JP2007248170A - Container for preparing fecal suspension - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fecal suspending/filtering container for recovering peeled cancerous cells in feces, rapidly, simply and safely. <P>SOLUTION: The container for suspending feces is a container which keeps a region for crushing and suspending feces, a filter for filtering the suspended feces, integrated with a region for recovering the filtrate passed through the filter and enables the use with the container that has sampled feces connected. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a stool collection kit for collecting stool used for colorectal cancer testing, and a stool processing kit for suspending and filtering the collected stool.

  In Western countries, colorectal cancer is always the top cancer mortality rate. In Japan, the number of patients with colorectal cancer has increased rapidly in recent years. This is thought to be due to the fact that the Japanese diet has become a Western type with a focus on meat. Approximately 60,000 people suffer from colorectal cancer every year in Japan, and the number of deaths by organ is the third largest after gastric cancer and lung cancer, and is expected to increase in the future. However, unlike other cancers, it is known that colorectal cancer is likely to be completely cured by surgery if it is an early stage cancer. Therefore, colorectal cancer is promising as a target for early cancer screening, and many examination methods have been devised.

  Currently, enema examination and endoscopy are performed as examination methods aiming at early detection of colorectal cancer. An enema examination is a method in which barium is injected into the large intestine, adhered to the mucosal surface of the large intestine, and the surface irregularities thereof are examined by X-rays. Endoscopy is a method of examining the large intestine directly with an endoscope. Endoscopy, in particular, has high sensitivity and specificity for the detection of colorectal cancer, and is a definitive test method. In addition, it has the advantage of removing early or premature polyps during the examination.

  However, these inspection methods are costly and burdensome to the subject, and are associated with a risk of complications. Furthermore, endoscopy requires skill in operation, and facilities that can perform the inspection are limited. Therefore, these methods are not suitable for colorectal cancer screening tests for asymptomatic general people.

  Thus, a fecal occult blood test that is simple and low in cost is widely used as a screening method for colorectal cancer. The fecal occult blood test is a method of diagnosing the presence or absence of bleeding in the intestine by examining the presence of hemoglobin contained in the stool, and indirectly examining the presence or absence of colorectal cancer.

  While it is a fecal occult blood test that is currently widely conducted, there are also questions about the usefulness of the test. There is a report that the sensitivity of the fecal occult blood test has a large variation of about 25%, and there is a high probability of overlooking the colorectal cancer patient. In addition, the positive predictive value is low, and the proportion of patients who are actually colorectal cancer patients in the fecal occult blood test-positive subjects is 10% or less, and many false positives are included. Therefore, development of a new inspection method with higher reliability is strongly desired.

  Therefore, as a new colorectal cancer testing method, attention is focused on a testing method using cancer cells detached in the stool. Compared to the fecal occult blood test method, which examines intestinal bleeding that occurs indirectly with colorectal cancer, this method directly examines the presence of cancer cells, so it can be a highly reliable test method in principle. Conceivable.

  In the above-described testing method using detached cells, nucleic acid or protein derived from cancer cells is directly extracted from the stool, and the strategy for making it the test target, and actually collecting the cancer cells themselves, the test target Two types of strategies are considered. The latter strategy of collecting cancer cells is advantageous in that cancer cell-derived products can be concentrated.

  As a method for collecting colon cancer cells, a collection method using Percoll (Non-Patent Documents 1, 2, and 3 below) and a method for collecting cancer cells from the surface of frozen stool have been devised (see below). Patent Document 1, Non-Patent Documents 4, 5, and 6). Further, a total system for stool processing relating to cell recovery from stool has been devised (Patent Document 2).

