Method for stabilizing hemoglobin and reagents for carrying it out

Abstract

A stool resuspension comprising a hemoglobin stabilizing reagent is provided.SOLUTION: In some embodiments, the hemoglobin stabilizing reagent may be an osmolyte, a multivalent cation, a sugar or polysaccharide, and optionally a multivalent cation, a protoporphyrin, or an HRP stabilizing component, and optionally a multivalent cation. Also provided are methods of stabilizing hemoglobin in a stool sample in a solution, and sample collection devices comprising the solution.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application is a continuation of U.S. Provisional Application No. 62 / 648,874, filed March 27, 2018, and and U.S. Provisional Application No. 62 / 685,248, filed June 14, 2018. No. 6,299,799, filed on Oct. 1, 2003, and which applications are incorporated herein by reference. [Background technology]

[0002] background Tests that detect hemoglobin (Hb) in stool samples are used for colorectal cancer screening. The typical procedure involves the use of a detection assay in the laboratory at the time of specimen collection. Use a specimen collection device containing a buffer to stabilize hemoglobin until the procedure is completed. The ability of the buffer to adequately stabilize hemoglobin is crucial for accurate test results. It is very important for the stability of hemoglobin during transportation under various environmental conditions. Increasing the buffer's ability to stabilize hemoglobin can, for example, allow for longer transport periods. This allows for faster turnaround times and provides more flexibility for patients and laboratories for sample processing. become. Summary of the Invention

[0003] Provided herein, among other things, is a stool resuspension solution that includes a hemoglobin stabilizing reagent. In some embodiments, the hemoglobin stabilizing reagent in solution is an osmolyte, a multivalent cation, or a hydroxyl group. a polysaccharide, a polyvalent cation, protoporphyrin, or HR P stabilizing component, and optionally, may be a multivalent cation.

[0004] This solution may be used in methods that can benefit from hemoglobin stabilization. In some embodiments, the method comprises mixing the stool sample with a stool resuspension solution to form a suspension. and maintaining the suspension for a period of time. In some embodiments, the method may include transporting the suspension to a remote location. The globin stabilizing reagent stabilizes hemoglobin, thereby reducing the amount of hemoglobin in the sample. This allows Robin to be measured more accurately after several days of transport.

[0005] Compositions, methods and devices that use fecal resuspension solutions are provided. DETAILED DESCRIPTION OF THE INVENTION

[0006] Description of exemplary embodiments Before describing the present invention in more detail, it is to be understood that the invention is limited to the specific embodiments described. It should be understood that the present invention is not limited to the above and may, of course, vary. The scope of the invention is limited only by the appended claims, and therefore the present specification The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should also be understood that this is not the case.

[0007] When a range of values ​​is given, each value in between is a value unless the context clearly dictates otherwise. Between the upper and lower limits of a range, to one-tenth of the unit of the lower limit, unless otherwise specified, and Any other stated or intervening value within the stated range is encompassed within the present invention. It is understood that the upper and lower limits of these smaller ranges are included within the scope of the smaller ranges. may be independently included in the range, subject to any specifically excluded limitation within the stated range. If the stated range includes one or both of the limits, In this case, a range excluding either or both of those included limitations is also included in the present invention.

[0008] Unless otherwise defined, all technical and scientific terms used herein are Scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. Although the present invention may also be used in clinical trials, preferred methods and materials are described below.

[0009] All publications and patents cited herein are incorporated by reference in their entirety. Patents are incorporated by reference as if they were specifically and individually indicated to be incorporated by reference. and the methods and / or materials for which such publications are cited in connection therewith, are incorporated herein by reference. The present application is hereby incorporated by reference to disclose and explain the subject matter herein. Any publication citation is in accordance with the filing date of the application. and the present invention is hereby expressly granted, by virtue of its disclosure prior to such publication by virtue of prior invention. This should not be construed as an admission that you have no legal right to claim that you have a right to use the product. Furthermore, the publication dates provided may differ from the actual publication dates, which should be independently confirmed. is required.

[0010] As used in this specification and the appended claims, the singular forms "a," "a" and "b" are used interchangeably. "n" and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the claims may be drafted to exclude any optional element. It should be noted that this statement may be applied to the elements of the claims. In connection with the use of a statement or a "negative" limitation, exclusionary terms such as "solely," "only," etc. It is intended to serve as a preliminary basis for the use of other terms.

[0011] As will be apparent to those skilled in the art upon reading this disclosure, each of the embodiments shown and described herein Each of the above may be implemented in several other ways without departing from the scope and spirit of the present invention. Individual components that can be easily separated from or combined with state features Any of the methods described may be carried out in the order of events described or in a manner consistent with the principles of the invention. It may be performed in any other order that is reasonably possible.

[0012] A stool resuspension solution containing one or more hemoglobin stabilizing reagents is provided. In some embodiments, one or more hemoglobin stabilizing reagents may be selected from the group consisting of, for example, osmolytes, multivalent cations, and the like. , a sugar or polysaccharide, and optionally a multivalent cation, protoporphyrin, The cations may be selected from a phosphate-soluble polymer and an HRP stabilizing component, and optionally a multivalent cation. Examples of such reagents and their concentrations in solution are given in the table below and in the Appendix.

