in-built collection and preservation device has the potential for many different genetic testing applications without requiring the donor to physically visit a laboratory, but such potential can be severely limited by the effectiveness of the composition and the quality of the preserved solution. Some non-limiting aspects of the present invention may seek to at least mitigate such problems.

[0252] Reference is made herein to improved sample collection devices described in International Publication No. WO2012 / 177656 and / or International Publication No. WO2015 / 112496. The contents of these applications are incorporated herein by reference as if fully reproduced herein.

[0253] The following presents a simplified summary of the disclosure of Annex 1 to provide a basic non-limiting understanding of the present disclosure.

[0254] On one side, for example, a (e.g., portable) collection device is described that is suitable for use by a sample donor without the need to visit a laboratory. This sample collection device comprises a container for receiving a naturally occurring body fluid, such as saliva or urine. The device further comprises a chamber containing a composition for extracting and storing nucleic acids in the collected sample, and the composition is effective in extracting and storing nucleic acids from organelles. The organelle may be mitochondria (although the same principle may be applicable to other organelles).

[0255] In addition or alternatively, on one side, the composition is described for use in a sample collection device for collecting a sample of a naturally occurring body fluid and extracting and storing nucleic acids in the collected sample, and the composition is effective in extracting and storing nucleic acids from organelles. The organelle may be mitochondria (although the same principle may be applicable to other organelles).

[0256] The nucleic acid may be DNA and / or RNA, as desired.

[0257] Mitochondrial DNA can have very different properties from the following nuclear DNA. (i) Mitochondrial DNA is very small compared to nuclear DNA. It has a minimal chromosome encoding only 36 genes, consisting of 16,600 bp. (ii) Mitochondrial DNA also has a relatively low concentration compared to nuclear DNA. There may be about 100 to about 10,000 separate copies of mitochondrial DNA per cell. (iii) Mitochondrial DNA is inherited only from the mother. (iv) Mitochondrial DNA is circular. (v) Mitochondrial DNA is highly susceptible to the effects of mutations. This is one factor that makes mitochondrial DNA an attractive target for research and analysis, as it can be mutated or marked more readily than nuclear DNA. For example, certain mutations may be useful markers or potential neurological disorders that may be difficult (or impossible) to detect from nuclear DNA.

[0258] However, extracting and preserving mitochondrial DNA using a sample collection device of the type disclosed herein has not been previously known. The above characteristics make it difficult to extract and preserve mitochondrial DNA. First, the susceptibility to mutations means that mitochondrial DNA is particularly vulnerable to damage and the effects of mutations by conventional processes used to lyse sample cells to extract DNA. Second, the low concentration and small size of mitochondrial DNA exacerbate the difficulty of obtaining a sufficient amount of undamaged mitochondrial DNA to provide a good-quality sample for downstream analysis.

[0259] In some embodiments, the composition may be configured to lyse organelles (e.g., mitochondria). Exemplary compositions are described hereinafter.

[0260] In addition to or as an alternative to any of the above, in one aspect, the composition is described for use in a sample collection device for collecting a sample of a naturally occurring body fluid and extracting and preserving nucleic acids in the collected sample, wherein (i) the composition has a significantly higher concentration of lysis and / or preservative than conventional compositions, and / or (ii) the composition has a volume that is not substantially larger than the volume of the body fluid sample for which the composition is intended.

[0261] In some embodiments, the composition may have a volume that is smaller than the volume of the body fluid sample for which the composition is intended.

[0262] By using a composition volume that is not substantially larger than the volume of the body fluid sample for which the composition is intended, the volume increase when mixing the composition with the collected sample can be equally small. Providing a relatively small volume reduces the effect of the volume increase on the concentration of nucleic acids in the resulting mixture and / or reduces the dilution effect of the composition volume with respect to the DNA concentration per unit volume.

[0263] For example, in one embodiment, the ratio of the volume of the composition to the volume of the collected body fluid sample may be about 1:1. Using such a ratio, the concentration of the extracted and preserved nucleic acids per unit volume is halved because the net volume is doubled. Thus, the concentration can remain relatively high. It is contemplated that by using a smaller volume of the composition, the effect of the volume increase can be further reduced. For example, if the volume of the composition is about half the volume of the body fluid sample, the concentration of the extracted and preserved nucleic acids per unit volume could be two-thirds of the original concentration instead of half. Thereby, the concentration per unit volume is increased by about 33% compared to the 1:1 embodiment.

[0264] Using an increased concentration of lysis and / or preservative agent can compensate for the reduced volume of the composition.

[0265] In addition or alternatively, using an increased concentration of lysis and / or preservative agent can provide rapid lysis and / or preservation action, and / or lysis of intracellular organelles, either or both of which can shorten the time during which mutations in organelle DNA (e.g., mitochondrial DNA) can occur.

[0266] In addition to or alternatively to any of the above aspects, the composition is described for use in a sample collection device for collecting a sample of naturally occurring body fluid and extracting and preserving nucleic acids in the collected sample, and the composition comprises at least one lysis agent and / or at least one preservative agent.

[0267] Optionally, the composition may contain at least two solubilizers, and optionally may contain at least three solubilizers.

[0268] In addition or alternatively, the composition may optionally contain at least one preservative, at least one chemical inhibitor and / or denaturant for blocking proteins and / or DNAase. Optionally, the composition may contain at least two such chemical inhibitors and / or denaturants, and optionally may contain at least three such chemical inhibitors and / or denaturants.

[0269] Details of exemplary compositions are further described below.

