Method for stabilizing hemoglobin and reagent for implementing method

Abstract

A fecal resuspending solution comprising a hemoglobin stabilizing agent is provided. In some embodiments, the hemoglobin stabilizing agent may be an osmotic agent, a multivalent cation, a sugar or a 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 fecal sample in a solution, as well as sample collection devices comprising the solution.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980022853.6 (PCT / US2019 / 022598), filed on March 15, 2019, and entitled “Method for stabilizing hemoglobin and reagents for implementing the method”.

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 648,874, filed on March 27, 2018, and U.S. Provisional Application Serial No. 62 / 685,248, filed on June 14, 2018, which are incorporated herein by reference.

[0004] background

[0005] Tests to detect hemoglobin (Hb) in stool samples are commonly used for screening for colorectal cancer. The usual procedure is to utilize a sample collection device that contains a buffer that stabilizes the hemoglobin from the time of collection until before the detection assay is performed in the laboratory. The ability of the buffer to properly stabilize the hemoglobin is critical for accurate test results, and the hemoglobin must be stable under a range of environmental conditions during transportation. For example, improving the ability of the buffer to stabilize the hemoglobin would allow for longer shipping times and provide greater flexibility for both the patient and the testing laboratory for sample handling. SUMMARY OF THE INVENTION

[0007] The present invention particularly provides a fecal resuspended solution comprising a hemoglobin stabilizing agent. In some embodiments, the hemoglobin stabilizing agent in the solution can be an osmotic fluid, a multivalent cation, a sugar or a polysaccharide, and optionally, a multivalent cation, a protoporphyrin or HRP stabilizing component, and optionally a multivalent cation.

[0008] The solution can be used in methods that may benefit from stabilizing hemoglobin. In some embodiments, the method may include combining a stool sample with a stool resuspension solution to produce a suspension, and maintaining the suspension for a period of time. In some embodiments, the method may include shipping the suspension to a remote location. The hemoglobin stabilizing agent in the solution may stabilize the hemoglobin, allowing for more accurate hemoglobin measurements several days after the sample has been shipped.

[0009] Compositions, methods, and devices for using fecal resuspension solutions are provided. DETAILED DESCRIPTION

[0010] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as they may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0011] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range, unless the context clearly dictates otherwise, is one-tenth the unit of the lower limit, and any other stated or intermediate values ​​in the range are included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also included in the present invention, subject to any explicitly excluded limitations in the range. Where the range includes one or two limits, ranges excluding one or both of those included limits are also included in the present invention.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0013] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference and are incorporated herein by reference to disclose and describe the methods and / or materials related to the publications cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0014] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0015] It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be performed in the order of events described or in any other order that is logically possible.

[0016] Provided is a solution of fecal resuspension comprising one or more hemoglobin stabilizing agents. In some embodiments, one or more hemoglobin stabilizing agents can be selected from, for example, osmotic agents, multivalent cations, sugars or polysaccharides, and optional multivalent cations, protoporphyrin and HRP stabilizing components and optional multivalent cations. Examples and concentrations of such agents in the solution are listed in the table below and in the appendix.

[0017] In some embodiments, the solution may include an osmotic agent, such as betaine. In these embodiments, the concentration of the osmotic agent (e.g., betaine) may be in the range of 2M to 5M.

[0018] In some embodiments, the solution may include a sugar, such as sucrose or trehalose. In these embodiments, the concentration of the sugar (e.g., sucrose or trehalose) may be in the range of 0.1 M to 0.5 M. In either case, the solution may optionally include a multivalent cation, Mg 2+ or Ca 2+ In these embodiments, the concentration of multivalent cations (eg, calcium or magnesium ions) may be in the range of 5 mM to 25 mM.

