CN111122849A - Detection kit for detecting tumor cells in pleural effusion sample of human body - Google Patents

Abstract

The invention provides a detection kit for detecting tumor cells in a pleural effusion specimen of a human body, which comprises a filter membrane, a cell fixture and periodic acid for immunohistochemistry, wherein filter holes of the filter membrane are straight channels penetrating through the thickness direction of the membrane, the aperture of the filter holes is 5-20 micrometers, the thickness of the filter membrane is less than 30 micrometers, and the cell fixture contains formaldehyde and/or paraformaldehyde. The kit disclosed by the invention is short in time consumption and high in detection speed, and can be used for rapid pathological detection; the detection steps are simple, and the pleural effusion sample does not need complex preliminary treatment; and the sample treatment capacity is large, the detection rate is improved, and the false negative is reduced.

Description

Detection kit for detecting tumor cells in pleural effusion sample of human body

Technical Field

The invention relates to the field of detection of tumor cells, in particular to a detection kit for detecting tumor cells in a pleural effusion sample of a human body.

Background

In some pathological conditions, such as infection, malignant tumor, etc., some body cavities of the human body may produce excessive body fluid to generate effusion, such as pleural cavity, abdominal cavity, etc. How to quickly, effectively and accurately judge whether the body fluid specimens are caused by malignant tumors has very important clinical significance. The gold standard for determining whether it is a malignant effusion is to find malignant cells in a body fluid specimen. Taking pleural effusion as an example, the pleural cavity is normally only provided with a small amount of lubricating fluid, and a large amount of fluid is generated under pathology to cause hydrops. The current methods for diagnosing whether pleural effusion is malignant are as follows: collecting pleural effusion samples, centrifuging, taking cell precipitates, fixing and embedding the cell precipitates into paraffin blocks for storage, and staining sections to find malignant tumor cells under a microscope. The method has excessive steps and long time consumption (3-7 days), and the tumor cells with extremely small number are easy to be missed for diagnosis.

Patent CN201810903264 discloses a dye method digital quantitative PCR kit and application and use method thereof. The method comprises the application of the copy number of the circular RNA hsa _ circ _0051778 as a detection index in a kit for identifying the lung adenocarcinoma and the tuberculous pleurisy, wherein the copy number of the circular RNA hsa _ circ _0051778 in the tuberculous pleurisy is higher than that in the lung adenocarcinoma; the kit comprises a circular RNA hsa _ circ _0051778 specific primer, a positive control, a negative control, a special PCR amplification premix for dye method digital PCR and sterile deionized water; the kit is used through the steps of sampling, PCR amplification system preparation, amplification reaction and identification. Thereby accurately and rapidly identifying the adenocarcinoma of the lung and the tuberculous pleurisy, and the detection result shows that: the sensitivity of the detection tool can reach more than 90 percent, and is higher than that of a single method clinically used at present. However, the kit can only be used for rapidly identifying the lung adenocarcinoma and the tuberculous pleurisy, but cannot be used for detecting the specific types of cancer cells in pleural fluid.

Therefore, there is a need in the art to provide a new detection kit for detecting tumor cells in pleural effusion samples of a human body.

Disclosure of Invention

We have developed a method for rapid enrichment of tumor cells based on membrane filtration technology for rapid isolation of tumor cells in enriched diagnostic fluid specimens. The principle is that the cells in the liquid specimen are directly separated by a membrane filtration technology, wherein the cells with larger volume such as epithelial cells, mesothelial cells and tumor cells are firmly adsorbed and fixed on a filtration membrane. The whole filtering process is completed in one step without a pretreatment step, and after the filtering is completed, various downstream stains (such as HE stain, namely hematoxylin-eosin stain, Papanicolaou stain and immunohistochemical stain) can be carried out on the filtering membrane.

The invention firstly provides a detection kit for detecting tumor cells in a pleural effusion specimen of a human body, which comprises a filter membrane, a cell fixture and periodic acid for immunohistochemistry, wherein filter holes of the filter membrane are straight pore channels penetrating through the thickness direction of the membrane, the pore diameter of the filter holes is 5-20 micrometers, the thickness of the filter membrane is less than 30 micrometers, and the cell fixture contains formaldehyde and/or paraformaldehyde.