[0270] In addition to or instead of any of the above aspects, in some embodiments, a composition is described that includes one, or any combination of two or three, of the following. At least one solubilizer for liberating nucleic acids from sample cells, selected from sodium dodecyl sulfate (SDS), Triton, Triton-X, Triton 100, Triton-X100, deoxycholate (e.g., sodium deoxycholate), cholate (e.g., sodium cholate), sodium lauroyl sarcosinate (and / or sarcosyl), maltoside (e.g., n-dodecyl-β-D-maltopyranoside and / or DDM), glycoside (e.g., digitonin), Tween (e.g., Tween 20 and / or Tween 80), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (and / or CHAPS), nonylphenoxypolyethoxylethanol (and / or Tergitol-type NP-40 and / or NP-40), sodium chloride (NaCl), lithium chloride (LiCl), potassium chloride (KCl), or any derivative of any of the foregoing (e.g., a commercially available derivative), including at least one, optionally at least two, optionally at least three. A buffer containing tris and / or methylenediaminetetraacetic acid (EDTA). At least one chemical inhibitor and / or denaturant for blocking proteins and / or DNAase, such as 2-mercaptoethanol, Ca 2+ At least one, optionally at least two, optionally at least three, selected from calcium ions, ethylene glycol tetraacetic acid (EGTA), methylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), iodoacetic acid, urea, etc., at least one chemical inhibitor and / or denaturant.

[0271] In addition to or instead of any of the above aspects, in some embodiments, a composition is described that includes at least one ionic surfactant (or ionic detergent), at least one non-ionic surfactant (or non-ionic detergent), and a salt. The composition may be a solution for lysing cells and / or organelles.

[0272] The combination of ionic and non-ionic surfactants has a synergistic stabilizing effect on micelle formation and may make micelle formation more resistant to high salt concentrations. This can avoid the undesirable precipitation of one or more of the surfactants even at relatively high surfactant concentrations and / or high salt concentrations. Allowing the solution to have both a high micelle surfactant concentration and a high salt concentration can enhance the cell and / or organelle lysis ability of the solution.

[0273] Examples of ionic surfactants or detergents include sodium dodecyl sulfate (SDS), deoxycholate (e.g., sodium deoxycholate), cholate (e.g., sodium cholate), sodium lauroyl sarcosinate (and / or sarcosyl), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (and / or CHAPS), and any one or more of the derivatives of the foregoing (e.g., commercially available derivatives).

[0274] In some embodiments, the ionic surfactant (or detergent) may include an amphoteric surfactant (or detergent), an example of which is CHAPS. In other embodiments, the ionic surfactant (or detergent) may optionally not include an amphoteric surfactant. For example, the ionic surfactant (or detergent) may include an anionic or cationic surfactant (or detergent).

[0275] Examples of non-ionic surfactants or detergents include Triton, Triton-X, Triton 100, Triton-X100, maltosides (e.g., n-dodecyl-β-D-maltopyranoside and / or DDM), glycosides (e.g., digitonin), Tweens (e.g., Tween 20 and / or Tween 80), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (and / or CHAPS), nonylphenoxypolyethoxylethanol (and / or Tergitol type NP-40 and / or NP-40), any one or more than one of derivatives (e.g., commercially available derivatives) of any of the foregoing.

[0276] Examples of salts include any one or more than one of sodium chloride (NaCl), lithium chloride (LiCl), and potassium chloride (KCl).

[0277] In another aspect, the annex 1 discloses a body fluid sample collection device for collecting a naturally occurring body fluid (e.g., saliva) comprising a composition for extracting and preserving nucleic acids in the collected sample. In some embodiments, the composition is effective in extracting and preserving nucleic acids from organelles. The organelle may be mitochondria. In some embodiments, the composition combines a plurality of lysing agents. In some embodiments, the composition includes at least one ionic detergent, at least one non-ionic detergent, and a salt.

[0278] Without limiting the present disclosure, a numbered item-by-item list of specific features and / or aspects disclosed in Attachment 1 follows:

