CN113567672A - Kit for detecting cancer cells in ascites or peritoneal lavage fluid - Google Patents
Kit for detecting cancer cells in ascites or peritoneal lavage fluid Download PDFInfo
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- CN113567672A CN113567672A CN202110844018.XA CN202110844018A CN113567672A CN 113567672 A CN113567672 A CN 113567672A CN 202110844018 A CN202110844018 A CN 202110844018A CN 113567672 A CN113567672 A CN 113567672A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention provides a kit for detecting cancer cells in ascites or peritoneal lavage fluid, and belongs to the technical field of peritoneal carcinomatosis detection. The kit comprises a nanometer microporous filter membrane filtering device for collecting epithelial cells in peritoneal lavage fluid, an IF-FISH reagent, a fluorescence in situ hybridization reagent and a marker for immunofluorescence staining reagent detection, wherein the marker comprises one or more of the following components: CEP8, CEP17 and EpCAM proteins. The kit is specially used for ascites or peritoneal lavage fluid samples with a small amount of free cancer cells, and has high detection sensitivity; simultaneously, two technologies of FISH and IF are adopted for dyeing and observation, so that the detection accuracy is improved, and the sample amount is saved.
Description
Technical Field
The invention belongs to the technical field of peritoneal carcinomatosis detection, and particularly relates to a kit for detecting cancer cells in ascites or peritoneal lavage fluid.
Background
Localized progression of abdominal and pelvic malignant tumors such as gastric cancer, colorectal cancer, primary peritoneal cancer, etc. is likely to form peritoneal surface tumors, commonly referred to as Peritoneal Carcinosis (PC) (peritoneal cancer for short). Examination of ascites or peritoneal lavage fluid cytology peritoneal free cancer Cells (CY) are the gold standard for the diagnosis of peritoneal micrometastases. It is well known that intraperitoneally free cancer cells are a prerequisite for the development of peritoneal metastases and are an independent poor prognostic factor for gastric cancer (Lee SD, Ryu KW Eom BW, et al, diagnostic signalling of cancer bathing cytology in tissues with gastric cancer [ J ]. Br J Surg,2012,99(3):397-403.DOI: 10.1002/bjs.7812.). The positive of the abdominal cavity free cancer cell can be used as an independent diagnosis index of the IV-stage gastric cancer. However, conventional techniques may not be able to achieve the desired purpose when only a small number of free cancer cells are present in the lavage fluid.
Disclosure of Invention
In view of the above, the present invention provides a reagent composition for detecting cancer cells in peritoneal lavage fluid, which is based on the difference in cell diameter, structure difference and deformability between free epithelial cells in the peritoneal cavity and other cells (mainly granulocytes) in the peritoneal cavity, and separates and enriches epithelial cells from the peritoneal lavage fluid by using a nano-microporous membrane filtration device, and then identifies tumor cells or heteroploid cells by using Fluorescence In Situ Hybridization (FISH) and immunofluorescence staining (IF) techniques to perform chromosome CEP8 and/or CEP17 labeling and specific protein labeling surface antigens on the separated gastrointestinal tumor cells to realize accurate detection of malignant epithelial cells, thereby realizing early diagnosis of peritoneal micrometastasis.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a kit for detecting cancer cells in ascites or peritoneal lavage fluid, which comprises a nano microporous filter membrane filtering device for collecting epithelial cells in the peritoneal lavage fluid and an IF-FISH reagent, wherein a detection marker of the IF-FISH reagent comprises one or more of the following components: CEP8, CEP17 and EpCAM proteins.
Preferably, the number of cancer cells in the ascites or peritoneal lavage fluid is less than or equal to 101。
Preferably, the nano microporous filter membrane filtering device is a circulating tumor cell separator.
Preferably, the IF-FISH reagent comprises a fixing solution, a perforating agent, a CEP8 probe, a CEP17 probe, a solution A, a solution B, a cell nucleus staining solution, a primary anti-CD 45 Antibody Cocktail (Mouse), a Secondary anti-Goat anti-Mouse IgG (H + L) high hly Cross-Adsorbed second Antibody, a primary anti-EpCAM Polyclonal Antibody (Rabbit)) and a Secondary anti-Goat anti-Rabbit IgG (H + L) high hly Cross-Adsorbed second Antibody.