[0013] In some embodiments, the solution contains an osmolyte (e.g., betaine). In these embodiments, the osmolyte (e.g., betaine) may be The concentration can range from 2M to 5M.

[0014] In some embodiments, the solution contains a sugar (e.g., sucrose or trehalose). In these embodiments, the sugar (e.g., sucrose or trehalose) may be The concentration can range from 0.1 M to 0.5 M. In either case, the solution contains a multivalent Thione, Mg 2+ or Ca 2+ In these embodiments, Multivalent cations (e.g., calcium or magnesium ions) are present in concentrations between 5mM and 25mM. The concentration can be in the range of M.

[0015] In some embodiments, the solution comprises a polysaccharide (e.g., an α-(1-4) linked D-galacturonate). Substituted or unsubstituted polygalacturons of α-(1-4)-linked D-galacturonic acid In these embodiments, the polysaccharide (e.g., substituted or unsubstituted polysaccharide) may contain an acid. Ligalacturonic acid) 0.005% to 0.5% (e.g., 0.01% to 0.125%) In these embodiments, the solution may optionally contain multivalent cations. In embodiments where the solution contains multivalent cations, the multivalent cations may be 5 In some embodiments, the solution may contain 5 mM to 25 mM of HCl. Substituted or unsubstituted polygalacturonic acid and polyvalent cations (e.g., For example, calcium salts or magnesium salts).

[0016] In some embodiments, the solution contains protoporphyrin (e.g., protoporphyrin IX), or its analogs (e.g., octaethylporphyrin complexed with a metal ion, containing tetraphenylporphyrin (H2OEP) or tetraphenylporphyrin (H2TPP) In these embodiments, the protoporphyrin can be fused to hemin (a coordinating chloride ligand). Ferric oxide (Fe 3+ ) ion-containing protoporphyrin IX) or hematoxylin In other embodiments, the protoporphyrin may be a divalent or trivalent cation. (e.g., Zn 2+ , Cr 3+ , or Co 3+ ) complexed with protoporphyrins In these embodiments, protoporphyrin may be 0.1 μM to 1 Some may be present in solution at concentrations ranging from 0 to 10 μM (e.g., 1 μM to 10 μM). In embodiments, the solution contains an HRP conjugate stabilizer (PN 85R-102; Fitzgerald d Industries), HRP conjugate stabilizer (PN SZ02; Surmodics), and HRP conjugate The compound may comprise an HRP stabilizing component selected from the group consisting of adjuvant stabilizer (PN ab171537; Abcam). Other HRP stabilizing components (e.g., AbGuard (BioRad PN: BUF052; BioRad Laboratories) Inc. 2000 Alfred Nobel Dr. Hercules, CA 94547)). If the formulation contains an HRP stabilizing component, the component may be present at 1% to 20% (e.g., 5% to 15% or The concentration can range from 5% to 20%).

[0017] In any embodiment, the solution contains a multivalent cation (e.g., calcium ions or maize ions). In these embodiments, the polyvalent cation may comprise 5 mM to 10 mM of ammonium hydroxide. The concentration may be in the range of 25 mM.

[0018] In any embodiment, the solution is a Tris buffer (e.g., 10 mM to 50 mM Tris , pH 7.5), bovine serum albumin (e.g., 5% to 20% BSA), polysorbate 20 (e.g., 0.05% to 0.2% polysorbate 20), sodium azide, etc. Preservatives (e.g., 0.05% to 0.2% sodium azide), salts such as sodium chloride (e.g., 50mM to 250mM sodium chloride), ethylenediaminetetraacetic acid, etc. rate inhibitors (e.g., 5 mM to 20 mM ethylenediaminetetraacetic acid), and gentamicin The solution further contains an antibiotic such as benzodiazepine (for example, 5 μg / mL to 50 μg / mL).

[0019] Also provided is a composition comprising (a) a stool sample and (b) the stool resuspension solution described above. The sample may be suspended in the solution. Some components of the stool may be dissolved in the solution, where Other insoluble components may be suspended but not dissolved in the solution. In these embodiments, the stool sample may contain hemoglobin, which may be dissolved in solution. In this condition, the presence of hemoglobin stabilizing reagents significantly reduces the risk of death in otherwise identical, unstressed animals. As described below, the actual or the amount of hemoglobin in the sample after exposure to simulated transport stress. In some embodiments, the use of a hemoglobin stabilization reagent can increase Control fecal samples suspended in an otherwise identical solution without the hemoglobin stabilizing reagent The amount of hemoglobin in the sample should be increased by at least 10% and at least 20% compared to the sample. The amount of hemoglobin in the sample can be increased by at least 30%. It can be determined by enzyme-linked immunosorbent assay (ELISA), although several other methods are publicly known. (Generally, Haug et al., Am J Gastroenterology 105: 682-690; Ahlquist et al., Ann Intern Med 1984;101:297-302; Ahlquist et al., JAMA 1993 269:1262-1267; Young et al., Dig Dis Sci. 2015 60: 609-622; and Harewood et al., Mayo Clin. Proc. 2002 77: 23-28 (See