[0279] Item number: 1. A composition for use in a sample collection device for collecting a sample of a naturally occurring body fluid sample, which is effective for extracting and storing nucleic acids in the collected sample and is effective for extracting and storing nucleic acids from organelles. 2. The composition according to item 1, wherein the organelle is mitochondria. 3. The composition according to item 1 or 2, wherein the nucleic acid comprises DNA. 4. The composition according to item 1, 2, or 3, wherein the composition comprises at least one lysing agent for lysing organelles. 5. The composition according to item 4, wherein the composition comprises at least two, optionally at least three, lysing agents. 6. The composition according to any of the preceding items, wherein the composition and / or the lysing agent comprises at least one selected from surfactants, detergents, salts. 7. The composition and / or the lysing agent comprises the following, including its derivatives; Sodium dodecyl sulfate (SDS), Triton, Triton-X, Triton 100, Triton-X100, deoxycholate (e.g., sodium deoxycholate), cholate (e.g., sodium cholate), sodium lauroyl sarcosinate (and / or sarcosyl), maltoside (e.g., n-dodecyl-β-D-maltopyranoside and / or DDM), glycoside (e.g., digitonin), Tween (e.g., Tween 20 and / or Tween 80), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (and / or CHAPS), nonylphenoxypolyethoxylethanol (and / or Tergitol type NP-40 and / or NP-40), sodium chloride (NaCl), lithium chloride (LiCl), potassium chloride (KCl) The composition according to any of the preceding items, selected from at least 1, optionally at least 2, optionally at least 3. 8. The composition according to any of the preceding items, comprising in combination (i) Triton or a derivative thereof, (ii) SDS or a derivative thereof, and (iii) a salt. 9. The composition according to any of the preceding items, comprising at least one chemical inhibitor and / or denaturant for blocking proteins and / or DNases. 10. The composition contains 2-mercaptoethanol, Ca 2+ ions, ethylene glycol tetraacetic acid (EGTA), methylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), iodoacetic acid, urea, selected from at least 1, optionally at least 2, optionally at least 3, of the composition according to any of the preceding items. 11. A composition for use in a sample collection device for collecting a sample of a naturally occurring body fluid sample, The composition is effective for extracting and preserving nucleic acids in the collected sample, The composition comprises at least one of the following, or any combination of two or three of them all, At least one lysing agent for releasing nucleic acids from sample cells, selected from sodium dodecyl sulfate (SDS), Triton, Triton-X, Triton 100, Triton-X100, deoxycholate (e.g., sodium deoxycholate), cholate (e.g., sodium cholate), sodium lauroyl sarcosinate (and / or sarcosyl), maltoside (e.g., n-dodecyl-β-D-maltopyranoside and / or DDM), glycoside (e.g., digitonin), Tween (e.g., Tween 20 and / or Tween 80), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (and / or CHAPS), nonylphenoxypolyethoxylethanol (and / or Tergitol type NP-40 and / or NP-40), sodium chloride (NaCl), lithium chloride (LiCl), potassium chloride (KCl) including derivatives thereof, at least one, optionally at least two, optionally at least three, including at least one lysing agent; A buffer containing Tris and / or ethylenediaminetetraacetic acid (EDTA); At least one chemical inhibitor and / or denaturing agent for blocking protein and / or DNAase, including 2-mercaptoethanol, Ca 2+ Ions, ethylene glycol tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), iodoacetic acid, urea, at least one, optionally at least two, optionally at least three, including at least one chemical inhibitor and / or denaturing agent selected therefrom. 12. The composition according to any of the preceding items, further comprising a pH buffer. 13. The composition according to item 12, wherein the buffer is Tris or contains it. 14. The composition according to any of the preceding items, having a pH greater than about 8. 15. The composition according to any of the preceding items, further comprising proteinase K. 16. The composition is the composition according to any of the preceding items, having a total salt concentration selected from 249 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM and exceeding at least one thereof. 17. The composition is the composition according to any of the preceding items, having a total salt concentration selected from 300 mM, 2000 mM, 1000 mM, 750 mM, 500 mM and not exceeding at least one thereof. 18. The composition is the composition according to any of the preceding items, having a volume selected from 5 ml, 4 ml, 3 ml, 2 ml, 1 ml, 750 μl, 500 μl, 250 μl, 100 μl and not exceeding at least one thereof. 19. The composition is the composition according to any of the preceding items, containing Tris, SDS, Triton, NaCl, and EDTA. 20. The composition according to item 19, further comprising one or more selected from proteinase K, sodium deoxycholate, and urea. 21. The concentration of the components in the composition is Tris: about (R + 1) * 10 mM SDS: about (R + 1) * 1% (v / v) Triton: about (R + 1) * 1% (v / v) NaCl: exceeding or equal to about (R + 1) * 250 mM EDTA: (R + 1) * 5 mM as defined by where R is the ratio Vs / V, Vs is a predetermined volume of sample Vs whose concentration is intended to be mixed during use, Vc is the volume of the composition, the composition according to item 19 or 20. 22. Optionally, a composition for lysing cell material according to any of the preceding items, the composition containing at least one ionic surfactant (or ionic detergent), at least one non-ionic surfactant (or non-ionic detergent), and a salt. 23. The composition according to item 22, wherein at least one ionic surfactant or detergent is selected from sodium dodecyl sulfate (SDS), deoxycholate (e.g., sodium deoxycholate), cholate (e.g., sodium cholate), sodium lauroyl sarcosinate (and / or sarcosyl), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (and / or CHAPS), any one or more than one of the derivatives of any of the foregoing. 24. The composition according to item 22 or 23, wherein at least one ionic surfactant or detergent is selected from Triton, Triton-X, Triton 100, Triton-X100, maltoside (e.g., n-dodecyl-β-D-maltopyranoside and / or DDM), glycoside (e.g., digitonin), Tween (e.g., Tween 20 and / or Tween 80), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (and / or CHAPS), nonylphenoxypolyethoxylethanol (and / or Tergitol-type NP-40 and / or NP-40), any one or more than one of the derivatives of any of the foregoing. 25. The composition according to item 22, 23, or 24, wherein the salt is selected from sodium chloride (NaCl), lithium chloride (LiCl), potassium chloride (KCl), any one or more than one of them. 26. The composition according to any one of items 22 - 25, having a total salt concentration of at least one selected from 249 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM. 27. The composition according to any one of items 22 - 26, wherein the ionic and non-ionic surfactants or detergents form a mixed micelle population and / or mixed micelles. 28. The composition according to any one of the preceding items, further comprising ethanol. 29. The composition according to any one of items 1 - 27, substantially free of ethanol. 30. A body fluid sample collection device for collecting a naturally expressed body fluid, comprising a container for receiving the naturally expressed body fluid and a chamber containing a composition for extracting and storing nucleic acids in the collected sample, wherein the composition is as defined according to any of the preceding items. 31. Optionally, the body fluid sample collection device according to item 30, for collecting a predetermined first volume of a naturally expressed body fluid, comprising a container for receiving a sample of the naturally expressed body fluid and a chamber containing a second volume of the composition for extracting and storing nucleic acids in the collected sample, wherein the second volume does not substantially exceed the first volume, optionally, is smaller than it. 32. The body fluid sample collection device according to item 31, wherein the second volume is defined by at least one selected from about 100% or less of the first volume; about 90% or less of the first volume; about 80% or less of the first volume; about 70% or less of the first volume; about 60% or less of the first volume; about 50% or less of the first volume; about 40% or less of the first volume; about 30% or less of the first volume; about 20% or less of the first volume; about 90% of the first volume; about 80% of the first volume; about 70% of the first volume; about 60% of the first volume; about 50% of the first volume; about 40% of the first volume; about 30% of the first volume; about 20% of the first volume; about 1 / 3 of the first volume; about 1 / 4 of the first volume. 33. The device according to item 31 or 32, wherein the first volume is defined by at least one of about 0.5 ml to about 2.25 ml, about 0.75 ml to about 2.25 ml, about 1 ml to about 2 ml, about 1 ml to about 1.5 ml, about 1 ml. 34. The device according to item 31, 32, or 33, wherein the first volume is about 1 ml and the second volume is about 250 μl. 35. A container for receiving a sample of a naturally occurring bodily fluid, and a chamber containing a composition for extracting and storing nucleic acids in the collected sample, for collecting a sample of a naturally occurring bodily fluid, optionally, the bodily fluid sample collection device according to any one of items 30 to 34, wherein the volume of the composition is from about 100 μl to about 1 ml, at least about 100 μl and less than 1 ml, from about 250 μl to about 750 μl, at least about 100 μl, at least about 250 μl, at least about 500 μl less than, less than 1 ml, less than about 750 μl, less than about 500 μl, about 100 μl, about 250 μl, about 500 μl, or about 750 μl, defined by at least one, a bodily fluid sample collection device. 36. Use of a composition according to any one of items 1 to 29 in a sample collection device for extracting and storing nucleic acids in a sample of a naturally occurring bodily fluid. 37. Use of proteinase K in a process for the extraction and storage of mitochondrial DNA using a sample collection device for home use.