[0019] In some embodiments, the solution may include a polysaccharide, such as a substituted or unsubstituted polygalacturonic acid, such as α-(1–4)-linked D-galacturonic acid. In these embodiments, the concentration of the polysaccharide (e.g., substituted or unsubstituted polygalacturonic acid) may be in the range of 0.005% to 0.5% (e.g., 0.01% to 0.125%). In these embodiments, the solution may optionally include a polyvalent cation. In embodiments where the solution includes a polyvalent cation, the concentration of the polyvalent cation may be in the range of 5 mM to 25 mM. In some embodiments, the solution may include a substituted or unsubstituted polygalacturonic acid and a polyvalent cation (e.g., a calcium salt or a magnesium salt) at a concentration of 5 mM to 25 mM.

[0020] In some embodiments, the solution may include a protoporphyrin, such as protoporphyrin IX or an analog thereof, such as octaethylporphyrin (H2OEP) or tetraphenylporphyrin (H2TPP), complexed with a metal ion. In these embodiments, the protoporphyrin may be a heme (containing a ferric iron ion (Fe3) with a coordinated chloride ligand). 3+In other embodiments, protoporphyrin can be a protoporphyrin IX) or hemoglobin with a divalent or trivalent cation (e.g., Zn 2+ ,Cr 3+ or Co 3+ ) complexed protoporphyrin IX. In these embodiments, the concentration of protoporphyrin in the solution can be in the range of 0.1 μM to 100 μM (e.g., 1 μM to 10 μM). In some embodiments, the solution may include an HRP stabilizing component selected from an HRP conjugate stabilizer (PN 85R-102; Fitzgerald Industries), an HRP conjugate stabilizer (PN SZ02; Surmodics), and an HRP conjugate stabilizer (PN abl71537; Abcam). Other HRP stabilizing components can be used, for example, AbGuard (BioRad PN: BUF052; BioRad Laboratories Inc. 2000 Alfred Nobel Dr, Hercules, CA 94547). If the solution includes an HRP stabilizing component, the concentration of the component can be in the range of 1% to 20%, for example, 5% to 15% or 5% to 20%.

[0021] In any embodiment, the solution may contain multivalent cations, such as calcium or magnesium ions. In these embodiments, the concentration of the multivalent cations may be in the range of 5 mM to 25 mM.

[0022] In any embodiment, the solution further comprises 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), a preservative such as sodium azide (e.g., 0.05% to 0.2% sodium azide), a salt such as sodium chloride (e.g., 50 mM to 250 mM sodium chloride), a chelating agent such as ethylenediaminetetraacetic acid (e.g., 5 mM to 20 mM ethylenediaminetetraacetic acid), and an antibiotic such as gentamicin (e.g., 5 ug / mL to 50 ug / mL).

[0023] A composition is also provided, comprising: (a) a stool sample and (b) a solution in which the stool is resuspended as described above. The stool sample can be suspended in the solution. Certain components of the stool may be dissolved in the solution, while other insoluble components may be suspended but not dissolved in the solution. In some embodiments, the stool sample may contain hemoglobin that can be dissolved in the solution. In these embodiments, the presence of a hemoglobin stabilizing agent can increase the amount of hemoglobin in the sample after the sample has been subjected to a real or simulated transport pressure stress as described below, compared to an otherwise identical untreated control sample (e.g., a "T0" sample). In some embodiments, the use of a hemoglobin stabilizing agent can increase the amount of hemoglobin in the sample by at least 10%, at least 20, or at least 30%, relative to a control stool sample that has been suspended in an otherwise identical solution that does not contain a hemoglobin stabilizing agent. The amount of hemoglobin in a sample can be determined by enzyme-linked immunosorbent assay (ELISA), although several other methods are known (see 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).

[0024] Also provided is a method for stabilizing fecal hemoglobin. In some embodiments, the method may include merging a fecal sample with a solution of fecal resuspension as described above, the fecal resuspension solution being a solution comprising one or more hemoglobin stabilizing agents selected from: osmotic agents, multivalent cations, sugars or polysaccharides, and optionally, multivalent cations; protoporphyrin; and HRP stabilizing components and optional multivalent cations to produce a suspension; and maintaining the suspension for a period of time. Detailed information on the characteristics of the reagents that may be present in the solution and their concentrations are described above, below, and in the attached appendix. In some embodiments, the method may include obtaining a fecal sample, for example, by scraping or digging out from a larger mass of feces using a fecal sampling tool.