[0020] Also provided are methods for stabilizing fecal hemoglobin. In some embodiments, the methods include , the stool sample is resuspended in a stool resuspension solution as described above, i.e., osmolytes, multivalent cations, a sugar or polysaccharide, and optionally a multivalent cation, protoporphyrin; and HRP a stabilizing component, and optionally one or more hemoglobins selected from multivalent cations; mixing the solution containing the amine stabilizing reagent to form a suspension; and This may include maintaining the solution for a period of time. and their concentrations are described above, below, and in the attached appendix. In this method, for example, a stool sampling device is used to scrape or This may involve obtaining a stool sample from a large volume of feces by stooling.

[0021] In some embodiments, the method further comprises transferring the sample to a remote location, e.g., another This may include transporting the sample to a town, city, state, or country. The product may be transported en route or may be stored in a remote location for several days (e.g., 3, 4, 5, 6, or 7 days) to be tested, thereby The sample is subjected to stresses that potentially reduce the amount of hemoglobin in the sample.

[0022] Also, (a) stool samples and (b) other substances, such as osmolytes, multivalent cations, sugars, etc. or polysaccharides, and optionally, multivalent cations, protoporphyrin, and HRP stabilization component, and optionally one or more hemoglobin stabilizers selected from multivalent cations. Also provided are methods that include receiving a composition comprising a stool resuspension solution that includes a stabilization reagent. Examples and concentrations of such reagents in the solution are described above, below and in the accompanying appendix. In certain embodiments, the method comprises: The method may further include performing an assay, e.g., measuring the concentration of hemoglobin in the solution. Methods for doing this are described above. The amount of one or more colorectal cancer tumor markers can be tested after the sample is received. (Generally, see Lech et al, World J Gastroenterol. 2016 22: 1745-1755 and Scherud ers et al, Curr. Treat. Options Gastroenterol. 2016 14: 152-162). These embodiments may also include transmitting test results, for example, by fax or email, to: or further including transmitting the information to a remote location by internet via a portal. so that the results can be obtained by a medical practitioner (e.g., an MD or nurse, etc.) and a diagnosis can then be provided to the patient from whom the stool sample was taken.

[0023] Also, (a) a sample collection container having an open end; (b) a stool resuspension solution ( wherein the solution contains osmolytes, multivalent cations, sugars, etc., as described herein. or polysaccharides, and optionally multivalent cations, protoporphyrin, and HRP stabilizing a hemoglobin-stabilizing component, and optionally one or more polyvalent cations selected from (c) a catheter for scooping and / or scraping the stool sample; A sample collection device is provided that includes a stool sampling rod that includes a tilted tip. In this device, the distal end of the sample collection rod is sized to be inserted into the sample collection container. the proximal end of the sample collection rod is adapted to connect with the open end of the sample collection container. The distal end of the sample collection rod is then sealed within the device. In this configuration, the sample collection container and the sampling rod are connected via a threaded fit. With the exception of the hemoglobin stabilizing reagent, the details of an example of such a sample collection device, selection Limbs and designs can be found, for example, in U.S. Pat. No. 9,211,112. In one embodiment, the device comprises: a) a body; at its distal end by a pierceable seal secured to a recessed sealing surface on the distal end of the body; a sample collection chamber bounded at its proximal end by a septum having an opening; a body having a pierceable seal, the pierceable seal being pierceable by a pipette tip or needle; a) a body, the body being passable through the sample; and b) a flexible sampling rod having a proximal portion and a distal portion. through the opening when the distal portion is within the sample collection chamber. a flexible sampling rod adapted to fit and seal with said distal end; The portion comprises: i) an asymmetric beveled tip at a distal end, the beveled tip being a flexible sampling A flexible sampling rod having a vertex at the circumference of the rod and bending the rod away from the collision. and / or configured to deflect impact with an inserted pipette tip or needle. a plurality of stacked layers for forming a plurality of metering ridges; a flexible sampling rod having a concentric truncated cone; and c) a sample collection tube. and the above-described fecal resuspension solution in a chamber. Embodiment Embodiment 1 (a) a stool sample; and (b) protoporphyrin; polyvalent cations; sugars or polysaccharides. and optionally a multivalent cation; an osmolyte; and an HRP stabilizing component, and optionally a composition containing one or more hemoglobin stabilizing reagents, optionally selected from polyvalent cations; A composition comprising a resuspension solution.

[0024] Embodiment 2 2. The composition of embodiment 1, wherein the stool sample comprises hemoglobin.

[0025] Embodiment 3 2. The composition of embodiment 1, wherein the solution comprises betaine.

[0026] Embodiment 4 3. The composition of embodiment 2, wherein the betaine is at a concentration ranging from 2M to 5M.

[0027] Embodiment 5 the solution comprising sucrose or trehalose, and optionally multivalent cations; 2. The composition of embodiment 1.