In the invention, the straight pore channels of the filter membrane refer to vertical pore channels or inclined pore channels with through holes on the membrane vertical to the membrane surface, and the filter pores are not bent pore channels or random pore channels.

In a specific embodiment, the kit comprises periodic acid solution, and the concentration of the periodic acid solution is 0.01-2 wt%, preferably 0.05-1 wt%, and more preferably 0.1-0.5 wt%.

In the present invention, the concentration of the periodic acid solution used is correlated with the reaction time at room temperature after the periodic acid solution is added. The reaction is optimized for 2 minutes at a concentration of 0.2% in periodic acid solution. When the kit of the present invention is used, the antigen may be affected by a high concentration of periodic acid solution, resulting in a weak positive reaction. For example, if the reaction is carried out for 1 minute at a concentration of 0.5% in a periodic acid solution, a satisfactory detection result may be obtained, but the positive reaction may be slightly weak. On the other hand, if the concentration of the periodic acid solution is 1% or 2%, the positive reaction is weaker. In addition, when the concentration of the periodic acid solution is low, the reaction time needs to be prolonged accordingly, and for example, when the concentration of the periodic acid solution is 0.1%, the reaction time needs to be prolonged to about 5 minutes accordingly.

In a specific embodiment, the kit further comprises a primary antibody matched with the tumor cells to be detected, preferably the primary antibody comprises at least one of TTF-1 and P40.

In a specific embodiment, the kit further comprises one or more of triton X100, a secondary antibody, and a concentrated DAB stain.

In a specific embodiment, the kit further comprises a filter comprising a filter column and a valve for controlling the flow of liquid.

In a particular embodiment, the filtration membrane is a track-etched membrane and the thickness of the track-etched membrane is 25 microns or less, preferably 20 microns or less, more preferably 15 microns or less.

In a specific embodiment, the filtering membrane is a track etching membrane with uniform pore diameter and pore diameter of 7-9 microns.

In a specific embodiment, the kit further comprises glycerol with the concentration of 20-60 wt% for the sealing sheet.

In a specific embodiment, the cell fixing solution is a cell fixing solution, and the cell fixing solution is a paraformaldehyde solution with a concentration of 2.5-20 wt% or a formaldehyde solution with a concentration of 4-50 wt%.

The invention also provides an application of the kit, which comprises the steps of filtering the pleural effusion by using the filtering membrane, adding the physiological saline for continuous filtering when the liquid level of the pleural effusion is lowered to be still kept above the membrane surface of the filtering membrane, adding the liquid cell fixture when the liquid level of the mixed solution of the pleural effusion and the physiological saline is lowered to be still kept above the membrane surface of the filtering membrane, fixing the cells on the filtering membrane, wherein the cell fixture fixes the cells for more than 1 minute, preferably 3-60 minutes, more preferably 5-20 minutes, taking the filtering membrane with the fixed cells out of the filter, performing immunohistochemical treatment on the filtering membrane, and performing microscopic examination; and the immunohistochemical treatment comprises adding a periodic acid solution on a filter membrane, incubating a primary antibody and a secondary antibody on the filter membrane in sequence, dyeing the primary antibody and the secondary antibody by using a DAB dye, and finally sealing the secondary antibody by using glycerol so as to send the secondary antibody for microscopic examination; preferably, after the fixation reaction is completed, continuing filtration so as to allow the mixed solution containing the cell fixing solution, the pleural fluid and the physiological saline to flow to the lower part of the filtration membrane, and taking out the filtration membrane on which the cells are fixed from the filter; the periodic acid solution is preferably added for a reaction time of 0.5 to 10 minutes, more preferably 1 to 5 minutes.

The invention has at least the following beneficial effects:

1. the time consumption is short, the detection speed is high, and the kit can be used for rapid pathological detection.

