T/CSBM 0044-2023 - Non-antibody-dependent magnetic separation detection method of circulating tumor cells in peripheral blood

Standard No.: T/CSBM 0044-2023
Language: Chinese, Available in English version
Release Date: 2023
Published By: Group Standards of the People's Republic of China
Lastest: T/CSBM 0044-2023

Scope: Principle of the method 4.1 Based on the difference in characteristics between CTCs and blood cells, non-antibody-dependent methods are used to capture CTCs and analyze and identify the captured cells through cell staining, immunocytochemistry, fluorescence in situ hybridization or reverse transcription polymerase chain reaction. 4.2 Non-antibody-dependent methods include label-free capture probe method (see 9.2.1), peptide capture probe method (see 9.2.2), membrane filtration method (see Appendix C) and microfluidic chip method (see Appendix D). Non-antibody-dependent magnetic separation methods include but are not limited to the following two: 　——Label-free capture probe method: After CTCs are separated by centrifugal force in a certain density gradient medium, label-free paramagnetic nanoparticles are used to capture CTCs, and CTCs are effectively separated and enriched from other blood components under the action of an external magnetic field; 　——Peptide capture probe method: Peptide-modified magnetic particles are used to capture CTCs in peripheral blood, and the peptide capture probes stably bind to CTCs in peripheral blood, and CTCs are effectively separated and enriched from other blood components under the action of an external magnetic field. 5 Test conditions The test conditions are as follows: 　——Temperature: 25℃±5℃; 　——Relative humidity: 30%～65%; 　——Pressure: standard atmospheric pressure. 6 Reagents, materials and instruments Reagents, materials and instruments should meet the requirements of Appendix A. 7 Test process 7.1 See Figure 1 for the CTC test process in peripheral blood. 7.2 See Appendix B for the CTC magnetic separation test operation. 8 Sample preparation 8.1 Blood sample collection 8.1.1 Prepare heparin/EDTA-K2 vacuum anticoagulation tubes before blood collection. 8.1.2 The blood collection volume should be 4mL～15mL. After the blood collection is completed, the blood collection tube should be immediately inverted and mixed for no less than 5 times. It should not be violently shaken to produce bubbles. 8.1.3 Blood samples should be stored at 4℃～8℃ and CTC detection should be performed within 72h. 8.2 Sample pretreatment 8.2.1 Density gradient centrifugation 8.2.1.1 Check the blood sample before centrifugation. The qualified sample is the one without hemolysis and clots. 8.2.1.2 Preheat the cell density gradient separation solution in a 37℃ water bath for 15 minutes. The water level of the water bath should be higher than the sample liquid level to ensure that the sample in the container reaches the target temperature. 8.2.1.3 After preheating, add them to 15mL centrifuge tubes in sequence. 8.2.1.4 Hold both ends of the blood collection tube and gently flip the tube 180° 8 to 10 times to fully mix the peripheral blood sample. 8.2.1.5 Take 1mL of the mixed blood sample and dilute it with 3mL 1×PBS in a ratio of 1:3. Use a pipette to slowly add the diluted blood to the upper layer of the density gradient separation solution. Note: PBS is phosphate buffered saline. 8.2.1.6 Place in a centrifuge at 400g to 450g and centrifuge for 30min to 40min. 8.2.1.7 After centrifugation, the interface of the layers is clear. After centrifugation, the solution is divided into 3 layers: the upper layer is the plasma layer, the middle layer is the buffy coat layer, and the lower layer is the red blood cell layer. The middle layer is the target cell collection layer. There is a white membrane zone in the middle of the target cell layer, which is mainly composed of mononuclear cells. The target cell retention rate is ＞85%. 8.2.1.8 After removing the upper plasma with a pipette, transfer all the middle target cell collection layer (between 2.5mL and 6.5mL) to a 15mL centrifuge tube, add PBS to make the volume to 12mL, cover the tube tightly, gently flip the centrifuge tube 180° several times, place it in a centrifuge at 300g~350g, and centrifuge for 5min~7min. 8.2.1.9 After centrifugation, retain about 100?g of liquid at the bottom. Add an appropriate amount of PBS to resuspend the cells. 8.2.2 Red blood cell lysis method 8.2.2.1 Sampling: It is best to operate at 4℃ and take 1mL of fresh anticoagulated blood into a centrifuge tube. 8.2.2.2 Lysis: Add 3mL of 1× red blood cell lysis buffer to the centrifuge tube at a ratio of 1:3 and lyse for 5min. 8.2.2.3 Centrifugation: Centrifuge at 500g for 5 minutes at room temperature and discard the red supernatant. 