CN111458515A - Method for detecting number of lung small cell tumor cells in peripheral blood - Google Patents
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Abstract
The invention provides a reliable internal control quality control method for detecting the lung small cells, accurately calculates the recovery rate of the cells while accurately knowing the upper limit of the detection technology to obtain the number of the whole circulating tumor cells, and greatly improves the detection reliability. The method comprises the following steps: 1) obtaining a peripheral blood sample, 2) injecting 200 artificially synthesized cell-like fluorescent round beads into the peripheral blood sample, 3) enriching peripheral blood small cells by adopting the following technology, 4) identifying and counting the remaining cell-like fluorescent round beads in the peripheral blood after enrichment under a microscope, and calculating the number of the lung small cells in the peripheral blood according to a round bead recovery rate calculation formula.
Description
The technical field is as follows:
the invention relates to a method for detecting the number of small lung cell tumor cells in peripheral blood.
Background art:
the small cell lung cancer (SC L C) accounts for about twenty to twenty-five percent of the lung cancer, is the most malignant and has the worst prognosis, so that the lung cancer needs to be diagnosed and treated as soon as possible if the symptom of the lung cancer is found in daily life, and the early diagnosis method comprises the step of detecting the number of the lung small cell tumor cells in peripheral blood.
The prior art discloses methods for enriching small lung cell tumor cells in peripheral blood, such as:
wu, H.Hao, &. lTtT transition = "L" &. gTt L &. lTt/T &. gTt. L i, X.ZHou, Z.Guo, &. lTtT transition &. L "&. gTt L &. lTt/T &. gTt. Zhang, X.Zhang, W.Zhang, H.Guo, R.Brenner, and P. L in (2009) clinical evidence of the clinical design of the detecting circulating cells, J.Thoraccol 4:30-6 documents disclose clinical detection of lung cancer and enrichment of circulating cells.
Chen, F.Ge, W.Cui, F.Wang, Z.Yang, Y.Guo, &lTtTtranslation = L "&gTtL &lTt/T &gTt. L i, R.Bremener, and P. L in (2013) L un Cancer Circulating Cells Isolated by the EpCAM-independent energy gradient with tissue activation 19-Derived CYFRA21-1 and medical staging. Clin Chim Acta 419:57-61 documents disclose non-EpCAM dependent enriched lung Cancer Circulating Tumor Cells.
Application Value and Evaluation of Circulating Tumor Cells (CTC) infection of Small Cell L un-g Cancer Patents GE Han-tie, WANG et al China and Foreign Medical Treatment 2017-22, which disclose the use and Evaluation of Circulating Tumor Cells in Small lung Cell disorders.
However, the prior art has the defect that the proportion and the accurate number of the captured cells in the total number of the whole lung small cells cannot be known, and the quantification cannot be realized.
As the number of the lung small cells is very high compared with other tumors, quantitative data needs to be provided, a standard of the proportion of the captured cells to the total number of the whole lung small cells is established, and the occurrence, development, treatment effect and the like of the small cell lung cancer are judged according to the standard, so that the method can be used for detecting the small cell lung cancer, eliminating false positives and judging the treatment effect of the medicine.
The purpose of the invention is as follows:
the invention provides a reliable internal control quality control method for detecting the lung small cells, accurately calculates the recovery rate of the cells while accurately knowing the upper limit of the detection technology to obtain the number of the whole circulating tumor cells, and greatly improves the detection reliability.
The detection method comprises the following steps:
1) obtaining peripheral blood samples
6ml of human peripheral blood was collected in any of blood collection tubes containing an anticoagulant (e.g., EDTA, heparin, ACD, etc.) (BD, New Jersey, USA).