2. The method has simple steps, and the pleural effusion sample does not need fussy preliminary treatment.

3. The visual field is clean, which is more beneficial to observation under a microscope and interference elimination. This separation process is very important in particular for conventional dyeing processes.

4. The sample treatment capacity is large, the detection rate is improved, and false negative is reduced.

5. All cells to be examined were uniformly distributed on a flat membrane, and conventional microscopic observation after sectioning from paraffin blocks was not required. The detection efficiency and sensitivity can be greatly improved for the sample with few positive cells.

6. The kit comprises a cell fixture, wherein the cell fixture comprises formaldehyde and/or paraformaldehyde, and when the cell fixture in the kit is used, a paraformaldehyde solution with the concentration of 2.5-20 wt% or a formaldehyde solution with the concentration of 4-50 wt% is specifically used as a cell fixing solution, and a specific cell fixing method is combined, namely, tumor cells on a filter membrane are not directly contacted with air before being completely fixed, namely, the cells before being fixed are kept in a liquid environment. This enables the kit and the corresponding method of operation according to the invention to successfully immobilize tumor cells on a filter membrane for microscopic examination.

7. The kit for detecting the pleural effusion sample comprises periodic acid for immunohistochemistry. The subsequent combined use of primary antibody, secondary antibody and DAB staining agent after periodic acid is used in the invention, so that the types of tumor cells in the pleural fluid sample or body fluid sample can be clearly identified. The use of periodic acid is extremely important for the kit, and the non-periodic acid treatment can cause serious non-specific staining and false positive results. The kit provided by the invention provides a very convenient and rapid mode for identifying the tumor cells.

8. For the application of the kit of the present invention, in order to keep the tumor cells in the liquid before fixing, the inventors previously used a microscope to perform online monitoring of the tumor cells on the filter membrane, but after all, the complexity and the operation difficulty of the device equipment are greatly increased. In the invention, when the tumor cells are in the liquid environment, namely before the filtration is finished, the fixing liquid is poured into the filter, and after the tumor cells are completely fixed in the liquid environment on the filtering membrane, the residual liquid is filtered and the filtering membrane is taken out, so that the tumor cells in the specimen can be efficiently and accurately detected.

Drawings

FIG. 1 is a diagram showing the results of detecting malignant tumor cells in pleural effusion of a suspected lung cancer patient according to example 1 by membrane filtration using a conventional staining method.

FIG. 2 is a graph showing the results of the detection of malignant tumor cells in pleural fluid of suspected lung cancer patients stained by immunohistochemical method and subjected to membrane filtration, wherein TTF-1 protein was used as the primary antibody.

FIG. 3 is a graph showing the results of the detection of malignant tumor cells in pleural effusion of a suspected lung cancer patient in example 1 by membrane filtration using immunohistochemical staining, wherein P40 protein is used as a primary antibody.

FIG. 4 is a graph showing the results of detecting malignant tumor cells in pleural fluid of a suspected lung cancer patient in comparative example 1 by membrane filtration using immunohistochemical staining, wherein TTF-1 protein was used as the primary antibody.

FIG. 5 is a graph showing the results of detecting malignant tumor cells in pleural fluid of a suspected lung cancer patient in comparative example 2 by membrane filtration using immunohistochemical staining, wherein TTF-1 protein was used as the primary antibody.

FIG. 6 is a graph showing the results of detecting malignant tumor cells in pleural fluid of a suspected lung cancer patient in comparative example 3 by membrane filtration using immunohistochemical staining, wherein TTF-1 protein was used as the primary antibody.

FIG. 7 is a schematic diagram showing the principle of tumor cell retention by a trace-etched membrane of 12 μm thickness in example 1.

FIG. 8 is a schematic diagram showing the principle that the tumor cells cannot be retained by the trace-etched membrane having a thickness of 32 μm in comparative example 4.

Detailed Description

The present invention is further described with reference to the accompanying drawings, but the scope of the present invention is not limited to the embodiments.

