CN116716250A - Novel separation method of malignant tumor cells in malignant pleural effusion - Google Patents
Novel separation method of malignant tumor cells in malignant pleural effusion Download PDFInfo
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- CN116716250A CN116716250A CN202310680496.0A CN202310680496A CN116716250A CN 116716250 A CN116716250 A CN 116716250A CN 202310680496 A CN202310680496 A CN 202310680496A CN 116716250 A CN116716250 A CN 116716250A
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- filter membrane
- pleural effusion
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- lung cancer
- effusion
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Abstract
The invention belongs to the field of biotechnology and clinical medicine, and particularly relates to a novel separation method of malignant tumor cells in malignant pleural effusion. The method involves filtering complex body fluids using a 50 μm upper filter membrane and a 15 μm lower filter membrane. The method has the advantages of low sample demand (about 10 ml), high positive rate (87.10%), short detection period (2 days), simple microscopic identification, large subsequent research potential and the like.
Description
Technical Field
The invention belongs to the field of biotechnology and clinical medicine, and particularly relates to a novel separation method of malignant tumor cells in malignant pleural effusion.
Background
Malignant pleural effusion (maligant pleural effusion, MPE) refers to pleural effusion caused by metastasis of malignant tumors that originate in the pleura or malignant tumors in other parts to the pleura. 50% of lung cancer patients develop pleural effusion, and 15% of patients combine with pleural effusion when first visit. The etiology of pleural effusion is complex, including cancers, lung infections, heart failure, etc., and it is important to determine the etiology of pleural effusion, especially the judgment of benign and malignant. For patients who have first been treated for a chest effusion, especially those who have a high degree of suspicion of malignant pleural effusion, pathological tissue biopsies, that is, traditional invasive detection methods, may cause deterioration of the condition, even intolerance in a part of patients with poor physical condition. It is an important tool to diagnose cancer by a non-invasive strategy, i.e. using pleural effusion as a sample. Patients with advanced lung cancer have a high probability of occurrence of pericardial effusion, but clinical detection means are consistent with pleural effusion.
The current gold standard for diagnosing malignant pleural effusion is to find malignant tumor cells, i.e. shed tumor cells, in hydrothorax or pleural biopsy tissue. The detection technology of the exfoliated tumor cells adopted clinically is mainly based on cytological examination. Cytological examination is a simple, minimally invasive examination method, and is even superior to blind pleural biopsies in detecting malignant lesions of the pleura. Cytological examinations include liquid-based cytology and wax block entrapment. However, both require a large amount of pleural effusion (recommended for one-pass effusion >75 ml), and the positive rate is very low, and the single diagnosis positive rate is only 9-44%, and although the positive rate can be improved by continuous multiple cytological examination, the improvement effect is limited. The cell loss amount is large in the operation process, and the method has great influence on pleural effusion with small cell amount. Meanwhile, the detection period is long, and the detection method is extremely psychological stress on patients and families thereof. In addition, due to technical reasons, the cells collected in the method are dead, and only a single research mode of immunohistochemistry can be combined later, so that living cell related research cannot be carried out.
The use of complex body fluids as test samples has disadvantages in both laboratory and clinical applications, mainly due to the complex composition, especially in malignant pleural effusions, where the various cells often surround and even mask the target tumor cells. This confusing background can interfere with observations and even lead to false positive or false negative cytological results.
Disclosure of Invention
In order to better extract the desquamated tumor cells with diagnostic significance from complex body fluid samples, the invention constructs a filtration method based on a 2.5D microporous array filter membrane. The method enriches the cytological examination of pleural effusion and pericardial effusion, reduces the economic cost and time cost of patients, and has obvious advantages for the detection of pleural effusion with small cell number. Most importantly, the captured exfoliated tumor cells in the pleural effusion can be subjected to subsequent research, have great potential in the research of tumor cell culture, tumor metabolism, tumor typing and the like, and perhaps can provide a new exploration method for exploring the occurrence, development and metastasis mechanisms of lung cancer.
In a first aspect, the present invention provides a double layer filtration device comprising an upper layer filter membrane having a pore size of 50 μm and a lower layer filter membrane having a pore size of 15 μm.
Preferably, the filter is a 2.5D microwell array filter.