[0028] Embodiment 6 The sucrose or trehalose has a concentration in the range of 0.1M to 0.5M, 6. The composition of embodiment 5, wherein the solution optionally comprises a multivalent cation.

[0029] Embodiment 7 The solution contains substituted or unsubstituted polygalacturonic acid, and optionally polyvalent cations. 2. The composition of embodiment 1, comprising:

[0030] Embodiment 8 The solution contains α-(1-4) linked D-galacturonic acid, and optionally a multivalent cation 8. The composition of embodiment 1 or 7, comprising:

[0031] Embodiment 9 The substituted or unsubstituted polygalacturonic acid is present at a concentration ranging from 0.005% to 0.5%. 8. The composition of embodiment 7.

[0032] Embodiment 10 The solution contains substituted or unsubstituted polygalacturonic acid and a polyvalent cation (e.g., cation 1. The method of claim 1, wherein the hydroxybenzoate comprises a hydroxybenzoate salt (sodium salt or magnesium salt) at a concentration ranging from 5 mM to 25 mM. The composition described in

[0033] Embodiment 11 2. The composition of embodiment 1, wherein the solution comprises protoporphyrin.

[0034] Embodiment 12 12. The composition of embodiment 11, wherein the protoporphyrin is hemin or hematin. thing.

[0035] Embodiment 13 The protoporphyrin may be fused to a divalent or trivalent cation (e.g., Zn 2+ , Cr 3+ or Co 3+ 12. As described in embodiment 11, the compound comprises protoporphyrin IX complexed with Composition of.

[0036] Embodiment 14 The protoporphyrin is 0.1 μM to 100 μM (for example, 1 μM to 10 μM). 14. The composition of any one of embodiments 11 to 13, wherein the concentration is in the range.

[0037] Embodiment 15 The solution contained HRP conjugate stabilizer (PN 85R-102; Fitzgerald Industries), H RP conjugate stabilizer (PN SZ02; Surmodics) and HRP conjugate stabilizer (PN 2. The composition of embodiment 1, comprising an HRP stabilizing component selected from the group consisting of:

[0038] Embodiment 16 16. The composition of embodiment 15, wherein the HRP stabilizing component is at a concentration ranging from 1% to 20%. Finished product.

[0039] Embodiment 17 The above solution contains iron ions, cobalt ions, chromium ions, zinc ions, calcium ions, any of the above embodiments, containing one or more cations selected from ammonium ions, magnesium ions, or ammonium ions; The composition described in the form.

[0040] Embodiment 18 18. The composition of embodiment 17, wherein the cation is at a concentration ranging from 5 mM to 25 mM. .

[0041] Embodiment 19 The composition of any preceding embodiment, wherein the stool sample is suspended in the solution.

[0042] Embodiment 20 The solution contains Tris buffer, bovine serum albumin, polysorbate 20, sodium azide, further comprising cephalosporin, sodium chloride, ethylenediaminetetraacetic acid, and gentamicin, The composition of any of the above embodiments.

[0043] Embodiment 21 A method for stabilizing hemoglobin in a stool sample, comprising: polyvalent cations; sugars or polysaccharides, and optionally polyvalent cations; osmolytes; and an HRP stabilizing component, and optionally one or more selected from a multivalent cation with a stool resuspension solution containing a hemoglobin stabilizing reagent to form a suspension; and maintaining the suspension for a period of time.

[0044] Embodiment 22 18. The method of embodiment 17, wherein the method comprises transporting the suspension to a remote location. Law.

[0045] Embodiment 23 (A) From a remote location, (i) a stool sample and (ii) protoporphyrin; multivalent cations; A sugar or polysaccharide, and optionally a multivalent cation; an osmolyte; and an HRP stabilizer component. and optionally one or more hemoglobin-stabilizing compounds selected from the group consisting of polyvalent cations. and a stool resuspension solution containing a reagent, wherein the stool sample is suspended in the solution. and (b) measuring the amount of hemoglobin in the composition. A method for analyzing a stool sample, including:

[0046] Embodiment 24 further comprising measuring the amount of one or more colorectal cancer tumor markers in the sample. 20. The method of embodiment 19.

[0047] Embodiment 25 a sample collection container having an open end; and a sample collection container, the sample collection container containing protoporphyrin; thiones; sugars or polysaccharides, and optionally multivalent cations; osmolytes; and HR P stabilizer component, and optionally one or more hemoglobin compounds selected from multivalent cations. Fecal resuspension solution containing a bottle stabilization reagent and a skimmed and / or scraped stool sample and a stool sampling rod having a beveled distal end for collecting the sample. , such that the distal end of the sampling rod is inserted into the sample collection container. The proximal end of the sampling rod is dimensioned to connect with the open end of the sample collection container. the distal end of the sampling rod is adapted to be inserted into the mounting hole. A sample collection device that seals within the device.

[0048] Embodiment 26 The sample collection container and the sampling rod are connected via a screw fit. 26. The sample collection device of embodiment 25, wherein the sample collection device is connected to

[0049] Embodiment 27 A method for stabilizing hemoglobin in a stool sample, comprising: subjecting the stool sample to a multivalent cationic surfactant; The suspension is then mixed with a fecal resuspension solution containing protoporphyrin IX complexed with porphyrin. and maintaining the suspension for a period of time.

