JP2005241543A - Excreta container, cell retrieval device, excreta preservation kit and cell retrieval method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an excreta container or the like for setting cytological examination as a series of processes wherein sampled ordinary excreta is mixed with buffer liquid, preserved and carried. <P>SOLUTION: This excreta container comprises a liquid bag part for storing the sampled ordinary excreta and the buffer liquid, and a sealing part provided on the periphery of an opening part of the liquid bag part. The excreta container has a stomacher part for mixing the ordinary excreta and the buffer liquid by hand rubbing or mechanical rubbing from the outside in the liquid bag part, and a cuttable or perforative excreta taking-out cutting part on the bottom part of the liquid bag. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a stool container, a cell collection device, a stool preservation kit, and a cell collection method using them for collecting all spontaneously excreted stool and performing a cancer cell test or the like.

  In Europe and the United States, colorectal cancer accounts for the top cancer mortality. In Japan, the number of patients with colorectal cancer has increased rapidly in recent years. This is thought to be caused by the fact that the Japanese diet is centered on Western-style meat eating. About 60,000 people suffer from colorectal cancer every year in Japan, and the number of deaths by organ is the third largest after stomach cancer and lung cancer, and further increases are expected in the future. However, it is known that colorectal cancer can be cured almost 100% by surgery if it is early cancer, unlike other cancers. Therefore, colorectal cancer has been the subject of early cancer screening, and numerous testing methods have been devised.

  Enema examination and endoscopy are performed as inspection methods for 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. These tests have high sensitivity and specificity for the detection of colorectal cancer. In addition, endoscopy has the advantage of removing early or premalignant polyps. However, these examination methods place a heavy burden on the subject and are expensive. In particular, endoscopy requires skill in operation and involves a risk of complications. Therefore, it is not suitable for colorectal cancer testing for asymptomatic general people.

  Therefore, fecal occult blood tests are widely used as a primary screening method for colorectal cancer in the general population. The fecal occult blood test is a method for diagnosing the presence or absence of bleeding in the intestine by examining the presence of hemoglobin contained in the stool and indirectly predicting the occurrence of colorectal cancer.

  There are two types of fecal occult blood testing methods: chemical testing and immunological testing. The chemical fecal occult blood test method uses the peroxidase activity of hemoglobin, and the guaiac contained in the filter paper is blue-green due to the active oxygen generated when hydrogen peroxide added as a substrate is decomposed. It uses a reaction that turns into oxidized guaiac. Hemocult II filter paper (Fujisawa Pharmaceutical), Shionogi B filter paper (Yoshio Shionogi) and the like are used for inspection as commercial products.

  The immunological fecal occult blood test mainly uses the binding of an anti-human hemoglobin antibody to specific human hemoglobin, and due to its high specificity, it is now becoming the mainstream fecal occult blood test method. Inverse passive hemagglutination method (Immea Hem-Sp, Fuji Leobi), magnetic particle aggregation gradient method (Magstream Hem-Sp, Fuji Leobi), latex aggregation method (Immunocult, Chugai Pharmaceutical) and the like are known.

  Although the fecal occult blood test is widely used for colorectal cancer tests, there are questions about the usefulness of the test. In order to be positive in a chemical fecal occult blood test using Hemocult II, 20 mg of bleeding per day is required in the large intestine, but the actual bleeding of patients with colorectal cancer is considered to be 10 mg or less. Therefore, the sensitivity of fecal occult blood reaction is about 26%, and only about 1/4 of actual colorectal cancer patients can be found, and there is a report that 3/4 is missed (Non-patent Document 1 below). Furthermore, only 8.3% of the positive test subjects actually had colorectal cancer, and many false positives were included.

  Therefore, development of a new primary screening test method with higher accuracy is eagerly desired. As a candidate for this, attention has been focused on a test method using cancer cells detached in feces. Compared with the fecal occult blood test method, which examines intestinal bleeding that occurs indirectly with colorectal cancer, this method directly examines cancer cells, and is considered to be a more reliable test method.

  As a test method for examining cancer cells in stool, a method for genetic diagnosis using nucleic acid directly extracted from stool is reported in Patent Document 1 below. Specific gene mutation detection methods such as sequencing, PCR-RFLP (polymerase chain reaction-restriction enzyme fragment length polymorphism), SSCP (single-stranded conformational polymorphism), and PTT (protein truncation test) have been developed. ing. In addition to detection of gene mutations, there are known methods for making a diagnosis by using microsatellite instability (MSI) and the appearance of long DNA (L-DNA) as indicators.