Preferably, the shape of the upper layer filter membrane can be designed into one or more different geometric shapes of a circle, a rectangle, a regular hexagon and the like according to the requirement.
Preferably, the shape of the lower-layer filter membrane can be designed into one or more different geometric shapes of a circle, a rectangle, a regular hexagon and the like according to the requirement.
More specifically, the double layer filter device of the present invention may be formed by assembly using commercially available filter devices, such as those available from Bai Mei Hangzhou, as used in the exemplary embodiment of the present invention.
In another alternative embodiment, the filter device is assembled by a teflon clamp and an Nd-Fe-B magnetic ring, and a 50 μm upper filter membrane is placed on a 15 μm lower filter membrane to form the filter device.
In another aspect, the invention provides the use of the bilayer filtration device of the invention for separating exfoliated cells.
Specifically, the shed cells are isolated from complex body fluids.
Preferably, the complex body fluid comprises clinical samples such as pleural effusion, pericardial effusion, alveolar lavage, urine, saliva, tissue digestive juice, cerebrospinal fluid, and peritoneal lavage.
More preferably, the complex body fluid comprises pleural effusion, pericardial effusion.
Specifically, when the exfoliated cells include tumor cells, the application is also called the application of the double-layer filtration device in separating the exfoliated tumor cells, namely exfoliated tumor cells (etc).
Preferably, the complex body fluid is collected from suspected patients, more particularly, pleural effusions may be collected from suspected patients with lung cancer, pneumonia, pulmonary infection; pericardial effusion may be collected from suspected patients with pericarditis, tumors, rheumatic heart disease.
Preferably, the lung cancer comprises lung squamous carcinoma or lung adenocarcinoma, non-small cell lung cancer or small cell lung cancer.
In the present invention, pleural effusion (pleurodesis) is a common clinical symptom characterized by pathological fluid accumulation within the pleural cavity. The pleural cavity is a potential gap between the visceral layer and the parietal layer pleura, 5-15 ml of liquid is arranged in the pleural cavity of a normal person, the pleural cavity has a lubricating effect during respiratory movement, 500-1000 ml of liquid is formed and absorbed in the pleural cavity each day, and the pleural effusion can be generated due to the increase or decrease of the absorption of the liquid in the pleural cavity caused by any reasons. When used as a biopsy sample, pleural effusion is also known as hydrothorax, pleural effusion. The detection of malignant tumor cells in pleural effusions is a gold standard for malignant pleural effusions (malignant pleural effusion, MPE).
Preferably, the exfoliated cells can be used for diagnosis of cancer by confirming whether or not they contain cancer cells (exfoliated tumor cells) by microscopic observation, immunodetection, nucleic acid detection, or the like.
Specifically, the immune detection and the nucleic acid detection are carried out aiming at specific markers of cancer cells, and the detected markers represent that the exfoliated cells contain exfoliated cells.
Preferably, the immunodetection comprises ELISA, RIA, immunoblotting, immunofluorescence and flow cytometry based immunofluorescence techniques. In the present invention, CEA was detected by immunofluorescence to confirm tumor cells.
Meanwhile, the invention provides application of the double-layer filtering device in preparing cancer diagnosis products.
Preferably, the present invention provides the use of said double layer filtration device for the preparation of a product for diagnosing cancer by collecting complex body fluids.
Preferably, the cancer is lung cancer, including lung squamous carcinoma, lung adenocarcinoma.
More preferably, the complex body fluid comprises pleural effusion, pericardial effusion.
In another aspect, the present invention provides a method of separating shed cells in a complex body fluid, the method comprising the step of filtering the complex body fluid using an upper filter membrane having a pore size of 50 μm and a lower filter membrane having a pore size of 15 μm.
Preferably, the method is of non-diagnostic interest.
Preferably, the properties of the upper filter membrane and the lower filter membrane can be set arbitrarily.
Preferably, the shape of the upper layer filter membrane can be designed into one or more different geometric shapes of a circle, a rectangle, a regular hexagon and the like according to the requirement.
Preferably, the shape of the lower-layer filter membrane can be designed into one or more different geometric shapes of a circle, a rectangle, a regular hexagon and the like according to the requirement.
More specifically, the double layer filter device of the present invention may be formed by assembly using commercially available filter devices, such as those available from Bai Mei Hangzhou, as used in the exemplary embodiment of the present invention.