Example 1

This example is the detection of cancer cells in pleural fluid. For most of patients, the pleural fluid specimens can be diagnosed as squamous carcinoma, adenocarcinoma or small cell carcinoma by immunohistochemistry without conventional staining. For the thoracic water standard of few patients, if only immunohistochemistry (e.g., TTF-1 negative and P40 negative) is done without routine staining, there may be missed detection because its routine staining may be positive. However, since this is very rare, conventional staining dyes may not be included in the kit of the invention.

However, for the breast water standard, if only routine staining is performed, but immunohistochemical staining is not performed, even if cancer cells exist in the breast water of a patient, the breast water standard cannot directly judge the type of cancer through the regular characteristics of histological morphology and cell arrangement because cells in body fluid are used in the invention, but tumor tissues of biopsy are not, and the breast water standard needs to be subjected to immunohistochemical detection to distinguish squamous cell carcinoma, adenocarcinoma or small cell carcinoma. That is, even after a pleural effusion specimen is subjected to a conventional staining test to find tumor cells, the type and typing of the tumor cells (squamous carcinoma, adenocarcinoma, small cell carcinoma, large cell lung cancer, malignant mesothelioma, etc., or pleural effusion formed by metastasis of other cancers into the lung) are still unknown, and the source and typing of the tumor cells are significant for the subsequent treatment. Therefore, it is necessary to perform immunohistochemical detection on the breast water standard, and conventional staining can achieve the effect of mutual complementary verification on immunohistochemical staining, and can also make up for the condition of missed detection (immunohistochemistry is negative, but conventional staining is positive) of a few patients, so that a more real and reliable result can be obtained. In addition, for the conventional pathological detection in the prior art, the paraffin embedding method is adopted to detect the tumor tissues of the patients, and in many cases, a pathologist can judge whether the cancer is squamous carcinoma, adenocarcinoma or other types of cancers according to the tissue morphology displayed by the conventional staining of the cancer tissues; for atypical or inconclusive, further confirmation is made by immunohistochemical staining. In this embodiment, the detection of cancer cells in pleural fluid of a suspected lung cancer patient is taken as an example, and the specific steps are as follows:

1. assembling a membrane filtering device, arranging a track etching membrane (the membrane thickness is 12 micrometers +/-1 micrometer, the pore diameter is 8 micrometers +/-1 micrometer) as a filtering membrane in the filter, connecting a needle cylinder on the filter for storing a filtering sample, connecting a three-way valve at the lower end of the filter for opening and closing the filtering device, closing the three-way valve after exhausting, and adding 3ml of physiological saline to wet the tube wall.

2. The pleural effusion sample is added into the needle cylinder, and the three-way valve is opened to slowly filter the pleural effusion. During the filtration, the liquid cannot be completely drained, and the liquid level cannot be lower than the filtering membrane so as to avoid the rupture of tumor cells trapped on the filtering membrane.

3. Physiological saline is added to clean the tube wall during filtration until the filtrate is clear. In this process, it is still necessary to ensure that the liquid cannot flow completely out and that the liquid level cannot be lowered below the filter membrane in order to avoid rupture of the tumor cells trapped on the filter membrane. For example, when the level of the pleural fluid is maintained at 10mm or more above the membrane surface of the filtration membrane, the addition of physiological saline is not required. When the liquid level of the pleural effusion or the liquid level of the mixed solution of the pleural effusion and the physiological saline is reduced to be below 10mm above the membrane surface of the filtering membrane, the physiological saline needs to be added. And cleaning each pleural effusion sample by using physiological saline for 3-4 times approximately so that the pleural effusion sample is completely filtered.

4. Namely, when the filtration is completed, 4% paraformaldehyde serving as a fixing solution is added for filtration and fixation, and about 10ml of paraformaldehyde is added in total, so that tumor cells trapped on the filtration membrane are sufficiently fixed. Before the fixing step is completed, air entering the filtering device is still avoided, namely, the liquid above the membrane surface of the filter cannot completely flow out, and the liquid level cannot be lower than the filtering membrane.