[0025] In some embodiments, the method may include shipping the sample to a remote location, such as to another town, city, state, or country, for testing. In these embodiments, the sample may wait for several days, such as 3, 4, 5, 6, or 7 days, to be shipped en route or to be tested at the remote location, thereby subjecting the sample to stress that may reduce the amount of hemoglobin in the sample.

[0026] A method is also provided, which includes receiving a composition comprising: (a) a stool sample and (b) a stool resuspended solution comprising one or more hemoglobin stabilizing agents selected from, for example, osmotic pressure, multivalent cations, sugars or polysaccharides, and optional multivalent cations, protoporphyrin and HRP stabilizing components and optional multivalent cations. Examples and concentrations of such agents in the solution are listed above, below and in the attached appendix. In certain embodiments, the method may further include measuring the sample after the sample has been received. For example, the concentration of hemoglobin in the solution can be measured, and the implementation method thereof is described above. In addition, the amount of one or more colorectal cancer tumor markers in the sample can be tested after receipt (see generally Lech et al., World J Gastroenterol. 2016 22: 1745-1755 and Scheruders et al, Curr. Treat. Options Gastroenterol. 2016 14: 152-162). These embodiments may further include forwarding the test results to a remote location by, for example, fax or email, or over the Internet via a portal so that the results can be obtained by a practitioner (e.g., an MD or nurse, etc.) before providing a diagnosis to the patient from whom the stool sample was obtained.

[0027] A sample collection device is also provided, comprising: (a) a sample collection container having an open end; and (b) a stool resuspended solution located in the container, wherein the solution comprises one or more hemoglobin stabilizing agents selected from the following: an osmotic agent, a multivalent cation, a sugar or polysaccharide, and optionally a multivalent cation, a protoporphyrin, and an HRP stabilizing component, and optionally a multivalent cation, as described herein; (c) a stool sampling rod comprising a distal beveled tip for digging and / or scraping a stool sample. In the device, the distal end of the sampling rod is sized to be insertable into the sample collection container, and the proximal end of the sampling rod is adapted to be connected to the open end of the sample collection container, thereby sealing the distal end of the sampling rod within the device. In some embodiments, the sample collection container and the sampling rod can be connected by threaded engagement. In addition to the hemoglobin stabilizing agent, the details, selection and design of an example of such a sample collection device can be found in, for example, US9211112. In some embodiments, the device may include: a) a body including a sample collection chamber, which is limited at the distal end by a penetrable seal fixed to a recessed sealing surface at the distal end of the body and limited at the proximal end by a septum containing a hole, the penetrable seal being pierceable by a pipette tip or needle; b) a flexible sampling rod including a proximal portion and a distal portion, the sampling rod being adapted to pass through and seal the hole when the distal portion is in the sample collection chamber, wherein the distal portion includes: i) an asymmetric beveled tip at the distal end, the beveled tip having an apex at the periphery of the flexible sampling rod and configured to bend the flexible sampling rod away from collision and / or deflect collision with an inserted pipette tip or needle; ii) a plurality of coaxial truncated cones of stacked cones to form a plurality of metering ridges; c) a feces resuspended solution as described above in the sample collection chamber as described in US9211112.

[0028] Implementation

[0029] Embodiment 1. A composition comprising: (a) a stool sample; and (b) a stool resuspended solution comprising one or more hemoglobin stabilizing agents selected from the group consisting of: protoporphyrin; polyvalent cations; sugars or polysaccharides, and optionally polyvalent cations; a permeate; an HRP stabilizing component, and optionally polyvalent cations.

[0030] Embodiment 2. The composition of embodiment 1, wherein the stool sample comprises hemoglobin.

[0031] Embodiment 3. The composition of embodiment 1, wherein the solution comprises betaine.