2) Injecting 200 artificially synthesized cell-simulated fluorescent beads into peripheral blood sample
3) Peripheral blood minicells were enriched using the following technique
After centrifugation (1000 × g for 15 minutes), plasma proteins were removed, 50. mu.l of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA) were added thereto, mixed by inversion for 10 times, incubated at room temperature for 20 minutes, 6ml of cell buffer was added, the specimen was gently transferred to a centrifuge tube containing 3 ml of a cell separation medium (the specific gravity of the cell separation medium at 20 ℃ was 1.07260 to 1.07650 g/ml, the cell separation medium contained the following components: colloidal silica coated with polyvinylpyrrolidone, polysaccharide and sodium diatrizoate; a sugar solution containing dextran; a nonionic polymer polymerized from sucrose and epichlorohydrin), the sample was centrifuged again (900 × g, 6 minutes), the supernatant was taken, the supernatant was centrifuged again for 1000. 1000 × g and 4 minutes, and the deposited cells obtained were enriched in non-limbal cells and fluorescent nucleated cells.
4) And identifying and counting the remained cell-like fluorescent beads in the enriched peripheral blood under a microscope, and calculating the number of the lung small cells in the peripheral blood according to a bead recovery calculation formula.
The following is an explanation of the term of the present invention:
lung small cell tumor cell, defined as lung small cell carcinoma (SC L C) is a kind of malignant epithelial tumor composed of small cells, tumor cell cytoplasm is rare, cell boundary is unclear, nuclear chromatin is fine granular, no nucleolus or not obvious, cell is circular, oval or fusiform, nuclear incisional track is obvious, necrosis is typically extensive, and nuclear division count is high.
Peripheral blood, defined as follows: peripheral blood is blood other than bone marrow
Artificially synthesized cell-simulated fluorescent beads are defined as follows: the density of the polystyrene fluorescent microspheres mixed with the fluorescent dye on the beads is 1.095-1.120 g/cm-3, and the polystyrene fluorescent microspheres are the same as the density of lymphocytes and mononuclear leukocytes, and can be widely applied to blood flow determination, tracing, in-vivo imaging, image calibration, flow cytometry and the like.
Enrichment of lung minicells, defined as follows: peripheral Blood Mononuclear Cells (PBMC) are separated from a whole blood sample, and then CD45+ cells in the PBMC cells of a tumor patient are removed through immunomagnetic beads coupled with an anti-human CD45 monoclonal antibody, so that Circulating Tumor Cells (CTCs) in a patient peripheral blood sample are effectively enriched.
Identification and enumeration of lung minicells is defined as follows: tumor cells were identified by immunofluorescent staining of cells by PE-labeled anti-human CD45 antibody (PE-anti-CD45) and DAPI by immunofluorescence cytochemistry, and cells negative for CD45 expression (DAPI +/CD45-/CEP8 heteroploids) were identified as CTCs, in addition to in situ hybridization of CEP8 fluorescent probes to the chromosome mitochondria.
The formula for calculating the bead recovery rate is defined as follows: fluorescent bead enrichment rate ═ number of fluorescent beads identified last by microscope/fluorescent beads known to be added to blood of healthy persons.
The method is obtained by screening after the following experiments:
respectively adding 10, 100, 400 and 1000 lung small cell strains to correspondingly extract 6m L blood of a healthy person to simulate the real environment of tumor cells, completing the whole enrichment process, identifying and counting the rest lung small cell strains under a microscope to obtain the experimental cell enrichment rate, thereby calculating the real tumor cell enrichment rate and the upper and lower limits of the detection technology.
The following is a detailed description of the experiment:
first, experiment purpose
Adding a known number of cell strains into 6m L blood, simulating a real detection environment, completing the whole enrichment process, and obtaining the cell enrichment rate.
Second, Experimental methods
After 6ml of human peripheral blood was collected in any blood collection tube (BD, New Jersey, USA) containing an anticoagulant (e.g., EDTA, heparin, ACD, etc.) and centrifuged (1000 × g, 15 minutes), plasma proteins were removed, 50. mu.l of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA) were added thereto, the mixture was mixed by inversion for 10 times, incubated at room temperature for 20 minutes, 6ml of cell buffer was added, the specimen was gently transferred above a 3 ml cell separation medium (the cell separation medium has a specific gravity of 1.07260 to 1.07650 g/ml at 20 ℃ C., and contains a polyvinylpyrrolidone-coated colloidal silica, polysaccharide and sodium diatrizoate; a sugar solution containing dextran; a nonionic polymer polymerized from sucrose and epichlorohydrin) and the specimen was centrifuged (900 g, 6 minutes) again, and the supernatant was collected and centrifuged again for 1000 minutes to obtain a supernatant, which was then 1000. about.64 minutes, i.e., cell supernatant was discarded.