[0050] Embodiment 28 28. The method of embodiment 27, wherein the method comprises transporting the suspension to a remote location.

[0051] Embodiment 29 (a) From a remote location, (i) a stool sample; and (ii) complexed with multivalent cations a stool resuspension solution containing protoporphyrin IX, wherein the stool sample is suspended in the solution; (b) receiving a composition comprising a turbid fecal resuspension solution; and 10. A method of analyzing a stool sample, comprising measuring the amount of hemoglobin therein.

[0052] Embodiment 30 The method further comprises measuring the amount of one or more colon cancer tumor markers in the sample. The method according to any one of embodiments 27 to 29.

[0053] Embodiment 31 The polyvalent cation is Cr 3+ or Co 3+ Any of embodiments 27 to 30, wherein The method described below.

[0054] Embodiment 32 32. The method of claim 27, wherein the solution has a pH in the range of pH 6.5 to pH 7.4. Any of the methods described above.

[0055] Embodiment 33 The polyvalent cation is Cr 3+ and the pH of the solution is pH 6.9 to pH 7.4. 33. The method of any one of embodiments 27 to 32, wherein the range is

[0056] Embodiment 34 The above multivalent cation is Co 3+ and the pH of the solution is in the range of pH 6.5 to pH 7.0. 33. The method of any one of embodiments 27 to 32, wherein the range is

[0057] Embodiment 35 an embodiment in which the protoporphyrin IX is at a concentration ranging from 0.5 μM to 10 μM; 27 to 34. A method according to any one of claims 27 to 34.

[0058] Embodiment 36 A sample collection vessel having an open end and a protoporphyrin complexed with a multivalent cation. A stool resuspension solution containing methicone IX and a syringe for skimming and / or scraping the stool sample a stool sampling rod having a beveled distal end, The distal end of the sampling rod is sized to be inserted into the sample collection container. and a proximal end of the sampling rod is connected to the open end of the sample collection container. and adapted to seal the distal end of the sampling rod within the device. , sample collection device.

[0059] Embodiment 37 The sample collection container and the sampling rod are connected via a threaded fit 37. A sample collection device as described in embodiment 36.

[0060] Embodiment 38 The polyvalent cation is Cr 3+ or Co 3+ 38. As described in embodiment 36 or 37, wherein Sample collection device.

[0061] Embodiment 39 39. The method of claim 36, wherein the solution has a pH in the range of pH 6.5 to pH 7.4. The sample collection device according to any one of the preceding claims.

[0062] Embodiment 40 The above multivalent cation is Cr 3+ and the pH of the solution is in the range of pH 6.9 to pH 7.4. 40. The sample collection device according to any one of embodiments 36 to 39, wherein the sample collection device is in the range of 100 to 1500 mm.

[0063] Embodiment 41 The above multivalent cation is Co 3+ and the pH of the solution is in the range of pH 6.5 to pH 7.0. 40. The sample collection device according to any one of embodiments 36 to 39, wherein the sample collection device is in the range of 100 to 1500 mm.

[0064] Embodiment 42 an embodiment in which the protoporphyrin IX is at a concentration ranging from 0.5 uM to 10 uM; 22. A sample collection device according to any one of 16 to 21.

[0065] The above-described invention has been described in some detail by way of illustrations and examples for purposes of clarity of understanding. Although described in detail, those skilled in the art will be able to understand the spirit and scope of the appended claims in light of the teachings of the present invention. It will be readily apparent that several changes and modifications may be made without departing from the scope of the present invention. It is. [Example]

[0066] Example 1 Simulated transportation stress To assess the stability of hemoglobin in stool samples, samples were thermally cycled. The animals were exposed to simulated transport stress conditions using a 3-day (72 h) The time and temperature profiles of the transport simulation are shown. An ELISA-based method was used to measure hemoglobin concentrations in samples before and after exposure to chlorhexidine. The stability of hemoglobin was determined by the amount of time the sample was exposed to the transport simulation. It is calculated as the recovery of hemoglobin after administration.

[0067] [Table 1]

[0068] Accelerated transport simulation, mimicking a three-day transport simulation but in a shortened time period. Table 2 shows the time and temperature profiles of the accelerated transport simulation. The sample exposed to the accelerated transport simulation showed a 3-day transport profile. The results showed a similar level of Hb stability as when exposed to the simulation.

[0069] [Table 2]

[0070] To assess the stability of hemoglobin in stool samples beyond 3 days, a 7-day A transport simulation was conducted. Table 3 shows the time and temperature of the 7-day transport simulation. Show the profile.

[0071] [Table 3]

[0072] As an additional challenge, extreme temperatures are used to represent "worst case" scenarios. Table 4 shows the results of the three-day extreme temperature transport simulation. The time and temperature profile of the

[0073] [Table 4]

[0074] Assay Results The performance of existing buffer solutions (listed below) was tested. Day 3 of a 7-day transport simulation Mean % hemoglobin recovery across all samples for days 1, 5, and 7 The results are shown below.