[0032] Embodiment 4. The composition of embodiment 2, wherein the concentration of betaine is in the range of 2M-5M.

[0033] Embodiment 5. The composition of embodiment 1, wherein the solution comprises sucrose or trehalose, and optionally a multivalent cation.

[0034] Embodiment 6. The composition of embodiment 5, wherein the concentration of sucrose or trehalose is in the range of 0.1 M to 0.5 M, and the solution optionally comprises multivalent cations.

[0035] Embodiment 7. The composition of embodiment 1, wherein the solution comprises substituted or unsubstituted polygalacturonic acid and optionally a multivalent cation.

[0036] Embodiment 8. The composition of embodiment 1 or 7, wherein the solution comprises α-(1–4)-linked D-galacturonic acid and optionally a multivalent cation.

[0037] Embodiment 9. The composition of embodiment 7, wherein the concentration of the substituted or unsubstituted polygalacturonic acid is in the range of 0.005% to 0.5%.

[0038] Embodiment 10. The composition of embodiment 1, wherein the solution comprises substituted or unsubstituted polygalacturonic acid and a multivalent cation (eg, calcium or magnesium salt) at a concentration ranging from 5 mM to 25 mM.

[0039] Embodiment 11. The composition of Embodiment 1, wherein the solution comprises protoporphyrin.

[0040] Embodiment 12. The composition of embodiment 11, wherein the protoporphyrin is heme or hemoglobin.

[0041] Embodiment 13. The composition of embodiment 11, wherein the protoporphyrin comprises a divalent or trivalent cation (e.g., Zn 2+ ,Cr 3+ or Co 3+ ) complexed protoporphyrin IX.

[0042] Embodiment 14. The composition of any one of embodiments 11-13, wherein the concentration of protoporphyrin is in the range of 0.1 μM-100 μM (eg, 1 μM-10 μM).

[0043] Embodiment 15. The composition of embodiment 1, wherein the solution comprises an HRP stabilizing component selected from the group consisting of: HRP conjugate stabilizer (PN 85R-102; Fitzgerald Industries), HRP conjugate stabilizer (PN SZ02; Surmodics) and HRP conjugate stabilizer (PN abl7l537; Abcam).

[0044] Embodiment 16. The composition of Embodiment 15, wherein the concentration of the HRP stabilizing component is in the range of 1% to 20%.

[0045] Embodiment 17. The composition of any of the preceding embodiments, wherein the solution comprises one or more cations selected from iron, cobalt, chromium, zinc, calcium or magnesium ions.

[0046] Embodiment 18. The composition of Embodiment 17, wherein the concentration of the cation is in the range of 5 mM to 25 mM.

[0047] Embodiment 19. The composition of any of the preceding embodiments, wherein the stool sample is suspended in the solution.

[0048] Embodiment 20. The composition of any of the preceding embodiments, wherein the solution further comprises Tris buffer, bovine serum albumin, polysorbate 20, sodium azide, sodium chloride, ethylenediaminetetraacetic acid and gentamicin.

[0049] Embodiment 21. A method for stabilizing hemoglobin in a fecal sample, comprising: combining the fecal sample with a fecal resuspension solution, wherein the fecal resuspension solution comprises one or more hemoglobin stabilizing reagents selected from the following: protoporphyrin; polyvalent cations; sugars or polysaccharides, and optionally polyvalent cations; osmotic agents; and HRP stabilizing components and optionally polyvalent cations; to produce a suspension; and maintaining the suspension for a period of time.

[0050] Embodiment 22. The method of Embodiment 17, wherein the method comprises transporting the suspension to a remote location.

[0051] Embodiment 23. A method for analyzing a stool sample, the method comprising: (a) receiving a composition from a remote location, the composition comprising: (i) a stool sample; and (ii) a stool resuspended solution, which comprises one or more hemoglobin stabilizing reagents selected from the following: protoporphyrin; polyvalent cations; sugars or polysaccharides, and optionally polyvalent cations; osmotic agents; and HRP stabilizing components, and optionally polyvalent cations, wherein the stool sample is suspended in the solution, and (b) measuring the amount of hemoglobin in the composition.