WO97 / 09600 Publication

Japanese Patent Application No. 2003-281978 Int.J.Cancer, Vol.52, 347-350, 1992 Gastroenterology, Vol.114, 1196-1205, 1998 International Journal of Molecular Medicine, Vol.13, 451-454, 2004 Clinical Cancer Research, Vol.4, 337-342, 1998 The Lancet, Vol.359, 1917-1919, 2002 APMIS, Vol.110, 239-246, 2002

  In order to collect colon cancer cells from stool, it is necessary to prepare a stool suspension first. In the current method, feces and a suspension solution are put in a bag, and a suspension is prepared by using a device called a stomacher that can pulverize solids mildly (Patent Documents 1 and 2). . In Patent Document 2, the stool suspension is further filtered through a funnel filter, and colon cancer cells are collected from the filtrate.

  However, in these methods, since the process of the stomacher processing of the collected stool or the filtration process of the suspension is an open system, problems such as leakage of odor to the surroundings and scattering of the stool suspension occur. Furthermore, since there are many manual operation steps, there is a problem that it is not suitable for processing a large amount of samples.

  In addition, when a conventional stool specimen collection or suspension container for stool occult blood test is used in this method, a stool amount necessary for cell collection cannot be obtained. The fecal occult blood test can be detected with a small amount of stool (0.1 g or less), but it is considered that at least 0.5 g or more of stool is required to collect cells from the stool.

  An object of the present invention is to provide a stool collection container for quickly, simply and safely collecting exfoliated cancer cells in stool.

  The present inventors have found that the above problems can be solved by a stool collection kit and a stool processing kit that integrate a series of processes of stool collection, pulverization, suspension, and filtration, and have reached the present invention.

  The stool collection kit of the present invention includes a stool collection container and a storage container including a storage cylinder having a coupling portion with the stool collection container. The stool processing kit of the present invention includes a stool collection container, a stool collection container, a coupling part of the stool collection container, a syringe storage part, a suspension part, a filtrate receiving container, and a filter.

  The stool processing kit has a structure in which a suspension part for crushing and suspending stool, a filter for filtering the suspended stool, and a filtrate receiving container for collecting the filtrate through the filter are integrated. Furthermore, it is possible to attach a stool collection container and push the collected stool into the stool processing kit body.

  The steps of pulverization, suspension, and filtration are performed simultaneously by shaking the stool processing kit. Furthermore, this step can be automated by mounting the stool processing kit on a shaking device.

  According to the present invention, which integrates a series of processes of stool collection, pulverization, suspension, and filtration, a series of operations for obtaining a stool filtrate can be performed simply and in a short time. Furthermore, since the stool can be suspended in a closed system, the risk of scattering of the sample is low, and the safety for the operator can be increased. In addition, a large amount of stool (0.5 g or more) necessary for recovering cancer cells from feces can be treated. Furthermore, it is possible to automate a series of these operations and perform multi-sample processing.

Hereinafter, the specific structure of the stool collection kit and the stool processing kit of the present invention and the method of use thereof will be described.
<Feces collection>
FIG. 1 is a cross-sectional view of a container portion constituting a stool collection and stool processing kit of the present invention. The stool collection container 1 has a syringe-like structure and has a handle 3 that holds the container when the collection container is used. The tip of the syringe 2 is a part that is attached to the stool, and the inside of the syringe 2 is hollow. The storage container 4 includes the other coupling part 5 that can be coupled to the stool collection container, and includes a storage cylinder 6 that can store the stool collection container.

  FIG. 2A is a diagram showing the state of stool collection. By inserting the tip of the syringe 2 of the stool collection container 1 into the stool 7, the stool can be pushed into the syringe 2 and collected. Further, by piercing the stool with the collection container a plurality of times, it is possible to collect stool from a plurality of sites and to collect 0.5 g or more of stool necessary for cell examination.

  FIG. 2B is a view showing an operation of mounting the stool collection container 1 from which the stool is collected to the storage container 4. The storage container 4 can store the stool collection container 1 therein. The stool collection container 1 can be screwed into the storage container 4 and the stool can be sealed.