Preferably, the filter is a 2.5D microwell array filter.
Preferably, the complex body fluid comprises clinical samples such as pleural effusion, pericardial effusion, alveolar lavage, urine, saliva, tissue digestive juice, cerebrospinal fluid, and peritoneal lavage.
More preferably, the complex body fluid comprises pleural effusion, pericardial effusion.
Preferably, the method further comprises the step of collecting complex body fluids. As used herein, the collection method of the complex body fluid is a method conventionally used in the art.
Preferably, the pleural effusion may be collected from suspected patients with lung cancer, pneumonia, pulmonary infection.
Preferably, the pericardial effusion may be collected from suspected patients with pericarditis, tumors, rheumatic heart disease.
Preferably, the lung cancer comprises squamous lung cancer or adenocarcinoma.
Preferably, the lung cancer comprises non-small cell lung cancer or small cell lung cancer.
In particular, when tumor cells are included in the exfoliated cells, the method is also referred to as a method of isolating the exfoliated tumor cells.
Preferably, the ex vivo time of the complex body fluid should be as short as possible, more specifically, the ex vivo time should not exceed 20 hours, such as 0 hours, 4 hours, 8 hours, 20 hours.
Preferably, the method is suitable for treating 10ml-15ml of complex body fluids.
More preferably, the separation device according to the embodiment of the present invention is most suitable for treating 10ml of complex body fluid. When the filter area is changed, the optimum sample volume will also change accordingly, and the corresponding optimum volume can be determined according to techniques conventional in the art.
More preferably, the complex body fluid is previously treated with an anticoagulant.
Preferably, the anticoagulant includes heparin, citrate (sodium citrate), edetate (EDTA salt), oxalate.
Preferably, the anticoagulant is sodium citrate, more particularly, the concentration of sodium citrate may be 1%, 2%, 3%, 4% or higher. The percentages are weight ratios and are working concentrations.
Preferably, the anticoagulant is 3% sodium citrate.
More preferably, the method further comprises the step of rinsing the filter membrane with 75% alcohol wet filter membrane and PBS liquid prior to filtration.
Most preferably, the method for separating exfoliated cells provided by the present invention is: 10ml of fresh pleural effusion was used, and after treatment with 3% sodium citrate, it was filtered through an upper microporous array membrane having a pore size of 50 μm and a lower microporous array membrane having a pore size of 15. Mu.m.
More specifically, when the filtration speed is reduced and the clogging phenomenon occurs, the filter paper is torn into a strip shape and curled, inserted into the bottom of the tool, or blown using a pipette, accelerating the filtration.
More specifically, the purpose of diagnosing cancer can be further achieved by performing cytological detection and genetic detection on the detached cells. The present invention also provides a method for diagnosing cancer.
The beneficial effects are that:
the double-layer filtering device provided by the invention can be used for treating complex body fluid to realize separation of the exfoliated cells in the complex body fluid with high flux and low damage. The detection accuracy rate in the pleural effusion is 84.2%, and the detection rate of malignant pleural effusion is as high as 87.10%. The method also has the advantages of low sample demand (about 10 ml), high positive rate (87.10%), short detection period (2 days), simple microscopic identification, large potential for subsequent research and the like.
Drawings
FIG. 1 is a single layer 2.5D microporous array filter membrane constructed in accordance with the present invention.
FIG. 2 is a graph showing the filtration results of a single layer 2.5D microporous array filter membrane.
FIG. 3 is a double layer 2.5D microporous array filter membrane constructed in accordance with the present invention.
FIG. 4 is a filtration result of a double layer 2.5D microporous array filter membrane.
FIG. 5 is a graph of the results of the influence of pleural effusion time on separation effect.
FIG. 6 is a graph of the results of the effect of anticoagulant on the separation.
FIG. 7 shows immunofluorescent staining of pleural effusion samples.
Fig. 8 is a graph of statistical results of clinical information.
FIG. 9 shows immunofluorescent staining of pericardial effusion samples.
FIG. 10 is a cell image obtained after culturing the isolated cells.