8.2.2.4 Secondary lysis centrifugation: If the red blood cell lysis is found to be incomplete, the lysis (see 8.2.2.2) and centrifugation (see 8.2.2.3) can be repeated once. 8.2.2.5 Washing: Add an appropriate amount of 1×PBS, gently mix and resuspend the precipitate, and centrifuge at 500g for 5 minutes at room temperature. 8.2.2.6 Precipitation: After centrifugation, remove the supernatant and resuspend the precipitated cells with an appropriate amount of 1×PBS. 9 Sample Testing 9.1 Test Preparation 9.1.1 CTC Capture Probe Activation and Dispersibility Evaluation 9.1.1.1 Clamp the tube wall with tweezers, tilt the centrifuge tube 45°, turn on the ultrasonic cleaner, and shake it vigorously from side to side for more than 1 minute. 9.1.1.2 Take 2μL of the activated CTC capture probe and spread it evenly on the slide. No obvious precipitation and floccules can be observed by naked eye. 9.1.1.3 Observe under a microscope when necessary. It is required that the probe can be evenly dispersed into fine particles suspended in the dispersion medium without precipitation or agglomeration. The activation of CTC capture probe is completed. Note 1: Activation is a process of reducing material agglomeration, improving material reaction activity, and chemically treating the material to improve material dispersibility. Note 2: Dispersibility refers to the ability of the material to be evenly dispersed into fine particles suspended in the dispersion medium without precipitation in water or other uniform liquid media. Note 3: Dispersibility evaluation refers to using an ultrasonic cleaner to fully ultrasonically disperse the probe, taking a small amount of the probe and using a particle size analyzer to detect the particle size distribution of the probe. The polydispersity index (PDI) is ≤0.3, indicating good dispersibility. 9.1.2 Smear placement Before centrifugal smearing, align and place them in the order of "slide + filter paper + smear funnel" in sequence, and align the smear funnel hole with the filter paper hole; clamp the slide on the smear machine with the front of the slide against the filter paper and the smooth side facing outward, and there should be no gaps. 9.2 CTC magnetic separation and capture 9.2.1 Label-free capture probe method 9.2.1.1 Resuspend the washed cell pellet in a 1.5mL centrifuge tube with a small amount of PBS and adjust the volume to 1mL. 9.2.1.2 Take an appropriate amount of magnetic nanoprobes and add them to a 1.5mL centrifuge tube. 9.2.1.3 Place the centrifuge tube with magnetic nanoprobes on a mini rotating incubator and mix at 4℃ for 6min~8min. 9.2.1.4 After taking out the centrifuge tube, immediately place it on a multifunctional magnetic separator and magnetically adsorb it at 4℃ for 6min~8min. Note: Magnetic adsorption is the phenomenon that magnetic materials spontaneously move from the liquid phase and adsorb to the magnet under the action of an external magnetic field. 9.2.1.5 Slowly aspirate the supernatant with a single-channel pipette, re-add an appropriate amount of PBS, invert and mix again, and immediately place it in a multifunctional magnetic separator and magnetically adsorb it at 4℃ for 8min~10min. 9.2.1.6 Use a single-channel pipette to slowly aspirate the supernatant, remove the centrifuge tube, add an appropriate amount of PBS, and gently blow and mix the cells captured by the CTC capture probe to prepare a cell suspension. 9.2.2 Polypeptide Nanoprobe Method 9.2.2.1 Take the mixed anticoagulated blood into a centrifuge tube and dilute the anticoagulated blood with PBS. 9.2.2.2 After vortexing the polypeptide nanoprobe, take 10 μL of polypeptide nanoprobe into 2 mL of diluted peripheral blood, mix well, and incubate at 37°C for 1 hour. 9.2.2.3 After the incubation is completed, place the centrifuge tube on a multifunctional magnetic separator and separate by magnetic adsorption for 30 minutes. 9.2.2.4 Add PBS to wash, place the centrifuge tube on a multifunctional magnetic separator, slowly aspirate the supernatant, and repeat 2 to 3 times. 9.2.2.5 Resuspend the enriched cells with an appropriate amount of PBS to make a cell suspension. Note: In addition to the magnetic separation method, there are also membrane filtration method, microfluidic chip method, etc. that are not antibody-dependent; see Appendix C for membrane filtration method and Appendix D for microfluidic chip method. 9.3 Cell preparation 9.3.1 Take out a clean slide, mark the sample information and detection time, and align and fit them in the order of "smear funnel + filter paper + slide" and put them into the centrifugal smear machine. 