5. Fully fixing at room temperature for 10min, and taking out the filter membrane. At the moment, the tumor cells on the filtering membrane are fixed, the liquid in the filter can be drained after the fixation is finished, the tumor cells cannot be influenced, and the tumor cells cannot be broken again. The removed filter membrane was placed in a six-well plate and washed 5min × 3 times on a shaker using PBST (phosphate tween buffer).

After fixing and rinsing the filter membrane, the subsequent step is a dyeing step. For pleural fluid standards, three filtration membranes are generally used, one of which is stained using a conventional staining method, such as papanicolaou staining or hematoxylin staining. The other two filter membranes were further confirmed by immunohistochemical staining.

The papanicolaou staining step in the conventional staining method is as follows.

A piece of filter membrane that had been defined and washed was stained with the pap stain kit staining agent for 5 minutes and then added to warm tap water to return blue for five minutes.

And 7A, adding a slurry dye for dyeing for 5 minutes, and sucking dry filter paper along the membrane edge.

And 8A, adding a color enhancer to the color enhancer for enhancing the color for 20 seconds, and directly observing by using a 40% glycerol seal without washing.

As known to those skilled in the art, a slurry dye, a nuclear dye, and a color enhancer, all of which are components of a commercially available Papanicolaou staining kit.

The hematoxylin staining procedure in the conventional staining method is as follows.

And 6B, adding hematoxylin staining solution into a fixed and cleaned filter membrane for staining for 5 minutes, and then adding warm tap water for returning blue for five minutes. Observed using 40% glycerol mounting.

FIG. 1 is a diagram illustrating the detection of malignant tumor cells by membrane filtration using conventional staining method for pleural effusion of a suspected lung cancer patient as shown in example 1. Normal cells are marked at the two boxes in fig. 1, and malignant cells are marked at the two circles. That is, the pleural fluid of the patient contains tumor cells as seen by conventional staining. The assay takes less than 30 minutes. However, if it is desired to further determine whether the malignant cells are squamous carcinoma, adenocarcinoma or other types of cancer, an immunohistochemical staining test is required for detection.

The immunohistochemical staining procedure is as follows.

6. PBST was aspirated off, 1ml of buffer solution 1 (0.25% triton X100) was added, and the mixture was left to react at room temperature for 15 min.

7. Buffer 1 was aspirated and washed on a shaker for 5min × 3 times with PBST.

8. PBST was aspirated off, 1ml of buffer 2 (0.2% periodic acid) was added, and the mixture was left to react at room temperature for 2 min.

9. Buffer 2 was aspirated and washed on a shaker for 5min × 3 times with PBST.

10. The filter membrane was removed, placed right side up in a wet box, and the prepared primary antibody solution was added to cover the filter membrane sufficiently (primary antibody diluted with PBST, primary antibody to TTF-1 dilution concentration 1: 400, primary antibody to P40 dilution concentration 1: 200). Incubate at 4 ℃ overnight or at room temperature for 30 minutes. The primary antibody is TTF-1 or P40, for example.

11. Recovering primary antibody, taking out the filter membrane, placing in a six-hole plate, and washing with PBST on a shaking table for 5min × 3 times.

12. Taking out the filter membrane, placing in a wet box with the right side facing upwards, adding 200ul of reaction enhancing solution to fully cover the filter membrane, and treating at room temperature for 20 min. The reaction-enhancing solution is used, for example, in a commercially available immunohistochemical kit.

13. The reaction enhancing solution was recovered, the filter membrane was removed, placed in a six well plate, and washed on a shaker for 5min × 3 times with PBST.

14. The filter membrane was removed, placed face up in a wet box, 200ul of HRP-labeled secondary antibody was added to cover the filter membrane sufficiently, and treated at room temperature for 25 min.

Wherein the primary antibody is a first antibody and the secondary antibody is a second antibody. The first antibody is an immunoglobulin that specifically binds to a specific antigen. Classes include monoclonal antibodies and polyclonal antibodies. The secondary antibody is an antibody capable of binding to the antibody, i.e., the antibody, while the secondary antibody is labeled with horseradish peroxidase (HRP), and its primary function is to detect the presence of the antibody, amplify the signal of the primary antibody, and introduce a chromogenic system.