Detailed Description
The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
The invention experiment main consumable
2.5-dimensional microporous array membranes were purchased from Hangzhou Baimei: pore size 50 microns (F-PAC 009), pore size 15 microns (F-PAC 002), pore size 10 microns (F-PAC 003), pore size 8 microns (F-PAC 001). Teflon jigs, and Nd-Fe-B magnet rings were provided by the university of Beijing micro-nano electronics group Wang Wei teaching subject group.
Universal experimental method
1. Immunofluorescence staining method
(1) The upper and lower filters were wetted with 1ml of 75% alcohol.
(2) The upper and lower filters were rinsed with 3ml of PBS solution.
(3) 10ml of pleural effusion was filtered sequentially from the upper 50 micron diameter filter and the lower 15 micron filter.
(4) The lower layer of the filter membrane with the diameter of 15 micrometers was removed with forceps, the filter membrane was placed in a centrifuge tube with a 1.5ml size with a clamp, and then fixed with 3ml tissue fixing solution for 15 minutes.
(5) After fixing, the filter membrane was removed from the jig, placed in a six-well plate, rinsed with PBS, and blotted three times, one minute each time.
(6) After rinsing, the PBS liquid was sucked dry, 1ml of 0.1% triton-X100 was added, and the mixture was left at room temperature for 8 minutes.
(7) The liquid was blotted off, washed 3 times with PBS, 1% BSA was added, and incubated for 30 minutes at room temperature
(8) Cutting a sealing film with proper size, dripping 20 microliters (1:200 and PBS configuration) of CEA antibody on the sealing film, clamping up the filter film by forceps, contacting the cell surface with the antibody, flattening, reducing bubbles as much as possible, rapidly covering a cover glass, and keeping the temperature at 4 ℃ away from light overnight.
(9) The next day the filters were removed and placed in six well plates, washed three times with PBS for 8 minutes each, and blotted dry.
(10) The filter cells were spread up on a slide glass, 20. Mu.l of red fluorescent murine secondary antibody (1:200 and PBS configuration) was added dropwise and incubated for 1 hour at room temperature.
(11) The filters were removed and placed in six well plates, washed three times with PBS for 8 minutes each, and blotted dry.
(12) The filter membrane was removed and the cells were placed face up on a slide glass, dropped with a drop of DAPI-containing caplet, covered with a cover glass, and air-dried for 3 hours.
(13) Observations and counts were made under a forward fluorescence microscope.
Example 1 construction of Single layer 2.5D microporous array Filter Membrane Filter device
1. Construction of a Filter device
Step 1, assembling the filter element
Before the experiment, the Teflon clamp and the Nd-Fe-B magnetic ring are taken out, and the magnetic ring is sleeved on the upper clamp and the lower clamp, so that the positive electrode and the negative electrode are opposite. The inner side surface of the clamp contacted with the filter membrane is cleaned by 75% alcohol, then one corner of the filter membrane is gently clamped by forceps, and the reverse side is upwards placed in the center of the lower clamp to cover the sample hole. And after the filter membrane is adjusted to be flat and completely covers the sample hole, rapidly and vertically placing the upper clamp to complete element encapsulation. ( Optionally, the teflon clamp and Nd-Fe-B magnetic ring may be replaced with a filter membrane with a mating snap; some commercially available filter membranes are provided with matched plastic clamps through buckles, so that the Teflon clamp and the Nd-Fe-B magnetic ring are completely replaced, and the two layers are stacked to complete assembly. )
Step 2, construction of a monolayer filter device
(1) A single-layer filtration separation device was constructed using a permanent magnet (magnetic ring) and a 2.5-dimensional microporous array membrane, together with four sets of devices with filter membrane pore diameters of 8 μm, 10 μm, 15 μm and 50 μm, as shown in fig. 1.
(2) The filters were wetted with 1ml of 75% alcohol.
(3) The filters were rinsed with 3ml of PBS liquid.
(4) 10ml of untreated pleural effusion was filtered separately using 4 sets of devices and the phenomena recorded and timed.
(5) When the filtering speed is reduced and the blocking phenomenon occurs, the filter paper is torn into strips and curled, and is inserted into the bottom of the clamp, or is blown by using a liquid transfer device, so that the filtering is accelerated.