[0052] Embodiment 24. The method of Embodiment 19, further comprising measuring the amount of one or more colorectal cancer tumor markers in the sample.

[0053] Embodiment 25. A sample collection device, comprising: a sample collection container having an open end; a stool resuspended solution located in the container, the stool resuspended solution comprising one or more hemoglobin stabilizing reagents selected from protoporphyrin; polyvalent cations; sugars or polysaccharides and optional polyvalent cations; osmotic agents; HRP stabilizing components and optional polyvalent cations; and a stool sampling rod comprising a distal beveled tip for scooping and / or scraping stool samples, wherein the distal end of the sampling rod is sized to be inserted into the sample collection container, and the proximal end of the sampling rod is adapted to be connected to the open end of the sample collection container, thereby sealing the distal end of the sampling rod within the device.

[0054] Embodiment 26. The sample collection device of embodiment 25, wherein the sample collection container and the sampling rod are connected by threaded fitting.

[0055] Embodiment 27. A method for stabilizing hemoglobin in a stool sample, comprising: combining the stool sample with a stool resuspension solution, wherein the stool resuspension solution comprises protoporphyrin IX complexed with multivalent cations to produce a suspension; and maintaining the suspension for a period of time.

[0056] Embodiment 28. The method of Embodiment 27, wherein the method comprises transporting the suspension to a remote location.

[0057] Embodiment 29. A method for analyzing a stool sample, comprising: (a) receiving a composition from a remote location, the composition comprising: (i.) a stool sample; (ii.) a stool resuspended solution comprising protoporphyrin IX complexed with a multivalent cation, wherein the stool sample is suspended in the solution; and (b) measuring the amount of hemoglobin in the composition.

[0058] Embodiment 30. The method of any one of Embodiments 27-29, further comprising measuring the amount of one or more colorectal cancer tumor markers in the sample.

[0059] Embodiment 31. The method of any one of Embodiments 27-30, wherein the multivalent cation is Cr 3+ or Co 3+ .

[0060] Embodiment 32. The method of any one of Embodiments 27-31, wherein the solution has a pH in the range of pH 6.5-pH 7.4.

[0061] Embodiment 33. The method of any one of Embodiments 27-32, wherein the multivalent cation is Cr 3+ , and the pH of the solution is in the range of pH 6.9-pH 7.4.

[0062] Embodiment 34. The method of any one of Embodiments 27-32, wherein the multivalent cation is Co 3+ , and the pH of the solution is in the range of pH 6.5-pH 7.0.

[0063] Embodiment 35. The method of any one of Embodiments 27-34, wherein the concentration of protoporphyrin IX is in the range of 0.5uM–10uM.

[0064] Embodiment 36. A sample collection device comprising: a sample collection container having an open end; a stool resuspension solution comprising protoporphyrin IX complexed with a multivalent cation; and a stool sampling rod comprising a distal beveled tip for scooping and / or scraping a stool sample, wherein the distal end of the sampling rod is sized to be inserted into the sample collection container and the proximal end of the sampling rod is adapted to connect to the open end of the sample collection container, thereby sealing the distal end of the sampling rod within the device.

[0065] Embodiment 37. The sample collection device of embodiment 36, wherein the sample collection container and the sampling rod are connected by threaded fitting.

[0066] Embodiment 38. The sample collection device of embodiment 36 or 37, wherein the multivalent cation is Cr 3+ or Co 3 + .

[0067] Embodiment 39. The sample collection device of any one of Embodiments 36-38, wherein the solution has a pH in the range of pH 6.5-pH 7.4.

[0068] Embodiment 40. The sample collection device of any one of Embodiments 36-39, wherein the multivalent cation is Cr 3+ , and the pH of the solution is in the range of pH 6.9-pH 7.4.