9.3.2 Add 0.2mL~0.5mL of cell suspension into the corresponding marked smear funnel sample tube, start the centrifugal smear machine, centrifuge at 800r/min for 5min at room temperature, and gently take out the slide after centrifugation. Note 1: After the sample is diluted (i.e. cell suspension), it is slightly turbid. If the cell concentration is too high, cells will stack up on each other, affecting observation and identification; if the cell concentration is too low, the cells in the smear will be sparse, which is easy to cause false negatives. Note 2: If the centrifugation speed is too low, the cell structure is not clear, and if the centrifugation speed is too high, the cells are easily damaged. 9.3.3 The preparation results show that the cells are in a single layer, evenly distributed, with clear cell morphology and complete structure. 9.3.4 The cell preparation is applicable to the label-free capture probe method, peptide nanoprobe method, and microfluidic chip method. The membrane filtration method does not require the use of cell preparation. 9.4 CTC identification 9.4.1 Cytopathological identification method (conventional staining) Cytopathological staining identification is carried out according to the following steps: a) Cell fixation: Dry the cell slices obtained after preparation at room temperature and fix them with cell fixative for 10 minutes to 15 minutes; b) Cell staining: Remove the cell fixative, dry at room temperature, and then stain with cytoplasm staining solution and cell nucleus staining solution in turn, with each staining solution staining for 1 minute to 2 minutes; c) Sealing: After cell staining, rinse with distilled water 2 to 3 times, dry at room temperature, and seal with sealing agent; d) Observation: Observe the cell morphology and staining under a 10x and 40x microscope. After the cytoplasm and cell nucleus structure are clear, there is no overlap between cells, and there is no or only a trace amount of residual staining solution in the non-cellular area; e) Identification: Under a 100x microscope, tumor cells are identified based on cytopathological characteristics. 9.4.2 Immunocytochemistry and Immunofluorescence Method 9.4.2.1 Immunocytochemistry shall be carried out in the following steps: a) Cell fixation: air-dry the cell slices obtained after preparation at room temperature, fix them with cell fixative for 10 min to 20 min, and wash them in PBS buffer for 3 times, 5 min each time; b) Permeabilization (for intracellular antigens): permeabilize the cells with 0.2% TritonX-100 for 10 min, and rinse them with PBS buffer for 3 times, 5 min each time; c) Blocking: block with endogenous peroxidase blocking solution according to the reagent instructions, and soak them in PBS for 2 times, 5 min each time; non-specific blocking: circle the reaction site with a histochemical pen, add 5% BSA blocking solution, and block for 30 min; d) Primary antibody incubation: pour out the BSA blocking solution, add the diluted primary antibody (CK, CD45) directly to the reaction site, incubate in a 37℃ wet box for 1 h to 2 h, discard the primary antibody, and rinse them with PBS buffer for 3 times, 5 min each time; 9.4.3 Fluorescence in situ hybridization method The fluorescence in situ hybridization method is carried out in the following steps: a) Cell fixation: Dry the cell slices obtained after preparation at room temperature, add cell fixative to fix, and dry at room temperature; b) Rinse: Rinse the slices in 2×SSC buffer solution twice, 5 minutes each time; Note: SSC is sodium citrate buffer. c) Digestion: Add appropriate amount of 0.04% pepsin solution, digest the cells at room temperature for 10 minutes to 20 minutes, and soak and wash in 2×SSC buffer solution twice, 5 minutes each time; d) Dehydration: Dehydrate in 70%, 85% and 100% gradient ethanol in sequence for 5 minutes each, and dry at room temperature; e) Hybridization: In a dark room, take out the probe (see Appendix E), let it stand at room temperature for 5 minutes, flick the bottom of the centrifuge tube with your finger, mix the probe and centrifuge briefly, add 10μL of probe solution to the hybridization area, cover with a cover slip immediately, spread the probe evenly under the cover slip, and seal the slide with sealing glue. Place the slide on the hybridization instrument and denature at 45℃ for 2h, or 88℃ for 2min; f) Washing: In the dark room, carefully tear off the sealing glue around the cover glass with tweezers to avoid sticking or moving the cover glass, immerse the slide in 2×SSC for about 0.5min, take it out, and gently peel off the cover glass with tweezers; Place the slide in 2×SSC and wash at room temperature for 1min~2min; Take out the slide and immerse it in the post-hybridization washing solution (0.4×SSC/0.3% NP-40) preheated at 68℃ for 2min~5min; Take out the slide and immerse it in preheated deionized water for 1min, and dry the slide naturally in the dark; g) Re-staining: After drying the slide in the dark room, drop 10μL of DAPI staining solution on the hybridization area, immediately cover it with a cover glass and place it in the dark for staining for 10min~20min. Slides that cannot be observed by fluorescence microscope immediately during the re-staining step should be stored at -20℃. 