2. Test filter device
500ml of fresh pleural effusion (a pleural effusion sample is collected from a malignant pleural effusion patient in a second hospital affiliated to university of Dalian medical science) of the same batch of patients is collected, and 4 groups of the patients are split into 15ml of each group after the patients are uniformly mixed. Filtration was performed using 2.5D single layer microporous array filters of different pore sizes (8 μm, 10 μm, 15 μm and 50 μm). 2.5D microporous array Filter membranes different pore sizes were referenced according to the reported blood CTCs capture method and filter membrane production specifications (FIG. 2A, results from which 3 independent replicates were run).
Experimental results: on a 2.5D microporous array filter membrane with the pore diameter of 8 mu m, the pleural effusion with the volume of about 1-2ml can be filtered; on a 2.5D microporous array filter with a pore size of 10 μm, a plug was also formed after filtration of a volume of about 3-4ml of pleural effusion (FIG. 2B); the filtration is smooth on a 2.5D microporous array filter membrane with the aperture of 15 mu m, and the filtration can be smooth in about 50 seconds; the filtration was also smooth with a 2.5D microporous array filter membrane having a pore size of 50. Mu.m, taking about 15 seconds. After the end of the experiment, the batch of pleural effusion was also filtered using a 300 mesh nylon filter screen (pore size about 48 microns) and the filter screen was checked for obvious floc (fig. 2C).
The pleural effusion can be filtered on a single-layer 2.5D microporous array filter membrane with the aperture more than or equal to 15 mu m; however, in the past, a 2.5D microporous array filter membrane with a pore size of 8 μm was used to successfully filter blood to obtain CTCs, and the filtration of pleural effusion was not performed at all. The single layer filtration mode is thus not suitable for pleural effusion, and it may be possible to filter pleural effusion by connecting two sets of single layer filter elements in series one above the other.
Example 2 construction of double-layer 2.5D microporous array Filter Membrane Filter device
1. Construction of a Filter device
Step 1, assembling the filter element
Before the assembly of the filter element is ready for experiments, the Teflon clamp and the Nd-Fe-B magnetic ring are taken out, and the magnetic ring is sleeved on the upper clamp and the lower clamp, so that the positive electrode and the negative electrode are opposite. The inner side surface of the clamp contacted with the filter membrane is cleaned by 75% alcohol, then one corner of the filter membrane is gently clamped by forceps, and the reverse side is upwards placed in the center of the lower clamp to cover the sample hole. And after the filter membrane is adjusted to be flat and completely covers the sample hole, rapidly and vertically placing the upper clamp to complete element encapsulation.
Step 2, construction of double-layer filtering device
(1) A double-layer filtering and separating device is built by using a permanent magnet and a 2.5-dimensional microporous array membrane, wherein the upper layer is the microporous array membrane with the aperture of 50 mu m, the lower layer is the microporous array membrane with the aperture of 15 mu m, and 2 groups of devices are arranged in total, as shown in figure 3.
(2) The upper and lower filters were wetted with 1ml of 75% alcohol.
(3) The upper and lower filters were rinsed with 3ml of PBS solution.
(4) 15ml of untreated pleural effusion was filtered separately and the phenomena recorded and timed.
(5) When the filtering speed is reduced and the blocking phenomenon occurs, the filter paper is torn into strips and curled, and is inserted into the bottom of the clamp, or is blown by using a liquid transfer device, so that the filtering is accelerated.
2. Test filter device
Through a single-layer filtration mode exploration experiment, the single-layer filtration mode difference is clear, and the filtration effect is enhanced along with the increase of the pore diameter. The pleural effusion is combined to filter on a single-layer filter membrane with the pore diameter larger than or equal to 15 mu m and uneven texture, so that a double-layer filter mode consisting of a filter membrane with the upper layer of 50 mu m and the lower layer of 15 mu m is adopted, and the pore diameter of a microporous filter membrane in a double-layer filter model is searched. A large amount of fresh pleural effusion collected by the same batch of patients is selected, and after being uniformly mixed upside down, the filtration of 15ml of pleural effusion is carried out by a double-layer filtration device consisting of a microporous array membrane with the upper layer aperture of 50 mu m and a microporous array membrane with the lower layer aperture of 15 mu m without any treatment, and the average value of time is obtained after three independent repeated experiments, and the experimental result is shown in figure 4. We can see: the double-layer filtration model can effectively complete the filtration of the pleural effusion, the average filtration time of the microporous array membrane of 50 μm on the upper layer is 42 seconds, the average filtration time of the pleural effusion after one filtration on the microporous array membrane of 15 μm on the lower layer is about 10 minutes, and the microporous array membrane of 10 μm fails to filter successfully.