FIG. 2C is a view in which the stool collection container 1 is fitted in the storage container 4. In this state, it can be stored and transported for the next operation. Transportation can be done by mail or courier in a normal envelope. For transportation, it is also possible to use a box dedicated to a stool collection container incorporating a cryogen. Since the dedicated box can be produced according to the shape of the stool storage container, it is possible to carry multiple specimens in a small space.
<Feces suspension and filtration>
FIG. 3 shows a cross-sectional view of the stool processing container main body 8, and the stool processing container main body 8 includes a syringe storage part 9, a suspension part 10, and a filtrate receiving container 11. The upper part of the syringe housing part 9 has a coupling part 12 for connection with the stool collection container 1, and the stool collection container 1 and the stool processing container main body 8 can be coupled in a screwed manner. In this case, it is possible to change the container so that only the stool is dropped directly into the stool processing container body 8 without connecting the stool collection container 1. The syringe housing portion 9 is a portion in which the syringe 2 of the stool collection container 1 is accommodated. The suspension portion 10 is a central portion of the main body that suspends feces. The connection part 13 between the suspension part 10 and the filtrate receiving container 11 is a part having a screw-like structure composed of a coupling part for fitting the filter 14 and connecting the part and the filtrate receiving container 11. The filter 14 is a polyamide mesh filter having a diameter of about 30 mm, which is used for filtering the suspension, and has a rubber ring structure on the outer periphery. The filter 14 is fitted into the connection part 13 of the suspension part 10. To do. The material of the filter part is not necessarily limited to the above, and other materials may be used as long as they satisfy the conditions of the filter and can be adapted to this operation. The diameter of the filter is preferably about 500 μm to 1000 μm, but can be changed from about 100 μm to about 2000 μm depending on conditions such as the amount of stool and the mixture in the stool. The filtrate receiving container 11 is connected to the connection part 13 of the suspension part 10 in a screwed manner. A 50 ml centrifuge tube commercially available from Falcon, Inc., which is called by name such as a conical tube, or a newly prepared tube is connected and used.

  FIG. 4A is a schematic view of the stool suspension / filtration operation after the stool collection container 1 is joined to the stool processing container body 8. There is a stool grinding ball 15 in the main body central portion 10 in advance. The material for the stool grinding balls 15 may be made of ceramics such as zirconia or metal such as stainless steel. The size is preferably about 10 mm in diameter, but can be used from about 5 mm to 25 mm in diameter. The standard number of three is used, but one to ten or more can be used. A suspension solution 16 is placed in the filtrate receiving container 11 in advance. The amount of the suspension solution 16 is preferably about 20 ml to 40 ml. Any kind of solution can be used as long as it is a solution used for washing or culturing cells, such as Hanks solution or DMEM culture solution. It is desirable to add serum or BSA having a function of stabilizing cells or suppressing nonspecific adsorption to the tube to the solution.

  The actual suspension / filtration operation is performed by shaking the container up and down. In the central part 10 of the main body, the stool 17 and the grinding balls 15 are mixed with the suspension solution 16 so that the stool is crushed. By repeating the shaking, the stool becomes more suspended. The direction of shaking can be changed at any time, corresponding to the state of stool suspension. The number of shakes is about 100 round trips. Depending on the state of the stool, the number of shakings can be changed from about 50 to 1000 times. Shaking is performed using a human hand or a shaking device. As the shaking device, a commercially available shaker or a shaking device that shakes in an arm type can be used. In addition, when a mesh-like structure is provided in the central part of the suspension part 10, the suspension of feces is promoted by friction, and at the same time, a part of the solid crushed material contained in the liquid is captured, and the filter Clogging can be reduced.

  4A is a filtrate after stool suspension, and shows a state after being filtered by the filter 14 and moved to the filtrate receiving container 11. The suspension can be completely transferred to the filtrate receiving container 11 by holding the suspension part 10 with one hand and tapping the filtrate receiving container 11 with the other hand. It is.