9.4.4 Reverse transcription polymerase chain reaction (RT-PCR) 9.4.4.1 RNA extraction is carried out according to the following steps: a) Cell homogenization: take the enriched cell suspension, centrifuge at 400g for 5 minutes, add an appropriate amount of Trizol and repeatedly blow to dissolve the cells; b) RNA isolation: add chloroform [chloroform: Trizol (1:5)], cover the EP tube, turn it upside down and shake it vigorously, and let it stand on ice for 5 minutes to 10 minutes, until the upper and lower layers are clear. At 4°C, centrifuge at 13400g for 15min. The sample will be divided into three layers: organic phase, middle layer and upper colorless aqueous phase (RNA is in the aqueous phase). Collect the supernatant. c) Precipitate RNA: Add an equal amount of isopropanol to the supernatant, invert 5 times, let stand at room temperature for 10min, centrifuge at 13400g for 15min at 4°C. A gelatinous precipitate will form on the side and bottom of the centrifuge tube after centrifugation. Discard the supernatant. d) Wash and desalt RNA: Add 75% ethanol (prepared with DEPC water) and gently tap the bottom to wash the precipitate. Centrifuge at 9390g for 5min at 4°C and discard the supernatant. Note: DEPC is diethyl pyrocarbonate. e) Drying and re-dissolving RNA: Remove the filter paper and place the open EP tube on the filter paper with the tube mouth facing the alcohol, but it should not be completely dry; dissolve in 20μL~30μL of DEPC water, take 2μL for electrophoresis to detect integrity (1.5% agarose gel electrophoresis, mainly observe whether the three bands of 28s, 18s and 5s are clear), 2μL for colorimetry with UV spectrophotometer, and the rest is frozen in a -70℃ refrigerator. 9.4.4.2 The reverse transcription reaction is carried out according to the following steps: a) RNA quantification: Determine the RNA concentration and quantify it using a UV spectrophotometer; b) DNA removal: Add 2 μL of total cell RNA, 2 μL of 250 pmoL random primers, 2 μL of 2.5 mmol/L dNTPs and 10 μL of RNase-free water to a 200 μL clean RNase-free test tube, mix well, centrifuge briefly, incubate at 70°C for 5 min, and quench in an ice bath; c) Reverse transcription: then add 2 μL of 10× reverse transcription buffer, 1 U/μL reverse transcriptase, 1 U/μL RNase inhibitor, mix well, centrifuge briefly, react at 42°C for 60 min, heat at 95°C for 10 min to inactivate the reverse transcriptase, and store at -20°C. 9.4.4.3 PCR amplification should be carried out according to the following steps: a) PCR amplification system (25μL): including DEPC water (high pressure double distilled water can be used) 17.5μL, 10×Taqbuffer 2.5μL, MgCl2 2.0μL, 10moL/L dNTPMix 0.5μL, upstream primer 0.5μL, downstream primer 0.5μL, Tap enzyme (5u/μL) 0.5μL, cDNA template 1.0μL; b) PCR amplification conditions: 94℃ pre-denaturation for 2min, 94℃ denaturation for 20s, 55℃ annealing for 30s, 60℃ extension for 40s, 45 cycles; c) After the cycle amplification reaction is completed, check the amplification curve and Ct value. 9.4.4.4 The corresponding primers are used to amplify the corresponding gene targets. The reverse transcription polymerase chain reaction targets are shown in Appendix F. 9.4.4.5 After conventional PCR amplification, the PCR amplification products should be analyzed by gel electrophoresis, nucleic acid probe hybridization and other methods. 9.4.5 Gene sequencing method 9.4.5.1 DNA extraction is carried out according to the following steps: a) Thaw 1 mL of ETA anticoagulated -20℃ frozen blood at room temperature and then lyse it with pre-cooled red blood cell lysis buffer, repeat once; b) Mix the precipitated white blood cell mass with STE buffer, add SDS nuclear proteinase K to lyse the cells and digest the protein; c) After lysis overnight, add an equal volume of Tris-saturated phenol solution, and rotate to mix the organic phase and aqueous phase; d) After centrifugation, transfer the aqueous phase with a large-mouthed pipette, and repeat the extraction with Tris-saturated phenol solution once; e) Add an equal volume of chloroform: isoamyl alcohol (24:1), centrifuge, carefully transfer and collect the aqueous phase, and repeat once; f) Add 1/5 volume of sodium acetate and mix well, then add 2 times volume of -70℃ pre-cooled anhydrous ethanol, mix well to form white flocs, and collect the precipitate by centrifugation; g) Wash twice with 2 mL of 70% ethanol, evaporate and dry at room temperature, and do not let the DNA dry completely; h) Add