15. The secondary antibody was recovered, filtered off, placed in a six-well plate, washed 5min x 3 times with PBST on a shaker.

16. Taking out the filter membrane, placing in a wet box with the right side facing upwards, adding DAB (fresh preparation means diluting concentrated DAB dye to appropriate concentration) color-developing supernatant, covering the filter membrane sufficiently, and treating at room temperature for 5 min.

17. The filter membrane was removed, placed in a six well plate, and washed on a shaker for 5min × 2 times with PBST.

18. The filter membrane was removed and observed with a 40% glycerol mounting.

FIG. 2 is a diagram of the detection of malignant tumor cells by membrane filtration of pleural fluid of the suspected lung cancer patient of example 1 stained by immunohistochemical method, wherein TTF-1 protein is used as the primary antibody. In FIG. 2, TTF-1 protein was detected by filtration membrane after filtration using immunohistochemistry. As can be seen in fig. 2, the tumor cell nuclei were stained brown, and the tumor cells on the defined filtration membrane were lung adenocarcinoma. The immunohistochemical detection takes 4-6 hours.

It will be appreciated by those skilled in the art that TTF-1 is a marker for lung adenocarcinoma, and that both P63 and P40 are markers for squamous carcinoma, and that P40 is relatively more specific and accurate than P63 and therefore is relatively better. Therefore, the inventor also detected malignant tumor cells by membrane filtration of pleural fluid of suspected lung cancer patients stained by immunohistochemical method as shown in example 1, wherein P40 protein was used as primary antibody, and the detection result is shown in FIG. 3. As can be seen in FIG. 3, this patient was negative for immunohistochemical P40 protein. This indicates that the tumor cells in the pleural fluid of the suspected lung cancer patient are lung adenocarcinoma rather than squamous carcinoma.

It will be appreciated by those skilled in the art that the conventional commercial immunohistochemical kit contains a secondary antibody, and a "reaction enhancing solution" for better detection and observation. In the present invention, a mixed secondary antibody and a reaction-enhancing solution in a commercially available immunohistochemical kit were used. In addition, primary antibodies are generally not included in immunohistochemical kits that are routinely purchased because they are too numerous. In addition, a DAB color development kit is commercially available whose main component is a concentrated DAB coloring agent.

In addition, the pleural effusion sample to be filtered and detected in the invention can be pretreated or not. For example, if too many blood clots are present in the pleural fluid, pretreatment is generally required. The initial height of the filtered liquid specimen in the filter column of the filter can be selected at will, for example, the initial height can be 1-5 cm high or 10-20 cm high, and the like. Of course, if the amount of the specimen is too large, the filtration time is correspondingly long, and the number of times of washing with the physiological saline to be used is also large. When the amount of the specimen is too small, it may be difficult to keep the liquid across the membrane surface of the filtration membrane, and the specimen may be too small to be retained by the tumor cells without filtration. In the filtering operation of the invention, normal saline is generally used for flushing for 3-5 times in the whole process, firstly, the normal saline is not needed to be added for flushing when the height of the specimen is reduced from 20cm to 10mm, and when the height of the specimen is reduced to a certain height, for example, 10mm, the three-way valve on the filter is closed, the normal saline is added for the first flushing and filtering. The filtration membrane is kept in a liquid (a mixture of the specimen to be examined and a physiological saline solution), and a fixing agent is added to the liquid. The filtering membrane is not required to be fished out before the operation, so that the tumor cells on the membrane are prevented from being broken by pressure.

Because PBST and physiological saline are common reagents in a laboratory, the kit does not comprise the PBST and the physiological saline, and the two components are added into the kit, so that the work of transporting, storing and the like of the kit becomes inconvenient.