  FIG. 4B shows another pattern of the suspension / filtration container. The adapter 19 can be inserted with the same filter as the above 14 at the center. Further, the above-described 13 screw-type connecting portion is present in both the upper and lower directions, and the suspension container 20 can be coupled to the opposite side of the filtrate receiving container 11. The suspension container 20 can use the same type of tube as the filtrate receiving container 11. A pulverizing ball is placed in the suspension container 20 in advance, and after dropping feces directly into the tube, the suspension container 20 is connected to the adapter 19 for use. The suspension / filtration operation can be performed by shaking as in FIG.

  FIG. 4C shows still another pattern of the suspension / filtration container. The stool collection container 1 can be screwed into a suspension-only container 22 that contains a suspension solution 21 in advance. After connection, shake as in the above to suspend feces. The solution 23 after suspension can be directly connected to the adapter 19 and filtered, or poured into the adapter 19 and filtered.

  By passing through the above procedure, the stool can be suspended and filtered for one stab (about 1-1.5 g) of the collection container. However, in actual examinations, there are cases where the stool viscosity and the amount collected vary depending on the subject, and the stool does not fall from the syringe 2 of the collection container 1 to the suspension part 10 when the stool is suspended or filtered. A sampling container for preventing these phenomena is shown schematically in FIG.

  The collection container of FIG. 5 (A) has a window 24 in the syringe part. When shaking, the suspension solution enters from the window and the feces in the syringe are pushed out.

  The handle of the collection container of FIG. 5 (B) has a lid 25, and a stool ball can be placed in the syringe before shaking. When shaken subsequently, the stool in the syringe is pushed out by the weight of the ball.

  The collection container shown in FIG. 5C has a structure in which the stool balls 26 are accommodated in advance in the syringe. When shaken, the film-like structure 27 holding the ball is broken, and the stool in the syringe is pushed down.

The stool suspension / filtrate prepared by any of the above methods is transferred to another container, and the cells present in this filtrate are collected and used for colorectal cancer testing.
<Cell recovery using magnetic beads>
The cancer cells contained in the filtrate are collected using a carrier having affinity for the cancer cells. As the carrier, magnetic beads with an antibody having affinity for cancer cells bound to the surface are used. Specifically, Ber-EP4 antibody-bound magnetic beads (Dynabeads Epithelial Enrich, Dynal) commercially available from Dynal are used. In addition to Ber-EP4, any antibody having affinity for colon cancer cells can be used. In addition to antibodies, aptamers and ligands that have affinity for colon cancer cells can be used.
<Colorectal cancer diagnosis>
The cells collected by the above method are subsequently used as a specimen for colorectal cancer determination. Cancer determination may be performed using cells themselves or substances extracted from cells. When using cells themselves, use them immediately after collection. It can also be stored in a cell fixative or the like. When using the extracted substance, the pellet can be stored frozen at -80 ° C.

  When the cells themselves are used, the cells are stained by Papanicolaou staining, and observed and determined with a microscope. When the ratio of the nucleus to the cytoplasm (N / C ratio) is high and atypical cells in which chromatin is aggregated can be confirmed, it is determined that the cell is a cancer cell. The staining method can be applied as long as it can identify cancer cells. In addition to general staining, immunostaining using a cancer cell-specific antibody can be applied.

DNA or RNA can be extracted from the cells and used for cancer determination. Nucleic acid extraction kits available from various companies can be used for DNA and RNA extraction. Specifically, DNA extraction includes Dynabeads DNA DIREIC Universal, Qiagen QIAampDNA Mini Kit, Sanko Junyaku Sepagene, and the like. Examples of RNA extraction include ISOGEN (Nippon Gene) and TRIzol Reagent (Invitrogen).

  When performing genetic diagnosis using the extracted DNA, methods such as sequence analysis, SSCP analysis, and DHPLC analysis are used for mutations in gene sequences such as K-ras, APC, p53, and Smad4, which are genes associated with colorectal cancer. Diagnose. Alternatively, colorectal cancer diagnosis can be performed by detecting methylation of promoter regions such as hMLH1, p14, p16, MGMT, APC, and SFRP1. When genetic diagnosis is performed using the extracted RNA, diagnosis can be performed by examining genes that are specifically expressed in colon cancer cells. Specifically, methods such as Northern blotting, RT-PCR, and microarray can be used.