[0075] [Table 5]

[0076] The results are shown in the table below.

[0077] [Table 6]

[0078] The desired % Hb recovery rate over 7 days is at least 70%. The solution does not achieve the desired level of stability beyond 3 days (72 hours).

[0079] In an effort to improve hemoglobin stability, several classes of compounds (Appendix A) into the base buffer (also referred to herein as the existing buffer) described above. It was evaluated as an additive for

[0080] These compounds were tested at various concentrations and in combination. A list of compounds tested using the assay and the results of these assays are provided in Appendix B. A list of compounds tested using a 3-day transport simulation and their results are provided. The results are presented in Appendix C. A list of the compound combinations tested is provided in Appendix D.

[0081] Among the compounds evaluated, several compounds have been shown to be effective as additives to existing buffer solutions for hemoglobin. These compounds were identified as having the potential to enhance bottle stability. It is listed.

[0082] [Table 7]

[0083] The most feasible method for improving Hb stability from accelerated transport and other simulations Compounds showing activity were analyzed in three fecal samples using several different transport simulations. The results are shown below.

[0084] In a 3-day (72-hour) transport simulation, the potential additives were compared with existing formulations (Hb This demonstrates improved stability over recovery buffer (see table below).

[0085] [Table 8]

[0086] The HRP (horseradish peroxidase) stabilizers tested and tabulated are: be.

[0087] HRP conjugate stabilizer (PN 85R-102), Fitzgerald Ins. dustries International, 30 Sudbury Road, Suite 1A North, Acton, MA, 01720 USA HRP conjugate stabilizer (PN SZ02), Surmodics, 9924 West 74th Street, Eden Prairie, MN 55344 USA HRP conjugate stabilizer (PN ab171537), Abcam, 1 K endall Square, Cambridge, MA 02139 USA In a 7-day transport simulation, the existing formulation containing protoporphyrin showed The stability was improved compared to the existing formulation without rotoporphyrin. Shown in the table below.

[0088] [Table 9]

[0089] In a 3-day extreme temperature transport simulation, the potential additives were significantly more effective than the existing formulation (H b) HRP stabilization buffer (recovery buffer) showing improved stability. Additives containing calcium chloride or magnesium sulfate are also used in conjunction with Polymedco buffers. This demonstrates improved Hb stability compared to the previous study. See table below.

[0090] [Table 10]

[0091] In the accelerated transport simulation, the additive polygalacturonic acid (containing calcium chloride) ) demonstrated improved Hb stability over existing formulations (Hb recovery buffers) at different concentrations. See the table below.

[0092] [Table 11]

[0093] Another class of compounds, called protoporphyrins, has been tested and added to existing buffer formulations. This can increase the stability of hemoglobin. The solubility of protoporphyrin is known to be pH sensitive, so several Note that the pH was altered in some experiments.

[0094] [Table 12]

[0095] In a 3-day (72-hour) transport simulation, the existing This formulation has improved stability over existing formulations without added protoporphyrin. The results are shown in the table below.

[0096] [Table 13]

[0097] In a 7-day transport simulation, the existing formulation containing protoporphyrin showed The stability was improved compared to the existing formulation without rotoporphyrin. is shown in the table below.

[0098] [Table 14]

[0099] Adding protoporphyrin to the existing formulation (buffer for Hb recovery) The stability of Hb is improved even in transport simulations at various temperatures. Additives containing porphyrin cobalt (Co) or protoporphyrin chromium (Cr) are Demonstrates improved Hb stability compared to Polymedco buffers. See table below. It refers to light.

[0100] [Table 15]

[0101] Further testing of protoporphyrin Protoporphyrin IX is available with a 7-day modified shipping schedule as described below. Various concentrations and pH were tested using simulated and other conditions.

[0102] [Table 16]

[0103] The following table shows the results of cobalt(III) at concentrations of 3-6 μM in the adjusted pH range of 6.4-7.0. ) Buffer solution containing PPIX (protoporphyrin IX) was used in a modified transport simulation for 7 days. These results demonstrate that the hydroxybenzoates provide increased Hb stability in the presence of hydroxybenzoates.

[0104] [Table 17]

[0105] The following table shows the results for Cobalt(III) PPIX (protoporphyrin IX) at 2.5-10 μM The buffer solution adjusted to pH 6.8 at a concentration of It has been shown to improve the stability of Hb.

[0106] The following table shows the chromium(III) PPI concentrations from 0.5 to 10 μM at adjusted pH 7.1. The buffer solution containing X (protoporphyrin IX) was used for transport simulations on days 3 and 7. These results show that Hb stability is improved by acetaminophen.

[0107] [Table 18]

[0108] The following table shows the results of cobalt ion precipitation at concentrations of 2.5-10 μM in an adjusted pH range of 6.2-7.4. (III) A buffer solution containing PPIX (protoporphyrin IX) was heated at 35°C for 16 hours. These results demonstrate that the HCl-containing ...

[0109] The following table shows the results of chromium ( III) A buffer solution containing PPIX (protoporphyrin IX) was challenged at 35°C for 16 hours. These results have been shown to provide increased Hb stability in a wide range of applications.