Preferably, the solution A is a mixed solution of sodium chloride and Tris-HCl, the solution B is an NP-40 solution, and the solution A and the solution B are used for washing the hybridization sample.
Preferably, the following reagents are also included: blocking reagent, absolute ethyl alcohol, methanol, paraformaldehyde solution with the mass concentration of 4%, PBS buffer solution and normal saline.
The invention has the beneficial effects that: the kit for detecting cancer cells in peritoneal lavage fluid provided by the invention comprises a nano microporous filter membrane filtering device for collecting epithelial cells in the peritoneal lavage fluid, and the device can maximally enrich the epithelial cells in the lavage fluid, so that peritoneal micrometastasis which cannot be detected due to small cell amount can be detected, and the kit is specially used for ascites or peritoneal lavage fluid samples with small amount of free cancer cells, and has high detection sensitivity; simultaneously, two technologies of FISH and IF are adopted for dyeing and observation, so that the detection accuracy is improved, and the sample amount is saved.
Drawings
FIG. 1 is a schematic diagram showing a filter membrane removing step of the nano-microporous filter membrane filtration apparatus according to the present invention;
FIG. 2 shows the IF-FISH detection results of a sample with the reference number 1, the left and right panels showing different regions of the sample;
FIG. 3 shows the IF-FISH detection result of the sample numbered 2;
FIG. 4 shows the result of IF-FISH detection (40 times magnification) of a sample number 2;
FIG. 5 shows the IF-FISH detection result of sample No. 5;
FIG. 6 shows the IF-FISH detection result of the sample No. 7;
FIG. 7 shows the IF-FISH detection result of the sample numbered 9;
FIG. 8 shows the IF-FISH detection results of a sample designated by reference numeral 8, the left and right panels showing different regions of the sample;
FIG. 9 shows the IF-FISH detection results of a sample with reference numeral 11, the left and right panels showing different regions of the sample;
FIG. 10 shows the IF-FISH detection result of a sample designated by reference numeral 10;
FIG. 11 shows the result of IF-FISH detection of a sample designated by reference numeral 13;
FIG. 12 shows the IF-FISH detection results of a sample designated by reference numeral 12, the left and right panels showing different regions of the sample;
FIG. 13 shows the IF-FISH detection result of sample No. 14;
FIG. 14 shows the IF-FISH detection results of a sample designated by reference numeral 15, the left and right panels showing different regions of the sample;
FIG. 15 shows the IF-FISH detection results of a sample numbered 18, the left and right panels showing different regions of the sample;
FIG. 16 shows the IF-FISH detection result of the sample designated by reference numeral 20, and the left and right panels show different regions of the sample.
Detailed Description
The invention provides a kit for detecting cancer cells in peritoneal lavage fluid, which comprises a nano microporous filter membrane filtering device for collecting epithelial cells in the peritoneal lavage fluid, and an IF-FISH reagent, wherein detection markers of the IF-FISH reagent comprise the following CEP8 (chromosome 8 centromere region) and/or CEP17 (chromosome 17 centromere region) and EpCAM protein.
In the present invention, the number of cancer cells in the ascites or peritoneal lavage fluid is preferably 10 or less. The kit provided by the invention is specially used for a sample with very low cancer cell number in ascites or peritoneal lavage fluid, so that the early screening of the peritoneal cancer is realized.
In the invention, the nano microporous filter membrane filtering device is preferably a circulating tumor cell separator. The circulating tumor cell separator is used for separating and enriching epithelial cells from ascites or peritoneal lavage fluid through the action of a nano microporous filter membrane based on different cell diameter sizes, structural differences and deformation capacities between free epithelial cells of the peritoneal cavity and other cells (mainly granulocytes) in the peritoneal cavity. In the present example, the circulating tumor cell separator is preferably available from YZYMED under the product model number YZYMED-A10.