  In contrast to the colon cancer cell recovery protocol using the stool collection kit and stool processing kit of the present invention, the conventional procedure for recovering colorectal cancer cells by the stomacher method differs in the procedure of specimen recovery and filtration. That is, in the present invention, stool collection is performed by a specific stool collection container, and suspension and filtration are performed by a stool processing container that can be combined with the stool collection container without using a stomacher. Suspension and filtration can be carried out in the same process and connected containers, which frees you from the trouble of handling stool, shortens work time, and reduces consumables. In addition, a mechanism for accurately moving viscous feces to the suspension site was incorporated into the collection container, making it easier to handle actual clinical samples.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these.
<Example 1: Production of stool collection container>
Each resin part of the stool collection kit and the stool processing kit of the present invention was produced by injection molding. The parts consist of a stool collection container (made of polyethylene), a storage container (made of polyethylene), a stool processing kit body (made of polyethylene), an adapter (made of polyethylene), and a filter (outer periphery: made of elastomer, filter part: made of nylon). See above for a description of each part.
<Example 2: Evaluation by simulated flight>
The use method of the stool collection container and the stool processing container was examined using simulated stool. The simulated stool was kneaded by mixing 2.5 g of fishing bait (Float Salmon, Marquee Co., Ltd.) as a base material and 1 g of flour (Cameriya strong wheat flour, Nissin Flour Milling) as a thickener, and then adding 2.5 ml of water. . Using simulated stool, an attempt was made to prepare a filtrate in each of the patterns (A), (B), and (C) of FIG. As a result, simulated stool filtrates could be produced in all patterns.
<Example 3: Evaluation of cell recovery rate>
The fecal suspension treatment was performed in a state where the simulated stool and the cultured cells were mixed, and the cell recovery rate was examined to evaluate the adaptability of the container to the actual sample.
Colon cancer cells HCT116 (ATCC, Sumisho Pharma) were cultured and used for experiments. Fetal bovine serum cell culture in Advanced DMEM medium (Invitrogen Corp.) as medium for culturing (FBS: f etal b ovine s erum, Invitrogen Corp.) is used which was added to a 10%, 5% _CO 2, The test was performed at 37 ° C. The cultured and expanded cells were collected by treatment with 0.25% trypsin-EDTA (Invitrogen), and the number of cells was counted using a hemocytometer (HIRSCHMANN) and used for the experiment.

Using the stool collection container shown in FIG. 5 (A), a simulated stool (about 1.3 g) for one stab was collected. Then collecting container 1.6x10 of 30ml containing the ^five HCT116 cells L-15 medium (Sigma-Aldrich, Inc.) + 10% BS (b ovine s erum, Invitrogen Corporation) and the container body center stool crushing ceramics It was set in the stool processing container body containing 3 balls (Nikkato). Next, this container was shaken with a special shaker (250 r / min, 1 minute) to prepare a suspension / filtrate of simulated stool.

Add 40μl (about 1.6x10 ⁷ pieces) of magnetic beads (Dynabeads Epithelial Enrich, Dynal) to this suspension / filtrate, set in a rotor (Macsmix, Daiichi Chemicals), and rotate for 30 minutes at room temperature. (About 12 rpm). Next, the tube was set on a magnet stand (Dynal MPC-1, Dynal), placed on a shaker (petit shaker MODEL224, Wakken Co., Ltd.) and shaken at room temperature for 15 minutes. After shaking, the solution was discarded by decantation while keeping the tube set on the magnet stand. Furthermore, in order to remove mock stool residue, the inside of the tube was washed twice with PBS (−) (Invitorogen). Subsequently, the tube was removed from the magnet stand, about 600 μl of L-15 medium was added, the magnetic beads were suspended, and transferred to a 1.5 ml tube (Assist) set on the magnet stand (Dynal MPC-S, Dynal). . After confirming that the magnetic beads were gathered on the magnet contact surface in the tube, the solution was removed. Further, the inside of the tube was washed three times with PBS (−). Finally, the tube was removed from the magnet stand and resuspended with 100 μl of L-15 medium.