[0110] [Table 19]

[0111] JPEG2026000956000020.jpg63121

[0112] Accordingly, the foregoing description merely illustrates the principles of the invention. Although not explicitly described or shown herein, any other technology embodying the principles of the present invention and its It will be understood that various arrangements can be devised which fall within the spirit and scope of the present invention. Furthermore, all examples and conditional language referred to in this specification is intended to be illustrative and not restrictive. To help the reader understand the principles of the present invention and the concepts to which the inventors have contributed to the advancement of the art. and is not limited to such specifically mentioned examples and conditions. Furthermore, the principles, aspects, and advantages of the present invention as described herein should be understood to be within the scope of the present invention. All statements reciting embodiments and specific examples thereof are intended to be illustrative and not restrictive of their structure and scope. Furthermore, such equivalents are not presently known and are intended to encompass all such equivalents. Both current and future equivalents, i.e., equivalents that perform the same function regardless of structure, It is intended to include any element developed that fulfills the above requirements. is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

[0113] Appendix A

[0114] [Table 20]

[0115] JPEG2026000956000022.jpg187125

[0116] JPEG2026000956000023.jpg186125

[0117] JPEG2026000956000024.jpg185125

[0118] JPEG2026000956000025.jpg155125

[0119] Appendix B

[0120] [Table 21]

[0121] JPEG2026000956000027.jpg183125

[0122] JPEG2026000956000028.jpg176125

[0123] JPEG2026000956000029.jpg182124

[0124] JPEG2026000956000030.jpg181125

[0125] JPEG2026000956000031.jpg182125

[0126] JPEG2026000956000032.jpg182125

[0127] JPEG2026000956000033.jpg186125

[0128] JPEG2026000956000034.jpg183125

[0129] JPEG2026000956000035.jpg186125

[0130] JPEG2026000956000036.jpg178124

[0131] JPEG2026000956000037.jpg187124

[0132] JPEG2026000956000038.jpg181125

[0133] JPEG2026000956000039.jpg185124

[0134] JPEG2026000956000040.jpg187125

[0135] JPEG2026000956000041.jpg179125

[0136] JPEG2026000956000042.jpg186124

[0137] JPEG2026000956000043.jpg187124

[0138] JPEG2026000956000044.jpg182124

[0139] JPEG2026000956000045.jpg178124

[0140] JPEG2026000956000046.jpg181125

[0141] JPEG2026000956000047.jpg186125

[0142] JPEG2026000956000048.jpg182125

[0143] JPEG2026000956000049.jpg187125

[0144] JPEG2026000956000050.jpg187124

[0145] JPEG2026000956000051.jpg186124

[0146] JPEG2026000956000052.jpg186125

[0147] JPEG2026000956000053.jpg185125

[0148] JPEG2026000956000054.jpg183124

[0149] JPEG2026000956000055.jpg184124

[0150] JPEG2026000956000056.jpg182124

[0151] JPEG2026000956000057.jpg183125

[0152] JPEG2026000956000058.jpg182125

[0153] JPEG2026000956000059.jpg182125

[0154] JPEG2026000956000060.jpg182124

[0155] JPEG2026000956000061.jpg181125

[0156] JPEG2026000956000062.jpg181124

[0157] JPEG2026000956000063.jpg186125

[0158] JPEG2026000956000064.jpg185124

[0159] JPEG2026000956000065.jpg182124

[0160] JPEG2026000956000066.jpg178125

[0161] JPEG2026000956000067.jpg182125

[0162] JPEG2026000956000068.jpg180125

[0163] JPEG2026000956000069.jpg186125

[0164] JPEG2026000956000070.jpg182124

[0165] JPEG2026000956000071.jpg182124

[0166] JPEG2026000956000072.jpg185124

[0167] JPEG2026000956000073.jpg183124

[0168] JPEG2026000956000074.jpg183124

[0169] JPEG2026000956000075.jpg181124

[0170] JPEG2026000956000076.jpg181124

[0171] JPEG2026000956000077.jpg182125

[0172] JPEG2026000956000078.jpg181124

[0173] JPEG2026000956000079.jpg183124

[0174] JPEG2026000956000080.jpg183124

[0175] JPEG2026000956000081.jpg180124

[0176] JPEG2026000956000082.jpg182125

[0177] JPEG2026000956000083.jpg182125

[0178] JPEG2026000956000084.jpg183125

[0179] JPEG2026000956000085.jpg186124

[0180] JPEG2026000956000086.jpg182124

[0181] JPEG2026000956000087.jpg186124

[0182] JPEG2026000956000088.jpg182124

[0183] JPEG2026000956000089.jpg184125

[0184] JPEG2026000956000090.jpg184125

[0185] JPEG2026000956000091.jpg183124

[0186] JPEG2026000956000092.jpg185125

[0187] JPEG2026000956000093.jpg184124

[0188] JPEG2026000956000094.jpg184125

[0189] JPEG2026000956000095.jpg183124

[0190] JPEG2026000956000096.jpg183124

[0191] JPEG2026000956000097.jpg183125

[0192] JPEG2026000956000098.jpg180124

[0193] JPEG2026000956000099.jpg17125

[0194] Appendix C

[0195] [Table 22]