  K-ras, APC, P53, DCC and the like are widely known as genes targeted for diagnosis of gene mutations. The search for genes that can be targeted for new diagnoses, focusing on the difference in gene expression levels, is still actively conducted using microarrays and the like. A method using a CD44 gene splicing variant expression pattern as a marker has also been proposed.

The problem with these test methods is that nucleic acids derived from various bacteria and normal cells are present in the stool, and the proportion of cancer cell-derived genes recovered from the stool is very small (about 0). .05%). This greatly hinders the examination of mutations in cancer cell-derived genes and subtle changes in expression patterns, making it difficult to put these methods to practical use.
Therefore, a method for collecting and diagnosing cancer cells directly from all feces has been considered for more reliable diagnosis of colorectal cancer.

Japanese translation of PCT publication No. 2002-515973 (WO97 / 28450) Jama, Vol.269,1262-7, 1993

  Large-scale colorectal cancer testing for the general population requires mass processing of multiple samples using an automated system. In the determination of colorectal cancer using cells collected from stool, a method of identifying colon cancer by performing cytological analysis is very effective.

  However, in the conventional stool collection method, each process from stool collection to cytological examination is separated, and the collected stool is mixed with buffer solution, stored and transported, and cytological examination is performed. Development of a stool container or the like for making a series of processes has been desired. More specifically, the present invention relates to a method for recovering colon cancer-derived cells from a stool using magnetic beads to which an epithelial cell-specific antibody is immobilized. It is an object of the present invention to provide a tool and a method up to introduction into a cell recovery apparatus.

In order to solve the above problems, a fecal container having a simple structure and operation has been developed.
That is, first, in the stool container of the present invention, the liquid bag part has a stomacher part that mixes naturally discharged stool and buffer liquid from the outside by hand or machine. The liquid bag portion is made of a flexible resin material so that it can be hand-rubbed or machine-rubbed. Similarly, the stool removal cutting part at the bottom of the liquid bag is made of a resin material so that it can be easily cut or punched.

The liquid bag portion is preferably opaque so that the inside cannot be seen when the subject and the inspector handle.
The buffer solution preferably contains serum.

  A label can be attached to the liquid bag portion. The label can be filled with the name, sample number, collection date and other information. Moreover, it is preferable to provide a memory line to the liquid bag portion. The amount of stored feces can be easily known by suspending the fecal collection container using the memory line. Furthermore, the liquid bag portion can have a stool outlet that can be cut with a cutter.

  The fecal container is sealed with a sealing lid to facilitate storage and transportation. Further, at the time of storage / transportation, the stool container is preferably stored by a transport outer cylinder combined with the sealing lid.

  The cell collection device for placing the stool container preferably has a mounting plate with a hole for projecting the stool removal cutting portion provided at the bottom of the liquid bag, and the stool withdrawing from the mounting plate with the hole is taken out The cutting part for cutting is cut with a rotary blade or scissors, or drilled with a cone or laser irradiation to collect the fecal liquid containing cancer cells.

  Secondly, the present invention is a stool preservation kit comprising a combination of the stool container and a buffer solution storage container. The buffer solution storage container preferably contains serum.

  Third, the present invention is a cell collection method using the stool container, the step of mixing the natural drainage stool and the buffer solution by scrubbing from the outside with the liquid bag part as a stomacher part, A process of storing and transporting the sealed stool container in an integrated transport outer cylinder combined with a sealing lid, and a stool removal cutting portion provided at the bottom of the liquid bag projecting the transport outer cylinder There are a step of placing on a cell recovery device having a mounting plate with a hole for making it, and a step of cutting or perforating the stool removal cutting part to recover the spontaneously discharged feces and the buffer liquid mixture. Here, the step of mixing the spontaneously discharged stool and the buffer solution by scrubbing from the outside with the liquid bag portion as the stomacher portion, and the transporting united by combining the sealed stool container with the sealing lid The order of storing and transporting in the external cylinder may be changed.

  Compared with the conventional stool collection method in which each step from stool collection to cytological examination was separated, the stool container of the present invention mixes the collected stool with a buffer solution, and stores and transports it. Thus, it is suitable for making cytological examination a series of steps. The stool collection container of the present invention can be effectively used for colorectal cancer testing. From the stool collected in the stool container of the present invention, it becomes possible to efficiently recover good live cancer cells from the stool at room temperature, and the recovered cells are cytologically, immunologically, biochemically recovered. Analysis can be performed with high accuracy. In addition, by using the stool container of the present invention, cells derived from early colon cancer, or cancer cells derived from the ascending colon, which are difficult to find by endoscopy, are collected by using all stool samples. This makes it possible to provide a highly reliable inspection method.