In the present invention, the IF-FISH reagent includes a fluorescence in situ hybridization reagent and an immunofluorescence staining reagent; specifically, the IF-FISH reagent comprises a fixing solution, a perforating agent, a CEP8 probe, a CEP17 probe, a solution A, a solution B, a cell nucleus staining solution, a primary anti-CD 45 Antibody Cocktail (Mouse), a Secondary anti-Goat anti-Mouse IgG (H + L) high hly Cross-Adsorbed second Antibody, a primary anti-EpCAM Polyclonal Antibody (Rabbit) and a Secondary anti-Goat anti-Rabbit IgG (H + L) high hly Cross-Adsorbed second Antibody. The solution A is a mixed solution of sodium chloride and Tris-HCl, the solution B is an NP-40 solution, and the solution A and the solution B are used for washing the hybridization sample. The fixative solution, punch, CEP8 probe, CEP17 probe, solution A, solution B and the staining solution for cell nuclei are preferably purchased from YZYMED. The primary anti-CD 45 Antibody Cocktail (Mouse) and Secondary anti-Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488, were used as control detection reagents. The IF-FISH reagent preferably comprises an anti-EpCAM Polyclonal Antibody (Rabbit) and the Secondary Antibody, Goat anti-Rabbit IgG (H + L) high Cross-Adsorbed Secondary Antibody, Alexa Fluor 546, which is used for immunodetection of EpCAM proteins.
In the present invention, the kit preferably further comprises the following reagents: blocking reagent, absolute ethyl alcohol, methanol, paraformaldehyde solution with the mass concentration of 8%, PBS buffer solution and normal saline. The blocking reagent is a PBS solution with the mass percentage of 5% BSA; absolute ethanol, preferably analytically pure and above, for cell dehydration; methanol for fixing the separated cells; a paraformaldehyde solution (PFA) with a mass concentration of 4% for fixing cells during pretreatment of a sample; PBS buffer for rinsing; saline, diluted sample. The specifications and uses of the reagents used in the practice of the invention are detailed in Table 1.
In the present invention, the method for detecting cancer cells in peritoneal lavage fluid by using the kit preferably comprises the following steps: collecting ascites or peritoneal lavage fluid with a nano microporous filter membrane filtering device, and rinsing to obtain collected cancer cells; and transferring the cancer cells to a slide for slide preparation, treating the cancer cells under the action of a fixing solution, a perforating agent and a sealing solution in sequence, then adding a primary antibody and a secondary antibody in sequence for immunoreaction, treating the cancer cells by using the fixing solution, the perforating agent and the sealing solution in sequence, adding a CEP8 probe and a CEP17 probe, carrying out binding reaction, sealing and detecting.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. The materials of which this example is mainly concerned are shown in Table 1.
TABLE 1 Main consumables and reagents
2. Sample collection and rinsing
1) Collecting samples:
a) a30 mL ascites or peritoneal lavage fluid specimen is first collected using a 50mL sterile tube.
b) The sample is stored without refrigeration at room temperature, and needs to be pretreated within 2 hours after collection (step 3). If the transfer detection can not be carried out within 2 hours, please vertically stand in a refrigerator at 4 ℃ for storage, and the storage time cannot exceed 24 hours. The samples were stored in a refrigerator at 4 ℃ and allowed to re-incubate at room temperature for at least 30 minutes prior to processing.
2) Rinsing of filters
a) The filter stopple and stopple were opened and the filter was placed on the rinse rack.
b) About 2mL of 75% medical grade alcohol was added to the filter using a pasteur tube until it was naturally filtered out of the lower mouth of the filter.
c) 4mL of physiological saline was added to the filter for rinsing, the residual alcohol in the filter was removed, and the physiological saline was naturally filtered out.
d) And c), repeating the step c), reserving a proper amount of normal saline to ensure that the filter membrane is wet, and installing a filter and a lower plug.
Note that: if there is a situation in which alcohol does not leach out naturally or leaches too slowly during the filter rinsing, please discard the filter immediately.