[0280] Although various aspects of the present disclosure are emphasized above, this does not limit the scope of the present disclosure. Protection is claimed for any novel features and / or concepts described herein and / or illustrated in the drawings, whether or not they are emphasized.

[0281] Non-limiting embodiments relating to Annex 1 are described below by way of example only with reference to Figures 4, 5, and 8 of the accompanying drawings.

[0282] Referring to FIGS. 4 and 5, a sample collection device 10 of a type that may be suitable for home use or at least does not require a visit to a laboratory is depicted. The sample collection device 10 may be portable, for example, and / or provided to a user at home (e.g., via postal delivery or courier) to provide a sample of a bodily fluid naturally expressed by the user. The bodily fluid may be, for example, saliva or urine. The following description focuses on saliva, but it can be understood that the same principles can apply to urine or another naturally expressed bodily fluid.

[0283] In the illustrated embodiment of Appendix 1, a composition 12 is provided to extract and / or preserve nucleic acids in the collected bodily fluid sample. In use, the composition is intended to be mixed with the collected bodily fluid sample, for example, when closing the device to seal it. The device 10 may then be returned to the sender or sent to a collection agent or laboratory for analysis, such as genetic analysis.

[0284] The function of the composition 12 may be to extract nucleic acids from the bodily fluid sample and / or to preserve the extracted nucleic acids (or the bodily fluid sample). The nucleic acids may be DNA and / or RNA.

[0285] In some embodiments, the composition 12 may be effective to lyse organelles (e.g., mitochondria) and / or preserve the extracted organelle nucleic acids.

[0286] In some embodiments, the composition 12 may avoid or reduce DNA mutations, damage, or other degradation in the collected sample, thereby providing sufficient time for the device to be returned for analysis. The composition 12 may be effective to preserve the extracted nucleic acids at room temperature or for at least one week, optionally at least two weeks, optionally at least three weeks, optionally at least one month, or even longer periods.

[0287] Composition 12 may be a liquid. Further details of examples of the composition are discussed hereinafter below.

[0288] Composition 12 may optionally be stored within the internal chamber 14 of the device and, for example, released to mix with a collected body fluid sample when the user seals the device or performs certain other operations of the device 10.

[0289] Figures 4 and 5 illustrate two alternative embodiments of the collection device 10. The device 10 generally has an opening 18 and may comprise a first body (e.g., a tube) 16 that defines a collection region 20 for naturally occurring body fluids introduced into the device through the opening 18. The device 10 may further comprise a second body (e.g., a sealing device) 22 that is attachable to or on the opening 18 so as to seal the device 10 after a provided sample has been introduced. In the embodiment of Figure 4, the chamber 14 containing the preservation solution 12 is provided within the second body (e.g., the sealing device) 22. In the embodiment of Figure 5, the chamber 14 containing the composition is provided within the first body (e.g., the tube) 16. In either embodiment, the chamber 14 is configured to be opened by a mechanism (schematically indicated at 24) to communicate with the collection region 20. The opening mechanism 24 may respond to the fitting of the second body (e.g., the sealing device) 22 to the first body (e.g., the tube) 16 or to certain other manual operations of the device 10. Various mechanisms 24 are envisioned including, but not limited to, one or more selected from internal closure or release of a cap, opening of a tap, rupture of a frangible wall, removal or displacement of an internal cover, relative rotation of a cap or nut.

[0290] Optionally, further details of the exemplary structures of the device 10 and the opening mechanism 24 are provided in the aforementioned International Publications WO2012 / 177656 and WO2015 / 112496, which are incorporated herein by reference.