  FIG. 1 shows an example of a stool container of the present invention. FIG. 1A is a cross-sectional view of a stool container, and FIG. 2B is a cross-sectional view showing a state in which a stomacher portion is held by hand after sealing with a sealing lid.

  The stool container of the present invention basically comprises a seal part, a stomacher part and a cutting part. A liquid bag part is a bag which accommodates the collected natural discharge stool. Feces are introduced with reference to a memory line provided on the side of the liquid bag using, for example, a dedicated spatula and funnel. Similarly, the buffer liquid is also introduced into the liquid bag portion with reference to a pre-weighed amount or a memory line provided on the side of the liquid bag (FIG. 1 (a)). Next, the liquid bag part is used as a stomacher part, and the above-mentioned spontaneously discharged stool and the buffer liquid are mixed with a hand or machine from the outside to obtain a liquefied specimen. At this time, the stool container is sealed with a sealing lid (FIG. 1B).

  The sealed stool container is combined with a sealing lid and stored in an integrated transportation cylinder and transported to an inspection department or an inspection organization (FIG. 2 (a)). At this time, only the collected stool may be transported, and the work after the introduction of the buffer solution may be performed in the inspection department or the inspection organization. At the time of the inspection, the carrying outer cylinder is placed on a cell collection device having a placing plate with a hole for projecting a stool take-out cutting portion provided at the bottom of the liquid bag. Next, the stool removal cutting part is cut using a rotary blade or scissors, or drilled using a cone or a laser to collect spontaneously discharged stool and a buffer liquid mixture (liquefied sample) (FIG. 2 ( b)).

  The liquid bag part can be opaque so that the inside can not be seen when the inspector handles it, but it is translucent, and the approximate amount of feces collected by the memory line provided on the side of the liquid bag You can also see the buffer volume.

  In order to avoid an inspection error, a label is affixed to the liquid bag portion, and the name, sample number, collection date and other information are written on the label.

  The stool collected by the stool collection container of the present invention is subjected to subsequent immunological, cytological, or biochemical tests. Hereinafter, an example of a standardized protocol for cell recovery from feces will be described according to the procedure.

(Step 1: Sample collection)
The feces used in the present invention uses human spontaneous excretion. Feces should be in solid form and diarrhea should not be used. Also, do not use feces excreted after the subject uses compulsory excretion drugs such as laxatives or barium for intestinal examination. There is no need for the subject to have any special dietary restrictions in advance.

  Feces collected from the subject are preferably used within 3 hours, but can be used up to about 10 hours. During this time, the sample stool can be stored at room temperature, and the sample stool need not be refrigerated or frozen.

  The amount of feces used is preferably about 5 to 80 g, but about 0.5 to 200 g can be used.

  Hanks solution is used for the buffer solution (medium). However, any buffer solution generally used in experiments involving cells can be used. Specific examples include PBS, PBS (−), various cell culture media (MEM, DMEM, RPMI) and the like.

  The amount of buffer solution to be added can be changed depending on the amount and state of stool. However, 1 ml or more per 1 g of stool is desirable. If 200 ml of buffer solution is added per bag of the stool container of the present invention, all the above stool volumes can be handled.

  Add serum to the buffer solution. The serum concentration is preferably 10%, but can be about 0.5 to 20%. The type of serum is preferably FBS (fetal bovine serum), but CS (calf serum) can also be used.

  The sample recovery step using the stool container of the present invention was performed at room temperature. However, if it takes time from sample collection to filtration, the sample may be stored in a cooler box or the like.

(Step 2: Filtration)
The suspension is filtered in a fume hood using a filtering device with a filter to remove residues.

(Step 3: Magnetic bead reaction)
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.

  Add 40 μl of magnetic beads per tube containing approximately 20-45 ml of dispensed filtrate. The amount of magnetic beads can be varied in the range of about 20 to 400 μl.

  The filtrate to which the magnetic beads have been added is mixed using a mix rotor, and the cells in the filtrate are bound to the magnetic beads. Mixing is desirably performed at room temperature or in a cold room at 4 ° C. The mixing time is preferably 30 minutes or more. This magnetic bead reaction step was performed at room temperature.