[0069] Embodiment 41. The sample collection device of any one of Embodiments 36-39, wherein the multivalent cation is Co 3+ , and the pH of the solution is in the range of pH 6.5-pH 7.0.

[0070] Embodiment 42. The sample collection device of any one of embodiments 16-21, wherein the concentration of protoporphyrin IX is in the range of 0.5uM-10uM.

[0071] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of the present invention that certain changes and modifications may be made therein without departing from the spirit or scope of the appended claims.

[0072] Example 1

[0073] Simulated delivery pressure

[0074] In order to evaluate the stability of hemoglobin in fecal samples, samples were placed under simulated transport pressure conditions using a thermal cycler. Table 1 shows the time and temperature profiles for 3 days (72 hours) of transport simulation. An ELISA-based detection method was used to determine the hemoglobin concentration of samples before and after exposure to transport simulation. The stability of hemoglobin was calculated as the recovery percentage of hemoglobin after sample exposure to transport simulation.

[0075]

[0076]

[0077] An accelerated shipping simulation profile was created to mimic the 3-day shipping simulation, but in a shortened time. Table 2 shows the time and temperature profile used for the accelerated shipping simulation. Samples exposed to the accelerated shipping simulation showed similar levels of Hb stability as those exposed to the 3-day shipping simulation.

[0078]

[0079] To evaluate the stability of hemoglobin in stool samples over 3 days, a 7-day transport simulation was created. Table 3 shows the time and temperature profile of the 7-day transport simulation.

[0080]

[0081]

[0082] As an additional challenge, a delivery simulation was created using extreme temperatures to represent a “worst case” scenario. Table 4 shows the time and temperature profile for the 3-day extreme temperature delivery simulation.

[0083]

[0084]

[0085] Measurement results

[0086] The performance of an existing buffer formulation (shown below) was tested. The average percent hemoglobin recovery for all samples was determined on days 3, 5 and 7 of the 7-day shipping simulation. These results are shown below.

[0087] Final concentration Components N / A water 20mM 1M Tris pH 7.5 10％ BSA 0.10％ 20% Tween 20 0.095％ 5% Sodium Azide 140mM Sodium chloride 10mM 0.5M EDTA 15 μg / mL 50mg / mL gentamicin

[0088] The results are listed in the table below.

[0089] Time %Hb recovery Day 3 71 Day 5 68 Day 7 63

[0090] The desired % Hb recovery at 7 days is a minimum of 70%. Thus, existing buffer formulations do not achieve the desired level of stability beyond 3 days (72 hours).

[0091] To improve the stability of hemoglobin, several classes of compounds (see Appendix A) were evaluated as additives to the above-mentioned base buffer formulation (also referred to herein as existing buffer formulation).

[0092] These compounds were tested at various concentrations and combinations. A list of compounds tested using the accelerated shipping simulation and the results of these assays is found in Appendix B. A list of compounds tested using the 3-day shipping simulation and the results are found in Appendix C. The combinations of compounds tested are shown in Appendix D.

[0093] Among the compounds evaluated, several were identified as having the potential to enhance hemoglobin stability as additives to existing buffer formulations. These compounds are listed in the table below:

[0094]

[0095] Compounds that showed the most potential to enhance Hb stability by accelerated transport and other simulations were tested on three stool samples in several different transport simulations. These results are shown below.

[0096] In a 3-day (72-hour) shipping simulation, the potential additive demonstrated improved stability over the existing formulation (Hb Collection Buffer). See the table below:

[0097]

[0098] The HRP (Horseradish Peroxidase) stabilizers tested and shown in the table are:

[0099] HRP conjugate stabilizer (PN 85R-102), Fitzgerald Industries International, 30 Sudbury Road, Suite 1A North, Acton, MA, 01720 USA

[0100] HRP conjugate stabilizer (PN SZ02), Surmodics, Inc., 9924 West 74 th Street,EdenPrairie,MN 55344USA