[0291] Device 10 may be configured for collecting a predetermined sample volume “Vs” of naturally occurring body fluid. The device may include, for example, a visual fill scale or fill line (e.g., indicated at 26) or other indicia to indicate when a predetermined sample volume Vs has been achieved. In some embodiments, the sample collection space may have a size equal to the predetermined volume Vs, or the sample collection space may be larger in volume. By way of example only, the predetermined volume Vs may, in some embodiments, be at least about 1 ml, or at least about 2 ml, or at least about 3 ml, or at least about 4 ml, or at least about 5 ml, or more. By way of example only, the predetermined volume Vs may, in some embodiments, be about 5 ml or less, or about 4 ml or less, or about 3 ml or less, or about 2 ml or less, or about 1 ml or less. By way of example only, in some embodiments, the predetermined volume Vs may, in some embodiments, be from about 1 ml to about 5 ml, optionally from about 1 ml to about 4 ml, optionally from about 1 ml to about 3 ml, optionally from about 1 ml to about 2 ml. By way of example only, the predetermined volume Vs may, in some embodiments, be about 1 ml, or about 2 ml, or about 3 ml, or about 4 ml, or about 5 ml.

[0292] In some embodiments, the volume "Vc" of the preservation solution 12 may not be substantially larger than a predetermined sample volume Vs, or optionally, may be smaller than that. By way of only one example, the composition volume Vc may be about 100% or less of Vs, or about 90% or less of Vs, or about 80% or less of Vs, or about 70% or less of Vs, or about 60% or less of Vs, or about 50% or less of Vs, or about 40% or less of Vs, or about 30% or less of Vs, or about 20% or less of Vs in some embodiments. By way of only one example, the composition volume Vc may be about 100% of Vs, or about 90% of Vs, or about 80% of Vs, or about 70% of Vs, or about 60% of Vs, or about 50% of Vs, or about 40% of Vs, or about 30% of Vs, or about 20% of Vs in some embodiments. By way of only one example, the composition volume Vc may be about one-third of Vs, or about one-fourth of Vs in some embodiments. The composition volume Vc may be the same as the internal space of the chamber 14 to fill the chamber 14, or the composition volume Vc may be smaller and partially fill the chamber 14.

[0293] By not making the composition volume Vc substantially larger than a predetermined sample collection volume Vs, or optionally, making it smaller, the volume increase when mixing the preservation solution 12 with the collected sample can be equally small. The volume increase can be equivalent to the dilution of the sample with respect to the DNA concentration per unit volume. FIG. 8 schematically illustrates the influence of dilution on the DNA sample concentration per unit volume depending on the composition volume Vc. The horizontal axis represents the ratio R of the sample volume Vs to the composition volume Vc over a range of ratios R = Vs / Vc from 1 / 1 to 5 / 1 (or R = 1 to 5). The vertical axis represents the ratio of the DNA concentration per unit volume after mixing with the composition volume compared to the initial sample volume. The values vary as the function Vs / (Vs + Vc) and are equivalent to the dilution by the composition volume.

[0294] In some embodiments disclosed herein, the ratio Vs / Vc may be about 1 / 1 (or R may be about 1). In FIG. 8, it can be seen that such a composition volume Vc reduces the DNA concentration per unit volume to up to 50% of the original amount, which was initially sufficient. However, by using a smaller composition volume Vc (equivalent to a larger ratio of sample volume Vs to composition volume Vc), it is thought that the concentration of DNA per unit volume can be increased compared to the 1 / 1 ratio embodiment. For example, using a composition volume that is half of the sample volume equal to a sample volume Vs to composition volume Vc ratio of 2 / 1 (R = 2), the DNA concentration can be increased, for example, by up to 66% of the original amount, which is a 30% improvement compared to the 1 / 1 ratio embodiment. Using a composition volume Vc that is one-third of the sample volume Vs equal to a sample volume Vs to composition volume Vc ratio of 3 / 1 (R = 3), the DNA concentration can be increased, for example, by up to 75% of the original amount, which is a 50% improvement compared to the 1 / 1 ratio embodiment. Using a composition volume Vc that is one-fourth of the sample volume Vs equal to a sample volume Vs to composition volume Vc ratio of 4 / 1 (R = 4), the DNA concentration can be increased, for example, by up to 80% of the original amount, which is a 60% improvement compared to the 1 / 1 ratio embodiment.

[0295] Achieving a desirably high DNA concentration per unit volume can provide important advantages for downstream analysis. First, modern automated assay procedures tend to use only small sample volumes. Ensuring that even small-volume samples contain good concentrations of DNA after extraction and storage is important in order to benefit from automated assays. Second, in collected samples, mitochondrial DNA is present at much lower concentrations compared to nuclear DNA, which is far more predominant. Improving the overall concentration of DNA per unit volume helps increase the sensitivity of automated assays for such small amounts of DNA. This can contribute to enabling the detection of mitochondrial DNA using automated assays of samples collected in relatively simple collection devices, which is understood to not be possible using currently commercially available devices.

[0296] In one specific example, a given sample volume Vs can be about 2 ml and the composition volume Vc can be about 2 ml. In another specific example, a given sample volume Vs can be about 1 ml and the composition volume Vc can be about 1 ml. In another specific example, a given sample volume Vs can be about 1 ml and the composition volume Vc can be about 250 μl.

[0297] Additionally or alternatively to any of the above, and by way of example only, the composition volume Vc can, in some embodiments, include (or optionally be less than 1 ml) from about 100 μl to about 1 ml. Additionally or alternatively, the composition volume Vc can optionally be at least about 100 μl, optionally at least about 250 μl, optionally at least about 500 μl. Additionally or alternatively, the composition volume can optionally be about 1 ml, optionally less than 1 ml, optionally less than about 750 μl, optionally less than about 500 μl. By way of example only, the composition volume Vc can, in some embodiments, be about 100 μl, or about 250 μl, or about 500 μl, or about 750 μl.