Third, the experimental procedure
Centrifuging (1000 × g, 15 minutes), removing plasma proteins, adding 50 microliters of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA), reversing and mixing uniformly for 10 times, incubating at room temperature for 20 minutes, adding 6 milliliters of cell buffer solution, gently transferring the sample to a centrifuge tube containing 3 milliliters of cell separation medium (the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/milliliter, the cell separation medium contains the following components of colloidal silica coated with polyvinylpyrrolidone, polysaccharide and sodium diatrizoate, a sugar solution containing dextran, a nonionic polymer polymerized from sucrose and epichlorohydrin), centrifuging the sample again (900 × g, 6 minutes), taking supernatant, centrifuging the supernatant again for 1000 × g, 4 minutes, discarding supernatant, and obtaining deposited cells, namely enriched cell strains, performing immunofluorescent staining on the cells enriched by the immunofluorescent cell strain, adding fluorescent antibody labeled with CD 829-CD 23, fluorescent antibody labeled with anti-CD 23, fluorescent antibody-CD 23, and fluorescent antibody-45 3 (DAP-P) in-CD 23), and performing in-situ hybridization on the deposited cells
Fourth, experimental data and results
Cells negative for CD45 expression (DAPI +/CD45-/CEP 8-heteroploid) were identified as cell lines. The number of identified cell lines was counted. The cell recovery was calculated using the following formula: cell line enrichment rate ═ cell line identified last/known number of cell lines added to blood of healthy persons
Fifth, conclusion
The known number of cell strains are added into peripheral blood of a healthy person of 6m L, the whole enrichment and identification process is completed according to the standard, and the obtained cell recovery rate reflects the real tumor cell recovery rate, so that the very high enrichment accuracy is obtained according to the recovery rates of 10, 100, 400 and 1000 cell strains.
According to the invention, a known number of lung small cell strains and a simulated artificial synthetic fluorescent round bead are added, after the whole enrichment process is completed, the rest lung small cell strains and the rest fluorescent round beads are identified and counted under a microscope, and the enrichment rates of the lung small cell strains and the fluorescent round beads are obtained. Multiple experiments prove that the enrichment rate of the fluorescent beads and the small lung cell strain is highly consistent and the fluorescent beads can replace the cell strain to be used as reliable quality internal control of detection. Multiple experiments prove that 200 artificially synthesized cell-like fluorescent beads are injected into detected peripheral blood to serve as internal control of a sample, after the whole enrichment process is completed, the rest beads can be easily identified and counted under a microscope, the number of whole lung small cells and the detection success rate are calculated according to the bead recovery rate, and meanwhile, the upper limit of detection is not touched.
The invention has the advantages that:
other existing lung small cell enrichment technologies lack effective internal control and cannot provide accurate cell recovery rate, and the upper limit of detection is unknown.
Compared with the prior art and the method, the method has the following results:
the specific implementation mode is as follows:
the invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.
Example 1
An example of a specific real detection process is provided:
injecting 200 cell-like beads into 6m L peripheral blood, checking under a microscope with 10-fold objective lens, identifying and counting the beads, and calculating the recovery rate according to the remaining formula, i.e., the number of identified beads/the total number of injected beads (recovery rate), and the recovery rate is used for calculating the number of total circulating tumor cells, i.e., the number of identified lung small cells/the recovery rate (total lung small cell number).
1) Obtaining peripheral blood samples
6ml of human peripheral blood was collected in any of blood collection tubes containing an anticoagulant (e.g., EDTA, heparin, ACD, etc.) (BD, New Jersey, USA).