[0101] HRP conjugate stabilizer (PN ab171537), Abcam, 1Kendall Square, Cambridge, MA02139, USA

[0102] In a 7-day shipping simulation, potential additives showed improved stability over the existing formulation (Hb Collection Buffer). See the table below:

[0103]

[0104] In a 3-day extreme temperature shipping simulation, potential additives showed improved stability over existing formulations (Hb Collection Buffer). Additives containing HRP stabilization buffer and calcium chloride or magnesium sulfate also showed improved Hb stability compared to Polymedco buffer. See the table below:

[0105]

[0106] In the accelerated transport simulation, the additive polygalacturonic acid (containing calcium chloride) showed improved Hb stability over the existing formulation (Hb Collection Buffer) at different concentrations. See the table below:

[0107]

[0108] Another class of compounds called protoporphyrins were tested and found to enhance the stability of hemoglobin when added to existing buffer formulations. The protoporphyrins tested are shown in the table below. Note that in some of the tested cases, the pH was changed because the solubility of protoporphyrin is known to be pH sensitive.

[0109]

[0110] In a 3 day (72 hour) transport simulation, the prior art formulation with added protoporphyrin had improved stability over the prior art formulation without any protoporphyrin. These results are shown in the table below.

[0111]

[0112] In a 7-day transport simulation, the existing formulation with added protoporphyrin had improved stability over the existing formulation without any protoporphyrin. These results are shown in the table below.

[0113]

[0114] The addition of protoporphyrin to the existing formulation (Hb Collection Buffer) also improved Hb stability in a 3-day extreme temperature shipping simulation. In addition, additives containing cobalt protoporphyrin (Co) or chromium protoporphyrin (Cr) demonstrated improved Hb stability compared to Polymedco buffer. See the table below:

[0115]

[0116] Further testing of protoporphyrin

[0117] Protoporphyrin IX was tested at various concentrations and pH values ​​using the 7-day altered transport simulation conditions listed below, as well as other conditions.

[0118]

[0119] The table below shows that buffers containing cobalt (III) PPIX (protoporphyrin IX) at concentrations of 3-6 μM provided improved Hb stability over the adjusted pH range of 6.4-7.0 in a 7-day altered transport simulation.

[0120]

[0121] The table below shows that buffers containing cobalt(III)PPIX (protoporphyrin IX) at concentrations of 2.5-10 μM provided enhanced Hb stability at an adjusted pH of 6.8 in 3- and 7-day transport simulations.

[0122] The table below shows that buffers containing chromium (III) PPIX (protoporphyrin IX) at concentrations of 0.5-10 μM provided improved Hb stability at an adjusted pH of 7.1 in 3- and 7-day transport simulations.

[0123]

[0124] The table below shows that buffers containing cobalt(III)PPIX (protoporphyrin IX) at concentrations of 2.5-10 μM provided increased Hb stability in the adjusted pH range of 6.2-7.4 during a 16-hour challenge at 35C.

[0125] The table below shows that buffers containing chromium (III) PPIX (protoporphyrin IX) at concentrations ranging from 1.25-5 μM provided increased Hb stability over an adjusted pH range of 6.2-7.4 during a 16-hour challenge at 35C.

[0126]

[0127] Therefore, the above only illustrates the principle of the present invention. It should be understood that those skilled in the art will be able to design various arrangements, although not explicitly described or shown here, they embody the principle of the present invention and are included in the spirit and scope of the present invention. In addition, all examples and conditional language listed herein are mainly intended to help readers understand the principle of the present invention and the conception that the inventor contributes to promote this field, and should be interpreted as not being limited to such specific examples and conditions. In addition, all statements of the principles, aspects and embodiments of the present invention described herein and their specific embodiments are intended to cover their structural and functional equivalents. In addition, it is intended that such equivalents include currently known equivalents and equivalents developed in the future, that is, any elements of the implementation of the same function developed, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. On the contrary, the scope and spirit of the present invention are embodied by the appended claims.