[0298] The concentration of the active ingredient of composition 12 may be variably adjusted as appropriate to compensate for the use of a small composition volume Vc. For example, when using a composition volume Vc that is about half of the reference, the concentration of the active ingredient of composition 12 may be 2 times or more than the concentration of the active ingredient of the reference composition 12. Varying the composition of the active ingredient can provide at least the same (or better) extraction and preservation ability despite the smaller composition volume Vc. A more detailed description of the component concentration will be discussed later.

[0299] In addition to, or alternatively to, the above-described feature of reducing dilution by using a small composition volume Vc (e.g., not substantially larger than or optionally smaller than the sample volume Vs), further features of the present disclosure may relate to the components of composition 12. These features regarding the components may be used regardless of the ratio of the sample volume Vs to the composition volume Vc, although the ratio may be useful for determining the preferred concentration of the components in the composition.

[0300] In some embodiments, the composition may include any one or more selected from the following. (i) At least one cell and / or organelle lysing agent for lysing cells and / or organelles to release nucleic acids therefrom. Suitable lysing agents may include one or more surfactants, and / or one or more detergents, and / or one or more salts. Exemplary surfactants may be sodium dodecyl sulfate (SDS), but in addition to, or alternatively, other surfactants may be used. Exemplary detergents may be Triton (e.g., Triton-X, Triton 100, Triton-X100), and / or deoxycholate (e.g., sodium deoxycholate), but in addition to, or alternatively, other detergents may be used. Exemplary salts may be selected from sodium chloride (NaCl), and / or lithium chloride (LiCl), and / or potassium chloride (KCl), but in addition to, or alternatively, other salts may be used. Additional surfactants and / or detergents are further described below. To avoid misunderstanding, where the context permits, the terms "surfactant" and "detergent" may be used synonymously and are intended to be read synonymously. Also, when a surfactant or detergent is described by name, the scope is intended to equally cover any derivatives (e.g., commercially available derivatives).

[0301] Surfactants and / or detergents can solubilize cells and membrane components to disrupt the membrane. Salts can adjust the osmolarity.

[0302] In some embodiments, at least two lysing agents (e.g., selected from the above) are used in combination, and optionally, at least three lysing agents (e.g., selected from the above) are used in combination.

[0303] In some embodiments, the salt concentration (in the combination of Composition 12 when combined with Composition 12 and / or a sample) can be at least 250 mM, optionally greater than 250 mM. This salt concentration can be the total salt concentration, or the concentration of at least one major salt component or more. In some embodiments, this salt concentration can be at least 300 mM, optionally in the range of 300 - 750 mM. Such concentrations are higher than those that would conventionally be used and can contribute to the organelle lysis ability of Composition 12.

[0304] In some embodiments, it is considered beneficial to use a non-ionic surfactant and / or detergent (e.g., Triton) in combination with an ionic surfactant and / or detergent (e.g., SDS). The ionic and non-ionic surfactants and / or detergents buffer each other and can stabilize surfactant aggregation such that micelles are formed desirably, even at relatively high salt concentrations (e.g., as in the previous paragraph). For example, Triton can have a stabilizing effect on SDS, preventing or reducing the risk of precipitation of SDS that could otherwise occur at relatively high salt concentrations. Additionally, or alternatively, for example, SDS can stabilize the formation of Triton micelles even in the presence of relatively high salt concentrations.

[0305] The stabilizing effect of the combination of ionic and non-ionic surfactants and / or detergents can be explained as follows, although it is not intended to limit the scope of the present disclosure in any way. In some embodiments, the composition can contain both (i) a surfactant / detergent and (ii) a salt component. The detergents are surfactants and they are used to disrupt cell membranes and thus assist in cell lysis and promote the release of intracellular substances in soluble form. Detergents disrupt protein-protein, protein-lipid, and lipid-lipid associations. Further, detergents denature proteins and various other macromolecules and prevent non-specific binding and protein crystallization in immunochemical assays.

[0306] Surfactants / detergents dissolved in solution may tend to aggregate to form so-called micelles. All detergents at a specific concentration form micelles, and this concentration is referred to as the critical micelle concentration (CMC). Once the CMC is reached, detergent monomers form micelles. At concentrations above the CMC, both micelles and monomers may be present in solution along with other non-micellar phases that may not be soluble in water.

[0307] Ionic surfactants / detergents (e.g., SDS) form ionic micelles. When these micelles (when alone) are exposed to high salt concentrations, the micelles break down, SDS coagulates and precipitates from the solution, and does not have the desired effect within the solution.

[0308] Triton X is another detergent, while SDS is ionic and Triton-X is non-ionic. When both SDS and Triton are dissolved in solution, the micelle population is one of both ionic (SDS) and non-ionic (Triton) micelles and is considered a mixed micelle population. This mixed micelle population has a much higher tolerance to salt concentration, and the non-ionic micelles buffer the effect of the salt on the ionic micelles.

[0309] Temperature, pH, and salt concentration can each affect the CMC of the solution (see below for a detailed explanation of each). Therefore, modifying any of the above can lead to the formation of precipitates. The concentration of detergent required to form micelles is specific. The exemplary buffer discussed herein contains additional sodium in the form of NaCl that reduces the critical micelle concentration and thus risks the precipitation of SDS by dramatically exceeding the CMC and coagulating and precipitating in the absence of the complementary ionic / non-ionic surfactant.