2) Injecting 200 artificially synthesized cell-simulated fluorescent beads into peripheral blood sample
3) Peripheral blood minicells were enriched using the following technique
After centrifugation (1000 × g for 15 minutes), plasma proteins were removed, 50. mu.l of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA) were added thereto, mixed by inversion for 10 times, incubated at room temperature for 20 minutes, 6ml of cell buffer was added, the specimen was gently transferred to a centrifuge tube containing 3 ml of a cell separation medium (the specific gravity of the cell separation medium at 20 ℃ was 1.07260 to 1.07650 g/ml, the cell separation medium contained the following components: colloidal silica coated with polyvinylpyrrolidone, polysaccharide and sodium diatrizoate; a sugar solution containing dextran; a nonionic polymer polymerized from sucrose and epichlorohydrin), the sample was centrifuged again (900 × g, 6 minutes), the supernatant was taken, the supernatant was centrifuged again for 1000. 1000 × g and 4 minutes, and the deposited cells obtained were enriched in non-limbal cells and fluorescent nucleated cells.
4) And identifying and counting the remained cell-like fluorescent beads in the enriched peripheral blood under a microscope, and calculating the number of the lung small cells in the peripheral blood according to a bead recovery calculation formula.
Example 2
Another example of a specific real detection process is provided:
injecting 200 cell-like beads into 6m L peripheral blood, checking under a microscope with 10-fold objective lens, identifying and counting the beads, and calculating the recovery rate according to the remaining formula, i.e., the number of identified beads/the total number of injected beads (recovery rate), and the recovery rate is used for calculating the number of total circulating tumor cells, i.e., the number of identified lung small cells/the recovery rate (total lung small cell number).
1) Obtaining peripheral blood samples
6ml of human peripheral blood was collected in any of blood collection tubes containing an anticoagulant (e.g., EDTA, heparin, ACD, etc.) (BD, New Jersey, USA).
2) Injecting 200 artificially synthesized cell-simulated fluorescent beads into peripheral blood sample
3) Peripheral blood minicells were enriched using the following technique
After centrifugation (1000 × g for 15 minutes), plasma proteins were removed, 50. mu.l of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA) were added thereto, mixed by inversion for 10 times, incubated at room temperature for 20 minutes, 6ml of cell buffer was added, the specimen was gently transferred to a centrifuge tube containing 3 ml of a cell separation medium (the specific gravity of the cell separation medium at 20 ℃ was 1.07260 to 1.07650 g/ml, the cell separation medium contained the following components: colloidal silica coated with polyvinylpyrrolidone, polysaccharide and sodium diatrizoate; a sugar solution containing dextran; a nonionic polymer polymerized from sucrose and epichlorohydrin), the sample was centrifuged again (900 × g, 6 minutes), the supernatant was taken, the supernatant was centrifuged again for 1000. 1000 × g and 4 minutes, and the deposited cells obtained were enriched in non-limbal cells and fluorescent nucleated cells.
4) And identifying and counting the remained cell-like fluorescent beads in the enriched peripheral blood under a microscope, and calculating the number of the lung small cells in the peripheral blood according to a bead recovery calculation formula.
Claims (4)
1. A method for detecting the number of small lung cell tumor cells in peripheral blood comprises the following steps:
1) obtaining peripheral blood samples
Collecting 6ml of human peripheral blood in any blood collection tube containing anticoagulant,
2) injecting 200 artificially synthesized cell-simulated fluorescent beads into peripheral blood sample
3) Peripheral blood minicells were enriched using the following technique
After centrifugation, removing plasma protein, adding 50 microliter of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA, reversing and uniformly mixing for 10 times, incubating at room temperature for 20 minutes, adding 6 milliliters of cell buffer solution, gently transferring the sample to a centrifuge tube filled with 3 milliliters of isolation solution above a cell separation medium, centrifuging the sample again, taking supernatant, centrifuging the supernatant again, discarding the supernatant, obtaining sedimentary cells which are enriched non-limbic nucleated cells and cell-like fluorescent round beads,
4) and identifying and counting the remained cell-like fluorescent beads in the enriched peripheral blood under a microscope, and calculating the number of the lung small cells in the peripheral blood according to a bead recovery calculation formula.