Comparative example 1

This example is the detection of cancer cells in pleural fluid. The detection method in comparative example 1 is similar to that of example 1, but lacks steps 1 to 3 of example 1, and in example 1, steps 4 to 5 and 6 to 18, i.e., the pleural fluid is not filtered by a filter membrane, but is directly fixed by paraformaldehyde, and then immunohistochemical staining is carried out, wherein TTF-1 antibody is used as a primary antibody. FIG. 4 is a diagram showing the results of detecting malignant tumor cells in pleural fluid of a suspected lung cancer patient in comparative example 1 by membrane filtration using immunohistochemical staining. As can be seen from a comparison of fig. 4 and fig. 2, there is a large amount of non-specific staining in fig. 4. It is apparent that the method shown in comparative example 1 cannot be used for accurately judging the condition of tumor cells in pleural fluid.

Comparative example 2

This example is the detection of cancer cells in pleural fluid. The detection method in comparative example 2 is similar to that of example 1, but step 4 in example 1 is omitted, or step 4 is implemented, and paraformaldehyde is added for fixation after the liquid on the filter membrane is filtered in step 4, while steps 1-3, 5 and 6-18 in example 1 are implemented, namely, the pleural effusion is not treated by fixing cells in the liquid environment by polyformaldehyde, and then immunohistochemical staining is carried out, wherein TTF-1 antibody is used as a primary antibody in the immunohistochemical staining. FIG. 5 is a diagram showing the results of detecting malignant tumor cells in pleural fluid of a suspected lung cancer patient in comparative example 2 by membrane filtration using immunohistochemical staining. As can be seen from a comparison of FIG. 5 and FIG. 2, the cells on the membrane in FIG. 5 are disrupted, the staining of the positive cells is significantly reduced, and only one circular hole of the track-etched membrane itself is visible in the field of view. It is obvious that the method shown in comparative example 2 cannot be used for accurate judgment of the tumor cells in the pleural fluid.

Comparative example 3

This example is the detection of cancer cells in pleural fluid. The detection method in comparative example 3 is similar to that of example 1, but lacks step 8 of example 1, and comprises steps 1-5, 6-7 and 9-18 of example 1, i.e., the pleural fluid is not subjected to periodic acid during immunohistochemical staining, and the primary antibody in immunohistochemical staining is subjected to TTF-1 antibody. FIG. 6 is a diagram showing the results of detecting malignant tumor cells in pleural fluid of a suspected lung cancer patient in comparative example 3, which was stained by immunohistochemical method and subjected to membrane filtration. As can be seen from a comparison of FIG. 6 and FIG. 2, false positive staining is evident in FIG. 6, and normal leukocytes are also stained with color. It is apparent that the method shown in comparative example 3 cannot be used for accurately judging the condition of tumor cells in pleural fluid.

Comparative example 4

This example is the detection of cancer cells in pleural fluid. The detection method in comparative example 4 is similar to that of example 1, but the thickness of the track-etched film in example 1 is changed from 12 μm + -1 μm to 32 μm + -1 μm. The experimental results show that the probability of tumor cell interception on the filtering membrane with the thickness is greatly reduced.

If the thickness of the trace-etched film in example 1 is changed from 12 μm + -1 μm to 25 μm + -1 μm. The experimental results show that tumor cells can be trapped on a filter membrane of this thickness, but the number of tumor cells trapped becomes small or the probability of trapping tumor cells becomes low. Further experiments have found that the greater the thickness of the track-etched membrane, the lower the probability that the filtration membrane will be able to retain the tumor cells. In this experiment, the filtration apparatus disclosed in the applicant's prior patent application CN201811172818.6, which can be used under a microscope, was used for observation. Specifically, when the membrane thickness, i.e., the length of the filtration pores, is smaller than the diameter of the target cells (tumor cells) to be filtered, the cells are deformed into a biconvex dumbbell-like structure and firmly fixed and clamped in the filtration pores of the filtration membrane (fig. 7), and to form the structure, the size and the length of the membrane pores are required to be smaller than or equal to the target cells to be filtered. Once the biconvex dumbbell-like structure is formed, the cells are firmly fixed in the membrane pores, and are difficult to elute even if various external forces are applied, such as back washing, back centrifugation (the centrifugal force is as high as 4000g), washing after dehydration and the like. When the thickness of the membrane is increased to significantly exceed the target cells, the cells are deformed to pass through the filtration pores directly (fig. 8), without forming a biconvex dumbbell-like structure to be fixed to the filtration membrane.