Example 3 separation method of tumor-shed cells in pleural effusion based on 2.5D microporous array Filter vs. time cause
Optimization of the element
In example 2, it was confirmed that a double-layer filtration device composed of a microporous array membrane having an upper pore size of 50 μm and a microporous array membrane having a lower pore size of 15 μm was capable of smoothly filtering a pleural effusion sample. However, the filtration time efficiency of 10 minutes is too low, so that the influence of the pleural effusion ex-vivo time on the filtration effect is obtained by using a double-layer filtration device to filter 15ml of untreated pleural effusion by taking the pleural effusion placement time as a variable.
The determination packet is: 0. groups 4, 8, 20, 24 and 30 hours 6. Fresh pleural effusion taken by the same patient in the same batch is selected, and after being mixed uniformly, 6 groups of the pleural effusion are split into 10ml groups. According to the above 6 groups, the pleural effusion samples were placed at room temperature in a laboratory (simulating clinical practice) and were subjected to filtration in a bilayer mode after being mixed up and down.
The experimental results show that: four groups of 0h, 4h, 8h and 20h can successfully filter 15ml of pleural effusion, but the required time increases with the longer placement time (fig. 5A); the two groups 24h and 30h were not evenly mixed and there was a jelly-like semi-solid visible to the naked eye (fig. 5B). During the experiment, we could find a significant flocculent semi-solid in the container placed for pleural effusion for more than 8 hours (fig. 5C), and after picking out the flocculent mass (fig. 5D), similar to the appearance of an upper layer on a 300 mesh nylon filter.
Therefore, the in vitro time of the pleural effusion is shortened, and the efficiency of detecting by using the double-layer separation device liquid can be improved. That is, we should examine the collected sample as soon as possible.
Example 4 anticoagulation factor based on separation method of shed tumor cells in 2.5D microporous array filter membrane pleural effusion
Optimization of (a)
After the double-layer filtering and separating device is established and the factor of the sample in-vitro time is studied, the best efficiency obtained by the method is about 40 seconds. Since the present laboratory previously used an 8 μm pore size to filter the same volume of blood sample only required about 20 seconds of filtration time, the filtration efficiency for pleural effusion remained insufficient. Considering that in the above experiments we found precipitation several times, consulting the literature, the aggregation of fibrin in pleural effusions was suspected, so that further investigation was carried out with the choice of sodium citrate anticoagulant. We collected the same patient with the same batch of pleural effusions and group experiments were performed with 15ml fresh pleural effusions treated with five concentrations of sodium citrate, 0%, 1%, 2%, 3% and 4% (fixed weight of pleural effusions were weighed, corresponding sodium citrate solids were dissolved in pleural effusions according to weight ratios of 1:100, 2:100, 3:100, 4:100).
The experimental results are shown in fig. 6: the use of the anticoagulant can improve the filtering efficiency, between 0% and 3%, the filtering time is obviously shortened along with the improvement of the anticoagulant concentration, but the influence of the anticoagulant concentration on the filtering effect tends to be stable after the anticoagulant concentration reaches 3%.
Example 5 separation of tumor-shed cells in pleural effusion based on 2.5D microporous array FilterEfficacy test of the ionization method
Syndrome/pattern of
Finally, a device with a double-layer 2.5D microporous array filter membrane with an upper layer aperture of 50 micrometers and a lower layer aperture of 15 micrometers is constructed, and a final and effective method for separating the fallen tumor cells in the pleural effusion is constructed through optimization of various parameters. The method comprises the pretreatment of 3% sodium citrate of the pleural effusion sample, and reduces the placement time and the core separation device, namely a double-layer filter device consisting of a microporous array membrane with the upper layer aperture of 50 mu m and a microporous array membrane with the lower layer aperture of 15 mu m. We performed fixation and fluorescent staining of the filtered lower 15 μm filter and cells attached thereto, and both captured cells were observed under a fluorescent microscope.