2. The detection method according to claim 1, comprising the steps of:
1) obtaining peripheral blood samples
Collecting 6ml of human peripheral blood in any blood collection tube (BD, New Jersey, USA) containing anticoagulant (such as EDTA, heparin, or ACD),
2) injecting 200 artificially synthesized cell-simulated fluorescent beads into peripheral blood sample
3) Peripheral blood minicells were enriched using the following technique
Centrifuging (1000 × g for 15 minutes), removing plasma protein, adding 50 microliters of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA), reversing and mixing uniformly for 10 times, incubating at room temperature for 20 minutes, adding 6 milliliters of cell buffer solution, gently transferring the sample to a centrifuge tube containing 3 milliliters of cell separation medium (the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/milliliter, the cell separation medium contains the following components of colloidal silica coated by polyvinylpyrrolidone, polysaccharide and sodium diatrizoate, sugar solution containing dextran, and nonionic polymer polymerized by sucrose and epichlorohydrin), centrifuging the sample again (900 × g for 6 minutes), taking supernatant, centrifuging the supernatant again for 1000 × g and 4 minutes, discarding supernatant, and obtaining sediment cells, namely enriched in the non-limbal cells and the fluorescent nucleated cells,
4) and identifying and counting the remained cell-like fluorescent beads in the enriched peripheral blood under a microscope, and calculating the number of the lung small cells in the peripheral blood according to a bead recovery calculation formula.
3. The detection method according to claim 1, comprising the steps of:
1) obtaining peripheral blood samples
Collecting 6ml of human peripheral blood in any blood collection tube (BD, New Jersey, USA) containing anticoagulant (such as EDTA, heparin, or ACD),
2) injecting 200 artificially synthesized cell-simulated fluorescent beads into peripheral blood sample
3) Peripheral blood minicells were enriched using the following technique
Centrifuging (1000 × g for 15 minutes), removing plasma protein, adding 50 microliters of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA), reversing and mixing uniformly for 10 times, incubating at room temperature for 20 minutes, adding 6 milliliters of cell buffer solution, gently transferring the sample to a centrifuge tube containing 3 milliliters of cell separation medium (the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/milliliter, the cell separation medium contains the following components of colloidal silica coated by polyvinylpyrrolidone, polysaccharide and sodium diatrizoate, sugar solution containing dextran, and nonionic polymer polymerized by sucrose and epichlorohydrin), centrifuging the sample again (900 × g for 6 minutes), taking supernatant, centrifuging the supernatant again for 1000 × g and 4 minutes, discarding supernatant, and obtaining sediment cells, namely enriched in the non-limbal cells and the fluorescent nucleated cells,
4) and identifying and counting the remained cell-like fluorescent beads in the enriched peripheral blood under a microscope, and calculating the number of the lung small cells in the peripheral blood according to a bead recovery calculation formula.
4. The detection method according to claim 1, comprising the steps of:
1) obtaining peripheral blood samples
Collecting 6ml of human peripheral blood in any blood collection tube (BD, New Jersey, USA) containing anticoagulant (such as EDTA, heparin, or ACD),
2) injecting 200 artificially synthesized cell-simulated fluorescent beads into peripheral blood sample
3) Peripheral blood minicells were enriched using the following technique
Centrifuging (1000 × g for 15 minutes), removing plasma protein, adding 50 microliters of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA), reversing and mixing uniformly for 10 times, incubating at room temperature for 20 minutes, adding 6 milliliters of cell buffer solution, gently transferring the sample to a centrifuge tube containing 3 milliliters of cell separation medium (the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/milliliter, the cell separation medium contains the following components of colloidal silica coated by polyvinylpyrrolidone, polysaccharide and sodium diatrizoate, sugar solution containing dextran, and nonionic polymer polymerized by sucrose and epichlorohydrin), centrifuging the sample again (900 × g for 6 minutes), taking supernatant, centrifuging the supernatant again for 1000 × g and 4 minutes, discarding supernatant, and obtaining sediment cells, namely enriched in the non-limbal cells and the fluorescent nucleated cells,
4) and identifying and counting the remained cell-like fluorescent beads in the enriched peripheral blood under a microscope, and calculating the number of the lung small cells in the peripheral blood according to a bead recovery calculation formula.
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