[0196] JPEG2026000956000101.jpg187125

[0197] JPEG2026000956000102.jpg187124

[0198] JPEG2026000956000103.jpg187124

[0199] JPEG2026000956000104.jpg186124

[0200] JPEG2026000956000105.jpg184125

[0201] JPEG2026000956000106.jpg187124

[0202] JPEG2026000956000107.jpg184124

[0203] JPEG2026000956000108.jpg186125

[0204] JPEG2026000956000109.jpg31125

[0205] Appendix D

[0206] [Table 23]

[0207] JPEG2026000956000111.jpg186119

[0208] JPEG2026000956000112.jpg125119

Claims

1. (a) a stool sample; and (b) a stool resuspension solution containing one or more hemoglobin stabilizing reagents selected from the following: protoporphyrin, multivalent cations, a sugar or polysaccharide, and optionally a multivalent cation; Osmolytes, and HRP stabilizing component, and optionally a multivalent cation A composition comprising:

2. The composition of claim 1 , wherein the stool sample comprises hemoglobin.

3. The fecal resuspension solution comprises protoporphyrin IX complexed with a multivalent cation.

3. The composition according to claim 1 or 2.

4. The polyvalent cation is Cr ３＋ or Co ３＋ The composition of claim 3, wherein

5. 5. The method of claim 3, wherein the solution has a pH in the range of pH 6.5 to pH 7.

4. The composition described above.

6. The multivalent cation is Cr ３＋ and the pH of the solution is in the range of pH 6.9 to pH 7.

4. The composition according to any one of claims 3 to 5, wherein the range is 0.01 to 0.

1.

7. The polyvalent cation is Co ３＋ and the pH of the solution is in the range of pH 6.5 to pH 7.

0. The composition according to any one of claims 3 to 5, wherein the range is 0.01 to 0.

1.

8. 3. The method of claim 2, wherein the protoporphyrin IX is at a concentration ranging from 0.5 μM to 10 μM.

8. The composition according to any one of claims 1 to 7.

9. The solution contains substituted or unsubstituted polygalacturonic acid and optionally a polyvalent cation. The composition of claim 1 comprising:

10. The substituted or unsubstituted polygalacturonic acid is present at a concentration ranging from 0.005% to 0.5%. The composition of claim 9, wherein the

11. The method of any one of claims 1 to 10, wherein the stool sample is suspended in the solution. composition.

12. The solution contains Tris buffer, bovine serum albumin, polysorbate 20, sodium azide, further comprising cephalosporin, sodium chloride, ethylenediaminetetraacetic acid, and gentamicin, 12. The composition of any one of claims 1 to 11.

13. 1. A method for stabilizing hemoglobin in a stool sample, comprising: protoporphyrin, multivalent cations, a sugar or polysaccharide, and optionally a multivalent cation; Osmolytes, and HRP stabilizing component, and optionally a multivalent cation A stool resuspension solution containing one or more hemoglobin stabilizing reagents selected from the group consisting of: mixing the sample with the water to form a suspension; and maintaining the suspension for a period of time. A method comprising:

14. 14. The method of claim 13, including the step of transporting the suspension to a remote location.

15. The fecal resuspension solution comprises protoporphyrin IX complexed with a multivalent cation.

15. The method according to any one of claims 13 to 14.

16. 1. A method for analyzing a stool sample, comprising: (a) (i) a stool sample, and (ii) protoporphyrin, multivalent cations, a sugar or polysaccharide, and optionally a multivalent cation; Osmolytes, and HRP stabilizing component, and optionally a multivalent cation A stool resuspension solution containing one or more hemoglobin stabilizing reagents selected from receiving from a remote location a composition comprising: Steps that are muddy; and (b) measuring the amount of hemoglobin in the composition A method comprising:

17. and determining the amount of one or more colon cancer tumor markers in said sample.

17. The method of claim 16, comprising:

18. The fecal resuspension solution comprises protoporphyrin IX complexed with a multivalent cation.

18. The method according to any one of claims 16 to 17.

19. 1. A sample collection device comprising: a sample collection container having an open end; protoporphyrin, multivalent cations, a sugar or polysaccharide, and optionally a multivalent cation; Osmolytes, and HRP stabilizing component, and optionally a multivalent cation A stool regenerating agent in the container, comprising one or more hemoglobin stabilizing reagents selected from a suspension solution; Fecal sump with a beveled distal end for scooping and / or scraping a fecal sample a ring rod; The distal end of the sampling rod is inserted into the sample collection container. The proximal end of the sampling rod is dimensioned to fit into the open end of the sample collection container. to connect the distal end of the sampling rod to A sample collection device that seals within the device.

20. The sample collection container and the sampling rod are connected via a threaded fit.

20. The sample collection device of claim 19.

21. The fecal resuspension solution comprises protoporphyrin IX complexed with a multivalent cation.

20. The sample collection device of claim 19.

Images