3) Sample pretreatment: adding 1000 mu L of 8% PFA into a 50mL centrifuge tube, adding 20mL of an ascites or peritoneal lavage fluid sample into the 50mL centrifuge tube, gently inverting and uniformly mixing for 8 times, standing at room temperature for 10min, and pre-fixing the cells.
Sample separation and slice preparation
1) Sample separation: the process of sample separation is semi-automatically completed by the instrument, and the specific steps are as follows:
a) the filter was placed in the very middle of the a10 support module, ensuring that the lower plug was pierced by the lower needle.
b) Adding about 10mL of the fixed sample into the filter by using a Pasteur tube, clicking to start, adding the rest 10mL of the sample into the filter by using the Pasteur tube after the sample is separated, and clicking to start again to finish the sample separation.
c) Add 4mL of PBS to the filter tube, click "start" and perform a sample rinse.
d) Repeating step c)2 times.
2) Methanol fixation: add 500. mu.L of methanol to the filter, fix at room temperature for 1min, and operate the device to drain the methanol by clicking the "start" button. If methanol remains, the filter is taken down from the machine, the lower rubber plug is opened, and the upper rubber plug is covered, so that the residual methanol naturally flows out.
3) Taking and sticking a film: the filter was removed as shown in FIG. 1 and the filter membrane was removed, 5. mu.L of methanol was dropped onto one end of the slide, the filter membrane was attached to the slide with the front side facing up, and air dried for 10 min.
4) Removing cells on the back of the filter membrane: dripping 10 μ L of methanol on the slide, removing the air-dried filter membrane with tweezers, and leveling to the slide on which methanol is dripped, at 50 deg.C for 10 min.
IF-FISH staining and reading
1) Assembling a pen and drawing circle: drawing a closed circle slightly larger than the filter membrane around the filter membrane with a grouping pen, and air drying for 2 min.
2) Cell fixation: 150 mul of stationary liquid (fluorescent in situ hybridization sample treatment kit stationary liquid) was dropped onto the filter membrane and fixed for 5min at room temperature. After completion, the PBS was rinsed 3 times for 3min each.
3) Cell penetration: 150 μ L of a punching agent (fluorescent in situ hybridization sample treatment kit punching agent) was dropped onto the filter and left at room temperature for 3 min. After completion, the PBS was rinsed 3 times for 3min each.
4) Blocking and primary antibody incubation: anti-EpCAM Polyclonal Antibody (Rabbit) and anti-CD 45 Antibody Cocktail (Mouse) were mixed (1:1, v/v) as shown in Table 1, diluted 1:100 with 5% BSA (in PBS), gently pipetted and mixed. 150 μ L of primary antibody was added dropwise to the filter, incubated overnight at 4 ℃ and then rinsed 5 times with PBS for 3min each time after incubation.
5) And (3) secondary antibody incubation: the secondary antibody Alexa Fluor 546Goat Anti-Rabbit and the secondary antibody Alexa Fluor 488Goat Anti-Mouse are mixed according to the volume ratio of 1:1, then diluted by PBS according to the volume ratio of 1:200, and after being uniformly blown, 150 mu L of secondary antibody is dripped on a filter membrane, incubated for 45min at 37 ℃, and rinsed for 5 times by PBS after the incubation is finished, and each time lasts for 3 min.
6) Secondary fixation: 150 μ L of stationary liquid was added dropwise to the filter membrane and incubated for 5min, and the membrane was rinsed with PBS 2 times for 3min each time.
7) Secondary penetration: 150 mul of perforating agent is dripped on the filter membrane and placed for 7min at room temperature, and PBS is rinsed for 3 times, 3min each time.
8) And (3) dehydrating: the slices are sequentially placed in 75%, 85% and 100% ethanol solutions for 2min respectively, and then placed in a dark and ventilated place to be dried.