[0310] The combination of SDS and Triton X is synergistic with respect to reducing the CMC and leading to the formation of larger mixed ionic / non-ionic micelles. The concentrations and ratios of the two detergents play a role in determining the stability of the micelles. Thus, these mixed detergent micelles are more stable in high salt concentration salt buffers and can thus prevent any precipitation of SDS. The combination of SDS and Triton-X probably forms larger mixed micelles with enhanced stability, as well as better water molecule association (due to the NaCl concentration), both contributing to the absence of SDS precipitation.

[0311] Synergistically, the presence of NaCl may also reduce the CMC, increase the size of the micelles formed, and cause micellization of Triton-X by a larger number of water molecules that bind non-specifically to the micelles. This may also be buffered by the mixed micelle population (i.e., the addition of ionic micelles) according to the principles disclosed herein.

[0312] Other detergents that can be used and do not modify biological activity include 2% Tween-20, NP40, and 0.1 - 1% deoxycholate.

[0313] The following are two non-limiting tables of exemplary detergents. Table 1 lists the categories of detergents and Table 2 lists the exemplary uses of each detergent as described herein.

Table 1

[0314]

Table 2

[0315] (ii) A buffer containing or consisting of, for example, Tris and / or methylenediaminetetraacetic acid (EDTA). The combination of Tris and EDTA can buffer the solution and exclude magnesium (inhibit DNase). This combination can provide a basic DNA storage buffer. EDTA can degrade RNA (if desired).

[0316] In some embodiments, the pH of both the sample and the composition can be slightly basic, for example, from about 7.4 to about 8, after being mixed together. The pH of Composition 12 alone (for example, prior to mixing with the sample) can be higher. The pH of Composition 12 alone can be at least about 8, optionally greater than 8, for example, about 8.3.

[0317] (iii) At least one chemical inhibitor and / or denaturant that blocks proteins and / or DNase. Suitable inhibitors and / or denaturants include one or more selected from 2-mercaptoethanol, Ca 2+ ions, ethylene glycol tetraacetic acid (EGTA), methylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), iodoacetic acid, urea.

[0318] As described above, EDTA is a chelating agent for divalent cations such as Mg 2+ in DNase nuclease. In some embodiments, EDTA functions simultaneously as a buffer and a denaturant.

[0319] (iv) Proteinase K for eliminating proteins. Proteinase K may be more effective when combined with SDS. For example, Proteinase K can be up to about 10 times more effective against denatured proteins, and SDS is effective as a protein denaturant. Proteinase K can be avoided from being inhibited by SDS or EDTA, other chemicals in the solution, and protein inhibitors. In some examples where the long-term stability of Proteinase K is regarded as a problem, this can generally be overcome by increasing the amount of Proteinase K in Composition 12, thereby compensating in advance for the expected partial loss over time. As only one example, the amount of Proteinase K in Composition 12 and / or in the combination of Composition 12 and the sample after mixing together may be about 30 - 70 mg / ml, optionally about 40 - 60 mg / ml, optionally about 50 mg / ml.

[0320] In some embodiments, the total salt concentration may be less than about 3000 mM, optionally less than about 2000 mM, optionally less than about 1000 mM, optionally less than about 750 mM, optionally less than about 500 mM. The salt concentration can be additive among all salt components in Composition 12 (e.g., including EDTA). An overly high total salt concentration can restrict the ability to isolate DNA in subsequent processing of collected and stored samples for genetic analysis. The salt concentration may be the concentration referred to when mixed with saliva or the concentration of the composition alone.

[0321] One specific example of Composition 12 may include Tris, SDS, Triton, NaCl, and EDTA. Optionally, the composition may include one or more selected from Proteinase K, sodium deoxycholate, and urea. In one example, the relative concentrations of the components, determined in the combination of both Composition 12 and the saliva sample when mixed together, may be as shown in Table 3 below.

Table 3

[0322] The concentration may be varied individually and independently within the scope of this specific embodiment, up to a maximum of 5%, optionally up to a maximum of 10%, and optionally up to a maximum of 20%.

[0323] As described above, the concentration is the desired concentration in a solution containing both Composition 12 and the saliva sample. The concentration of the components in Composition 12 alone may depend on the ratio R (R = Vs / Vc) of the sample volume Vs to the composition volume Vc, as previously described herein. The concentration may vary according to a concentration multiplier of (R + 1) or ((Vs / Vc) + 1). General values and some specific examples are illustrated in Table 4 below. [Table 4]

[0324] Again, the concentration may be varied individually and independently within the scope of these specific embodiments, up to a maximum of 5%, optionally up to a maximum of 10%, and optionally up to a maximum of 20%.

[0325] The present disclosure also contemplates the following within its scope. (a) A step of varying the proportion of SDS, including up to 0% (omitting SDS). (b) A step of varying the proportion of Triton, including up to 0% (omitting Triton). (c) A step of adding KCl and / or LiCl (salts that maintain the ionic strength of the buffer), either as a substitute for NaCl (omitting NaCl) or in combination with NaCl as a complement. (d) A step of varying the individual and / or total salt concentration. (e) For example, the step of adding proteinase K in an amount as described above. (f) The step of adding 0.1 - 1% sodium deoxycholate, which is a biological detergent capable of solubilizing membranes and cellular components and can increase organelle lysis. Sodium deoxycholate may be used to replace SDS and / or Triton, or may be used in combination with both. The relative proportions of SDS and / or Triton may also be varied. (For example, the ranges cited above may refer to the composition when mixed with saliva. The concentration may be increased in the composition alone according to the factor R + 1 described herein.) (g) The step of adding urea. Urea may increase protein denaturation and increase cell lysis. The proportions of SDS and / or Triton and / or other denaturing agents may be varied. (h) The step of using one or more other ionic and / or non-ionic surfactants and / or detergents in addition to or as an alternative to SDS and / or Triton.