10 μl of the resuspension was mixed with 10 μl of trypan blue solution (Sigma-Aldrich), and the number of cells was counted using a hemocytometer. The counted number was divided by the number of cells initially added (1.6 × 10 ⁵ cells) to obtain the cell recovery rate. As a result of three experiments, it was found that an average recovery rate of 32.2% (SD 5.7) was obtained, and cells could be recovered using this method.

  The stool collection kit and stool processing kit of the present invention facilitated a method for collecting colon cancer cells. This contributes to early detection of colorectal cancer.

It is sectional drawing which shows the stool collection container of this invention. It is sectional drawing which shows the stool collection method by the stool collection container of this invention. It is sectional drawing which shows the stool processing container main body of this invention. It is the figure which showed the pattern of the feces suspension and filtration processing method of this invention. It is the figure which showed the improvement pattern of the stool collection container of this invention.

Explanation of symbols

1: stool collection container, 2: syringe, 3: handle, 4: storage container, 5: binding site, 6: storage cylinder, 7: stool, 8: stool processing container body, 9: syringe storage part, 10: suspension Turbidity part, 11: Filtrate container, 12: Coupling part, 13: Connection part, 14: Filter, 15: Ball for stool crushing, 16: Solution for suspension, 17: Collected stool, 18: Filtrate after suspension of stool , 19: Adapter, 20: Suspension container, 21: Suspension solution, 22: Dedicated suspension container, 23: Solution after suspension, 24: Window, 25: Lid, 26: Ball for crushing feces, 27 : Membrane structure.

Claims (11)

  A stool collection kit comprising a stool collection container, a stool collection container main body that can be coupled to the stool collection container, and a means for moving the stool collected from the stool collection container to the stool collection container main body, (a) the stool collection The container comprises a syringe having a capacity capable of collecting 0.5 g or more of stool by piercing the stool, a stool collection port, and a handle provided on the outer periphery of the syringe at the other end of the stool collection port, (b ) The stool processing container main body includes a syringe storage unit that stores the syringe, a suspension unit that is connected to the syringe storage unit and suspends stool, and is connected to and separated from the suspension unit. A stool processing kit comprising: a filtrate receiving container for receiving a filtered stool filtrate; and a filter provided at a connection part of the suspension part and the filtrate receiving container.   The stool processing kit according to claim 1, wherein the stool processing is performed by shaking.   The stool processing kit according to claim 1, wherein the syringe has a window for drawing a water flow into the syringe when stool is suspended.   The stool collection container has a structure in which an upper part is openable and closable, and a stool crushing ball is inserted to push down stool in a syringe when stool is suspended. Fecal treatment kit.   The stool collection container has a stool crushing ball storage structure at the top of the syringe, and has a structure in which the ball is released into the syringe by shaking during stool suspension and the stool in the syringe can be pushed down. The stool processing kit according to claim 1, which is characterized.   The stool processing kit according to claim 1, wherein the member for the stool grinding ball storage structure is a thin film that can be pierced by shaking.   A stool processing kit comprising a stool processing container, a filtrate receiving container, and an adapter for connecting both containers. The stool processing kit according to claim 7, wherein the adapter has a ring-shaped structure having a filter, and the stool processing container and the filtrate receiving container can be connected to face each other in two directions. The stool processing kit according to claim 7, wherein the stool processing container and the filtrate receiving container connected to the adapter have the same shape.   A stool processing kit comprising: a filtrate receiving container and the adapter connected, and filtration is performed by adding a suspension to a filter portion in the adapter. A stool processing kit having a structure in which a stool collection container is directly connected to a filtrate receiving container.

Images