9) And (3) probe covering: and (3) taking out the CEP8/CEP17 probe, standing for 5min at room temperature, mixing the probes uniformly, centrifuging for a short time, dripping 10 mu L of the mixture into the center of the filter membrane, immediately covering the filter membrane with a cover glass (the probes are uniformly distributed under the cover glass and have no bubbles, and excessive squeezing of the cover glass is avoided as much as possible), and completely sealing the periphery of the cover glass by Fixogum mounting adhesive.
10) And (3) denaturation hybridization: the slide glass is placed on an in situ hybridization instrument, denatured at 95 ℃ for 2min, and hybridized at 42 ℃ for 1 h.
11) Washing a hybridization sample: carefully tearing off the mounting glue around the cover glass by using forceps to avoid sticking or moving the cover glass;
a) diluting the solution A (the fluorescence in situ hybridization sample treatment kit solution A) and deionized water according to the proportion of 1:4, putting the glass slide into the diluted solution, soaking for 1min, and naturally falling off the cover glass;
b) taking the solution A, the solution B (the fluorescence in situ hybridization sample treatment kit solution B) and deionized water according to the weight ratio of 1: 1: 3, preheating in a 52 ℃ water bath for at least 30min, and soaking the glass slide in the diluted solution (52 ℃) for 5 min;
c) and then putting the glass slide into the preheated deionized water at 37 ℃ for soaking for 10s, and naturally drying the glass slide in a dark place.
12) Sealing and detecting: dripping a cell nucleus staining solution (a cell nucleus staining solution of a fluorescence in situ hybridization sample processing kit) into the center of the filter membrane, sealing the membrane by a cover slip, placing the membrane under a fluorescence microscope, and confirming a cell area under a low-power objective lens (10 x); the cell was moved to a position of 40 Xobjective lens to find a uniform distribution of cells, and then the IF-FISH result of the cell nucleus was observed under a high power objective lens (100X).
13) And (4) storing the sample subjected to the experiment and microscopic examination at-20 ℃ in a dark place.
Attached: the volume of the dropwise added liquid in the experiment can be added according to the minimum amount capable of covering the filter membrane, so that the waste of the reagent is avoided.
Note that: IF-FISH procedure to prevent filter drying and fluorescence signal quenching, the whole incubation process should be done in a light-tight wet box; the whole operation process should be carried out in the dark.
The 48 samples were tested as described above and the results are shown in Table 2.
Table 248 samples of test results
According to the embodiment, the kit provided by the invention can realize early screening of peritoneal micrometastasis, the simultaneous detection of the three detection markers is favorable for improving the detection accuracy, and the kit provided by the invention can detect single-digit cancer cells, so that the detection sensitivity is greatly improved.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A kit for detecting cancer cells in ascites or peritoneal lavage fluid, which is characterized by comprising a nano microporous filter membrane filtering device for collecting epithelial cells in the peritoneal lavage fluid and an IF-FISH reagent, wherein a detection marker of the IF-FISH reagent comprises one or more of the following components: CEP8, CEP17 and EpCAM proteins.
2. The kit of claim 1, wherein the number of cancer cells in the ascites or peritoneal lavage fluid is 10 or less1。
3. The kit of claim 1, wherein the nano-microporous membrane filtration device is a circulating tumor cell separator.
4. The kit of claim 1, wherein the IF-FISH reagent comprises a fixative solution, a perforant, a CEP8 probe, a CEP17 probe, a solution a, a solution B, a nuclear staining solution, a primary anti-Mouse CD45 anti-body Cocktail, a Secondary anti-goal anti-Mouse IgG (H + L) Highly Cross-absorbed Secondary Antibody, a primary anti-goal EpCAM Polyclonal Antibody, and a Secondary anti-goal anti-Rabbit IgG (H + L) Highly Cross-absorbed Secondary Antibody.
5. The kit of claim 4, wherein the solution A is a mixed solution of sodium chloride and Tris-HCl, the solution B is an NP-40 solution, and the solution A and the solution B are used for washing the hybridization sample.
6. The kit according to any one of claims 1 to 5, further comprising the following reagents: blocking reagent, absolute ethyl alcohol, methanol, paraformaldehyde solution with the mass concentration of 4%, PBS buffer solution and normal saline.
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