[0128] Appendix A

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[0136] Appendix B

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[0237] Appendix C

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[0252] Appendix D

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Claims

1. A composition comprising: (a) stool samples; and (b) a stool resuspended solution comprising one or more hemoglobin stabilizing agents selected from the group consisting of: Protoporphyrin; Multivalent cations; a sugar or polysaccharide, and optionally a multivalent cation; Penetrants; and An HRP stabilizing component, and optionally a multivalent cation.

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

3. The composition of any of the preceding claims, wherein the feces resuspended solution comprises protoporphyrin IX complexed with multivalent cations.

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

5. The composition according to claim 3 or 4, wherein the solution has a pH in the range of pH 6.5-pH 7.

4.

6. A composition according to any one of claims 3 to 5, wherein the multivalent cation is Cr 3+ , and the pH of the solution is in the range of pH 6.9-pH 7.

4.

7. A composition according to any one of claims 3 to 5, wherein the multivalent cation is Co 3+ , and the pH of the solution is in the range of pH 6.5-pH 7.

0.

8. The composition according to any one of claims 3 to 7, wherein the concentration of protoporphyrin IX is in the range of 0.5uM to 10uM.

9. The composition of claim 1, wherein the solution comprises substituted or unsubstituted polygalacturonic acid and optionally a multivalent cation.

10. The composition according to claim 9, wherein the concentration of the substituted or unsubstituted polygalacturonic acid ranges from 0.005% to 0.5%.

11. The composition of any one of the preceding claims, wherein the stool sample is suspended in the solution.

12. The composition of any of the preceding claims, wherein the solution further comprises Tris buffer, bovine serum albumin, polysorbate 20, sodium azide, sodium chloride, ethylenediaminetetraacetic acid and gentamicin.

13. A method for stabilizing hemoglobin in a stool sample, comprising: The stool sample is combined with a stool resuspension solution comprising one or more hemoglobin stabilizing agents selected from: Protoporphyrin; Multivalent cations; a sugar or polysaccharide, and optionally a multivalent cation; Penetrants; and an HRP stabilizing component, and optionally a multivalent cation; to produce a suspension; and The suspension is maintained for a period of time.

14. A method according to claim 13, wherein the method comprises transporting the suspension to a remote location.

15. The method according to any one of claims 13-14, wherein the stool resuspended solution comprises protoporphyrin IX complexed with multivalent cations.

16. A method for analyzing a stool sample, the method comprising: (a) receiving from a remote location a composition comprising: (i.) fecal samples; and (ii.) a stool resuspended solution comprising one or more hemoglobin stabilizing agents selected from the following: Protoporphyrin; Multivalent cations; a sugar or polysaccharide, and optionally a multivalent cation; Penetrants; and an HRP stabilizing component, and optionally a multivalent cation, wherein the stool sample is suspended in the solution; (b) measuring the amount of hemoglobin in the composition.

17. The method according to claim 16, further comprising measuring the amount of one or more colorectal cancer tumor markers in the sample.

18. The method according to any one of claims 16-17, wherein the stool resuspended solution comprises protoporphyrin IX complexed with multivalent cations.

19. A sample collection device comprising: a sample collection container having an open end; A stool resuspended solution in a container, wherein the stool resuspended solution comprises one or more hemoglobin stabilizing agents selected from the group consisting of: Protoporphyrin; Multivalent cations; a sugar or polysaccharide, and optionally a multivalent cation; Penetrants; and an HRP stabilizing component, and optionally a multivalent cation; and A stool sampling rod comprising a distal beveled tip for scooping and / or scraping a stool sample, wherein the distal end of the sampling rod is sized to be inserted into the sample collection container and the proximal end of the sampling rod is adapted to connect with the open end of the sample collection container, thereby sealing the distal end of the sampling rod within the device.

20. The sample collection device according to claim 19, wherein the sample collection container and the sampling rod are connected by threaded fitting.

21. The sample collection device of claim 19, wherein the stool resuspended solution comprises protoporphyrin IX complexed with multivalent cations.

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