(Step 4: Magnetic separation)
The mixed filtrate-containing tube is placed on a magnetic stand and then shaken for 15 minutes to collect the magnetic beads on the side of the tube. The shaking time is preferably 10 minutes or more. The shaking method is not a problem as long as the filtrate is gently mixed, such as seesaw motion, rotation, and rotation.

  After the magnetic beads are attached to the wall, the filtrate is removed. After removing the filtrate, remove the tube from the magnetic stand, wash with the buffer solution, and collect the bead washing solution. The buffer solution is used in an amount of 500 μl per tube, but the amount can be arbitrarily changed assuming the next experiment. This magnetic separation step was performed at room temperature.

(Step 5: Magnetic separation, Eppendorf tube)
The cleaning solution is collected in an Eppendorf tube that is smaller than the previously used tube. The tube containing the washing solution is immediately placed on a dedicated magnetic stand. After collecting magnetic beads on the side wall of the Eppendorf tube, the supernatant is removed to obtain a pellet of the cell-bead complex. The magnetic separation and Eppendorf tube steps were performed at room temperature.

<Colorectal cancer diagnosis>
The pellet collected by this standardized protocol is then 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. 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 Nippon Gene's ISOGEN and Invitrogen's TRIzol Reagent. The extracted nucleic acid can be used in various methods as shown in the section of the prior art.

An example of the stool container of this invention is shown. It is the figure which stored the stool container of this invention in the outer cylinder, and the figure which mounted the stool container of this invention in the cell collection | recovery apparatus.

Claims (14)

  A stool container comprising a liquid bag part for storing collected natural drained stool and buffer liquid, and a seal part provided around an opening of the liquid bag part, wherein the liquid bag part is a natural drained stool and a buffer. A stool container comprising a stomacher part for mixing liquid from the outside by hand or machine, and a stool removal cutting part that can be cut or perforated at the bottom of the liquid bag.   The stool container according to claim 1, wherein the liquid bag portion is opaque.   The stool container according to claim 1, wherein the buffer solution contains serum.   The stool container according to claim 1, wherein a label on which at least one of a name, a sample number, and a collection date and time is written is attached to the liquid bag part.   The stool container according to claim 1, wherein the liquid bag portion has a memory line that serves as a measure of the amount of stool and the amount of buffer solution.   A stool container in which a sealing lid is fitted to the seal part of the stool container according to claim 1.   The stool container according to claim 6, wherein the stool container is stored by a transport outer cylinder integrated with the sealing lid.   It is a cell collection | recovery apparatus which mounts the stool container of Claim 7, Comprising: It has a mounting plate with a hole for making the cutting part for stool extraction provided in the bottom part of the said liquid bag protrude. Cell collection device.   The cell recovery apparatus according to claim 8, further comprising a rotary blade or a hook-shaped cutting means for cutting the stool removal cutting part.   9. The cell recovery apparatus according to claim 8, further comprising a cone-shaped perforating unit or a laser irradiation unit for cutting the stool removal cutting part.   A stool preservation kit comprising a combination of the stool container according to claim 1 and a buffer solution storage container.   12. The stool preservation kit according to claim 11, wherein the buffer solution storage container contains serum.   Introducing the collected spontaneously discharged feces into the liquid bag portion of the stool container; introducing the buffer solution into the liquid bag portion of the stool container; sealing the stool container with a sealing lid; and the liquid bag A step of mixing the spontaneously discharged stool and the buffer solution by mixing the hand with a hand or a machine from the outside as a stomacher part, and combining the sealed stool container with the sealing lid into an integrated transport outer cylinder A step of storing and transporting, a step of placing the outer cylinder for transportation on a cell recovery device having a placing plate with a hole for projecting a cut portion for removing stool provided at the bottom of the liquid bag, and A method for recovering cells, comprising a step of cutting or perforating a stool removal cutting section to recover the spontaneously discharged stool and the buffer solution mixture.   The step of introducing the collected spontaneously discharged stool into the liquid bag portion of the stool container, the step of introducing the buffer solution into the liquid bag portion of the stool container, the step of sealing the stool container with a sealing lid, and the sealing The step of storing and transporting the stool container in the transport outer cylinder combined with the sealing lid, and the hand bag or the machine from the outside with the liquid bag portion as a stomacher portion, the spontaneously discharged stool and the buffer A step of mixing the liquid, a step of placing the outer cylinder for transportation on a cell recovery device having a mounting plate with a hole for projecting a cut portion for removing stool provided at the bottom of the liquid bag; A method for recovering cells, comprising a step of cutting or perforating a stool removal cutting section to recover the spontaneously discharged stool and the buffer solution mixture.

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