From the comparison between example 1 and comparative examples 1 to 4, it can be seen that in order to obtain a detection kit capable of accurately determining whether tumor cells exist in pleural fluid, the present invention includes a filtration membrane for filtering pleural fluid, a cell-fixing material for cell fixation, periodic acid for immunohistochemical staining, and a filtration membrane having a specific thickness, and these conditions are not satisfactory. Even in the present invention, a special cell fixing method for fixing cells in a liquid must be employed.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions and substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (10)

1. A detection kit for detecting tumor cells in a pleural effusion specimen of a human body comprises a filter membrane, a cell fixture and periodic acid for immunohistochemistry, wherein filter holes of the filter membrane are straight channels penetrating through the thickness direction of the membrane, the aperture of each filter hole is 5-20 micrometers, the thickness of the filter membrane is less than 30 micrometers, and the cell fixture contains formaldehyde and/or paraformaldehyde.

2. The kit according to claim 1, wherein the kit comprises periodic acid solution, and the concentration of the periodic acid solution is 0.01-2 wt%, preferably 0.05-1 wt%, and more preferably 0.1-0.5 wt%.

3. The kit according to claim 1, further comprising a primary antibody that is matched to the tumor cells to be tested, preferably wherein the primary antibody comprises at least one of TTF-1 and P40.

4. The kit of claim 1, wherein the kit further comprises one or more of triton X100, secondary antibody, and concentrated DAB stain.

5. The kit of claim 1, further comprising a filter column and a valve for controlling the flow of the liquid.

6. Kit according to claim 1, wherein the filtration membrane is a track-etched membrane and the thickness of the track-etched membrane is below 25 microns, preferably below 20 microns, more preferably below 15 microns.

7. The kit according to claim 6, wherein the filtration membrane is a track-etched membrane with uniform pore size of 7-9 μm.

8. The kit according to claim 1, wherein the kit further comprises glycerol at a concentration of 20 to 60 wt% for the mounting.

9. The kit according to claim 1, wherein the cell fixative is a cell fixative solution, and the cell fixative solution is a paraformaldehyde solution with a concentration of 2.5 to 20 wt% or a formaldehyde solution with a concentration of 4 to 50 wt%.

10. The use of the kit according to any one of claims 1 to 9, wherein the pleural fluid is filtered by using a filter membrane, the physiological saline is added to continue filtering when the liquid level of the pleural fluid is lowered to still be kept above the membrane surface of the filter membrane, the physiological saline is added to continue filtering for one or more times, then the cell fixture in a liquid state is added when the liquid level of the mixed solution of the pleural fluid and the physiological saline is lowered to still be kept above the membrane surface of the filter membrane, the cells on the filter membrane are fixed for 1 minute or more, preferably 3 to 60 minutes, more preferably 5 to 20 minutes, then the filter membrane with the fixed cells is taken out of the filter, and the filter membrane is subjected to immunohistochemistry treatment and then microscopic examination; and the immunohistochemical treatment comprises adding a periodic acid solution on a filter membrane, incubating a primary antibody and a secondary antibody on the filter membrane in sequence, dyeing the primary antibody and the secondary antibody by using a DAB dye, and finally sealing the secondary antibody by using glycerol so as to send the secondary antibody for microscopic examination; preferably, after the fixation reaction is completed, continuing filtration so as to allow the mixed solution containing the cell fixing solution, the pleural fluid and the physiological saline to flow to the lower part of the filtration membrane, and taking out the filtration membrane on which the cells are fixed from the filter; the periodic acid solution is preferably added for a reaction time of 0.5 to 10 minutes, more preferably 1 to 5 minutes.

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