[0326] Optionally, according to the intended use, composition 12 may further contain ethanol. Alternatively, optionally, composition 12 may be substantially free of ethanol.

[0327] In some embodiments, composition 12 is effective for extracting and preserving nucleic acids from organelles. The organelles may be, for example, mitochondria.

[0328] It is known that it is difficult to extract and preserve mitochondrial DNA. Mitochondrial DNA is highly susceptible to mutations by conventional lysis techniques and has a very low concentration compared to nuclear DNA. This makes it particularly difficult to extract and preserve such DNA in sufficient quantities and without damage for downstream analysis using sample collection devices of the type used in the home. In a laboratory environment different from the sample collection devices used in the home, the general knowledge for obtaining mitochondrial DNA is to very slowly and delicately lyse cells under controlled conditions that do not present significant environmental effects so as to reduce the risk of damage to mitochondrial DNA.

[0329] Some embodiments of the present disclosure present different approaches. Each, within a high range of the proposed uses, by combining a plurality of lysis agents, for example, at least two lysis agents, optionally at least three lysis agents, or more lysis agents, not only the cell membrane but also the organelle membrane can be lysed to rapidly release nucleic acids from the organelles, thereby shortening the time during which lysis damage can occur.

[0330] In addition or alternatively, each, within a high range of the proposed uses, by combining a plurality of chemical inhibitors and / or denaturing agents, for example, at least two such drugs, optionally at least three such drugs, the extracted nucleic acids can be rapidly protected from damage by surrounding environmental substances, thereby reducing the amount of damaged organelle DNA.

[0331] Both of the above techniques offer surprising advantages compared to the conventional laboratory knowledge of a slow and delicate approach under controlled conditions that do not present significant environmental effects on organelle DNA. Using the above techniques in combination can provide surprising effectiveness.

[0332] The compositions described herein can find use in many different applications for lysing cellular material to extract cellular components. Preferred embodiments are described in the context of DNA extraction, such as organelle DNA extraction, but the compositions are not limited to such uses only. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for diagnosing and / or monitoring a medical condition, comprising: obtaining a sample of a naturally occurring body fluid; contacting the sample with a preservation solution for stabilizing cells and / or extracellular components in the sample; and analyzing the stabilized cells and / or extracellular components from the sample to identify, diagnose, or monitor the medical condition. A method comprising the above steps. (Item 2) The method according to item 1, wherein the naturally occurring body fluid comprises one or more selected from the group consisting of oral fluid, saliva, sputum, urine, and lung aspirate fluid. (Item 3) The method according to item 1, further comprising contacting the sample with a pretreatment agent prior to contacting the sample with the preservation solution, the pretreatment agent being configured to stimulate the release of one or more factors indicative of the medical condition. (Item 4) The method according to item 1, further comprising separating at least certain cells from at least certain extracellular components. (Item 5) The method according to item 1, wherein the analyzing step comprises flow cytometry. (Item 6) The method according to item 1, wherein the analyzing step comprises fluorescence-activated cell sorting (FACS). (Item 7) The method according to item 1, wherein the analyzing step comprises immunohistochemistry. (Item 8) The step of analyzing is the method according to item 1, including molecular analysis. (Item 9) The step of analyzing is the method according to item 1, including the step of identifying HIV virus infection prior to the presence of detectable antibodies in the sample. (Item 10) The step of analyzing is the method according to item 1, including the step of identifying the HIV virus in T cells, optionally CD4+ T cells. (Item 11) The step of analyzing is the method according to item 1, optionally including the step of assessing the amount of CD4+ T cells to identify immunodeficiency indicating HIV infection and / or AIDS. (Item 12) The step of analyzing is the method according to item 1, including the step of identifying biomarkers for (i) indicating COPD and / or (ii) indicating ACOS and / or (iii) differentiating between ACOS and non-ACOS types of COPD. (Item 13) The step of analyzing is the method according to item 1, including the step of performing at least one of electrolyte measurement, lipid measurement, vitamin measurement, hepatitis detection, iron deficiency profile measurement, liver function test, kidney profile measurement, diabetes screening test, blood picture measurement, thyroid profile measurement. (Item 14) The preservation solution is hypertonic with respect to blood, the method according to item 1. (Item 15) After the step of contacting the sample with the preservation solution, the combination of the sample mixed with the preservation solution is substantially isotonic, and the tonicity is defined with respect to blood, the method according to item 1. (Item 16) A solution for preserving cells and / or extracellular components in naturally occurring body fluids for further downstream analysis and / or for diagnosis of a medical condition, the solution being hypertonic with respect to blood. (Item 17) The solution according to item 1, wherein the naturally occurring body fluid includes one or more selected from oral fluid, saliva, sputum, urine, and fluid of lung aspirate. (Item 18) A fluid sample for downstream analysis of cells and / or extracellular components in a fluid sample, the fluid sample comprising a naturally occurring hypotonic body fluid to be mixed with a preservation solution, the sample fluid being substantially isotonic, and the tonicity being defined with respect to blood. (Item 19) A sample collection device for collecting a sample of a naturally occurring body fluid, the sample collection device containing or including a solution for preserving cells and / or extracellular components in the naturally occurring body fluid, the solution being hypertonic with respect to blood. (Item 20) A sample collection device containing a fluid sample, the fluid sample including (i) a naturally occurring hypotonic body fluid to be mixed with (ii) a preservation solution, the sample fluid being substantially isotonic, and the tonicity being defined with respect to blood.

Claims

【Claim 1】 An article, method or system described in this specification and the drawings.

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