By testing 5 samples of pleural effusion from patients diagnosed with malignant pleural effusion for exfoliated tumor cells, we found fluorescent-lit exfoliated tumor cells in both 10ml and 15ml groups. The detection efficiency of the method in the diagnosis of malignant pleural effusion patients is up to 100%. The effect of 10ml pleural effusion was found to be better than 15ml pleural effusion by 5 comparisons, as shown in figure 7: the cell density is too high, the overlapping is serious, the dyeing effect is poor and the cell density is difficult to identify after the filtered 15ml pleural effusion is observed under a lens; the filtered 10ml pleural effusion was observed under a microscope.
Example 6 separation method of tumor-shed cells in pleural effusion based on 2.5D microporous array Filter Membrane in pleural effusion
Clinical application in fluids
The cell separation method was determined by the above specific examples: a microporous array membrane with the upper layer aperture of 50 μm and a microporous array membrane with the lower layer aperture of 15 μm. In the second part, 3% sodium citrate is used for preprocessing the pleural effusion sample, so that the placing time is shortened; a final isolation procedure was constructed and efficacy verified. The detection effect of samples with different volumes is compared by a double-layer filtering device consisting of microporous array membranes, and the observation effect can be obtained by finding that 10ml of pleural effusion
Clinical pleural effusion samples were tested by the method described above. The study included a total of 38 patients with first symptoms of pleural effusion, 19 men and 19 women; 31 patients with malignant pleural effusion and 7 patients with pleural effusion due to other causes. Among them, 31 malignant pleural effusion patients, including 1 lung squamous cancer patient, 30 lung adenocarcinoma patients and 7 non-tumor-induced pleural effusion patients, were included. Clinical information of 31 tumor patients, including gender and age, is shown in fig. 8.
For 38 patients in the group, the overall assay accuracy was 84.2% (32/38). The false negative rate of the overall test was 12.9% (4/31), and the false positive rate was 28.5% (2/7). Wherein the detection positive detection rate can reach 87.10 percent (27/31) for 31 patients with the malignant pleural effusion. Their clinical information and the results of the ETCs detection are shown in Table 1.
Example 7 separation of tumor-shed cells in pleural effusion on pericardial effusion based on 2.5D microporous array Filter
Clinical application in fluids
To explore the feasibility of this method in other body fluid tests, we also collected 1 pericardial effusion sample (collected from a affiliated second hospital at university of Dai-Litsea medical university) and tested. This patient was 21# patient with a pleural effusion test group, diagnosed with advanced lung cancer. We successfully found shed tumor cells in pericardial effusion specimens of patient # 21, as shown in figure 9.
Example 8 cultivation of isolated cells
After the clear capture of ETCs, we removed the filter and placed it in 1640 medium containing 10% fetal bovine serum, 1% penicillin streptomycin, recombinant human epidermal growth factor EGF (20 ng/mL) and cultured. After 12 days of culture we obtained cell clumps from one cell division as shown in figure 10.
The culture method comprises the following steps:
(1) RPMI1640A medium was prepared and contained 10% fetal bovine serum, 1% penicillin streptomycin, and recombinant human epidermal growth factor EGF (20 ng/mL). The following steps are all carried out on an ultra-clean bench, so that aseptic operation is ensured.
(2) The upper and lower filters were wetted with 1ml of 75% alcohol.
(3) The upper and lower filters were rinsed with 3ml of PBS solution.
(4) 10ml of pleural effusion was filtered sequentially from the upper 50 micron diameter filter and the lower 15 micron filter.
(5) Removing the lower layer of filter membrane with diameter of 15 μm with forceps, placing the filter membrane in the prepared culture medium, and placing under 5% CO 2 Incubate in a cell incubator at 37℃and saturated humidity.
(6) Cells in the dishes were checked daily on a regular basis.
Example 9 ratio of the method of separating tumor-shed cells from Complex body fluid according to the invention to clinically conventional detection methods
Comparative analysis
We constructed the method of this study and three conventional clinical detection methods of pleural effusion shed cells: comparative analysis was performed for hydrothorax cytology, hydrothorax liquid-based cytology and wax block embedding. The method has the advantages of small required sample size; the detection time is short; the single detection positive rate is high; the detection result is simple to judge; and has significant advantages in practical application for pleural effusion detection with a small cell count, and can also be applied to other complex body fluids, such as pericardial effusion. The most important point is that the ETCs obtained by the separation still have biological activity, so the ETCs have great potential for subsequent research and analysis and are more suitable for clinical and laboratory researches (as shown in table 1).
Table 1: comparison of the research method with the clinical routine detection method
Wherein the pleural effusion cytology analysis determines that the percentage of cases diagnosed as malignant pleural effusion averages 55%, but varies greatly in different studies, but the highest positive rate does not exceed 60%. Other common clinical testing formats also vary widely in positive rate from study to study, and the data in the tables are from the references.
Claims (10)
1. A method of separating shed cells in a complex body fluid, the method comprising the step of filtering the complex body fluid using an upper filter membrane having a pore size of 50 μm and a lower filter membrane having a pore size of 15 μm;
preferably, the method is of non-diagnostic interest;
preferably, the complex body fluids include pleural effusion, pericardial effusion, alveolar lavage, urine, saliva, tissue digestive fluids, cerebrospinal fluid, and peritoneal lavage;
more preferably, the complex body fluid comprises pleural effusion, pericardial effusion.
2. The method of claim 1, wherein the upper filter membrane is one or more of circular, rectangular, and regular hexagonal;
preferably, the shape of the lower-layer filter membrane can be designed into one or more of a round shape, a rectangular shape and a regular hexagon shape according to the requirement;
preferably, the filter is a 2.5D microwell array filter.
3. The method of claim 1, wherein the complex body fluid should have an ex vivo time of no more than 20 hours;
more preferably, the ex vivo time is selected from 0h, 4h, 8h, 20h.
4. The method of claim 1, which is suitable for treating 10ml-15ml of complex body fluids.
5. The method of claim 1, wherein an anticoagulant is added to the complex body fluid;
preferably, the anticoagulant comprises heparin, citrate, edetate, oxalate;
preferably, the anticoagulant is sodium citrate, more particularly, the concentration of sodium citrate is 1%, 2%, 3%, 4% or higher;
preferably, the anticoagulant is 3% sodium citrate;
preferably, the method further comprises the step of rinsing the filter membrane with 75% alcohol wet filter membrane and PBS liquid.
6. A double-layer filtration device comprising an upper filter membrane having a pore size of 50 μm and a lower filter membrane having a pore size of 15 μm;
preferably, the filter membrane is a 2.5D microporous array filter membrane;
preferably, the shape of the upper layer filter membrane is one or more of a circle, a rectangle and a regular hexagon;
preferably, the shape of the lower layer filter membrane is one or more of a circle, a rectangle and a regular hexagon.
7. Use of the double-layer filtration device of claim 6 for separating exfoliated cells;
specifically, the shed cells are isolated from complex body fluids;
preferably, the complex body fluid comprises clinical samples such as pleural effusion, pericardial effusion, alveolar lavage, urine, saliva, tissue digestive juice, cerebrospinal fluid, and peritoneal lavage;
more preferably, the complex body fluid comprises pleural effusion, pericardial effusion.
8. The use of claim 7, wherein the complex body fluid is collected from a suspected patient;
preferably, the pleural effusion is collected from suspected patients of lung cancer, pneumonia, pulmonary infection;
preferably, the pericardial effusion is collected from suspected patients of pericarditis, tumors, rheumatic heart disease;
preferably, the lung cancer comprises squamous lung cancer or adenocarcinoma;
preferably, the lung cancer comprises non-small cell lung cancer or small cell lung cancer.
9. Use of the double-layer filtration device of claim 6 in the manufacture of a cancer diagnostic product;
preferably, the product is a cancer product diagnosed by isolating shed cells;
preferably, the shed cells are isolated from complex body fluids;
more preferably, the complex body fluid comprises pleural effusion, pericardial effusion;
preferably, the cancer is lung cancer;
preferably, the lung cancer comprises squamous lung cancer or adenocarcinoma;
preferably, the lung cancer comprises non-small cell lung cancer or small cell lung cancer.
10. The use according to claim 9, wherein diagnosis of cancer is performed by confirming whether or not the exfoliated cells contain cancer cells by performing any one or more of microscopic observation, immunodetection, and nucleic acid detection on the exfoliated cells;
in particular, the immunodetection includes ELISA, RIA, immunoblotting, immunofluorescence, and flow cytometry analysis based on immunofluorescence techniques.
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