sterile TE (pH7.4-8.4) buffer solution 29.4.5 Gene sequencing method 9.4.5.1 DNA extraction is carried out according to the following steps: a) Thaw 1 mL of ETA anticoagulated -20℃ frozen blood at room temperature and then lyse it with pre-cooled red blood cell lysis buffer, repeat once; b) Mix the precipitated white blood cell mass with STE buffer, add SDS nuclear proteinase K to lyse the cells and digest the protein; c) After lysis overnight, add an equal volume of Tris saturated phenol solution, and rotate to mix the organic phase and aqueous phase; d) After centrifugation, transfer the aqueous phase with a large pipette, and repeat the extraction with Tris saturated phenol solution once; e) Add an equal volume of chloroform: isoamyl alcohol (24:1), centrifuge, carefully transfer and collect the aqueous phase, and repeat once; f) Add 1/5 volume of sodium acetate and mix well, then add 2 times volume of -70℃ pre-cooled anhydrous ethanol, mix well to form white floccules, and collect the precipitate by centrifugation; g) Wash twice with 2 mL of 70% ethanol, evaporate and dry at room temperature, and do not let the DNA dry completely; h) Add sterile TE (pH7.4-8.4) buffer solution 29.4.5 Gene sequencing method 9.4.5.1 DNA extraction is carried out according to the following steps: a) Thaw 1 mL of ETA anticoagulated -20℃ frozen blood at room temperature and then lyse it with pre-cooled red blood cell lysis buffer, repeat once; b) Mix the precipitated white blood cell mass with STE buffer, add SDS nuclear proteinase K to lyse the cells and digest the protein; c) After lysis overnight, add an equal volume of Tris saturated phenol solution, and rotate to mix the organic phase and aqueous phase; d) After centrifugation, transfer the aqueous phase with a large pipette and repeat the extraction with Tris saturated phenol solution once; e) Add an equal volume of chloroform: isoamyl alcohol (24:1), centrifuge, carefully transfer and collect the aqueous phase, repeat once; f) Add 1/5 volume of sodium acetate and mix well, then add 2 times volume of -70℃ pre-cooled anhydrous ethanol, mix well to form white floccules, and collect the precipitate by centrifugation; g) Wash twice with 2 mL of 70% ethanol, evaporate and dry at room temperature, and do not let the DNA dry completely; h) Add 2μL to 50μL of sterilized TE (pH7.4-8.4) buffer solution to dissolve DNA and store at -20℃. 9.4.5.2 PCR amplification: Same as 9.4.4.3. 9.4.5.3 Sequencing: Sequence the purified PCR product according to the sequencer manual and analyze the sequencing results. It is advisable to select the sequencing method according to different cancer types. For sequencing categories, see Appendix G. ～50μL to dissolve DNA and store at -20℃. 9.4.5.2 PCR amplification: Same as 9.4.4.3. 9.4.5.3 Sequencing: Sequence the purified PCR product according to the sequencer manual and analyze the sequencing results. It is advisable to select the sequencing method according to different cancer types. For sequencing categories, see Appendix G. 10 Validity of the test results Cytopathological identification method 10.1.1 Different types of tumor cells in pathology mainly have the following six morphological characteristics: 　——Cell long diameter > 15 μm; 　——Cell nucleus-to-cytoplasm ratio > 0.8; 　——Common vacuoles or fat particles in the cytoplasm; 　——Cell nuclei are darkly stained and unevenly stained, with obvious nucleoli; 　——Nuclear morphology varies, and can be giant, binuclear or multinucleated; 　——Cell surface wrinkles or clear boundaries. 10.1.2 If the test result is clearly not a normal leukocyte in peripheral blood, and meets 3 or more of the above characteristics, the test result is determined to be valid; otherwise, the test result is determined to be invalid. 10.2 Immunocytochemistry and immunofluorescence method 10.2.1 The validity of the test results is as follows: 　——Positive control: The positive control can be used as an indicator of correct sample preparation and appropriate staining technique. Each staining should be compared with the positive control photo under the same test conditions. Positive controls are only used to monitor the correct execution of steps and the testing of reagents, and are not used to help describe the clear diagnosis of samples. If the positive results cannot show appropriate positive staining, the results of the test samples in this batch of experiments are considered invalid; 　——Negative control: Each staining should be compared with the blank control reagent under the same test conditions. The blank reagent is used to stain the slide instead of the antibody to determine nonspecific staining and provide a better explanation for the specific staining of the antigen site. The incubation time of the blank control reagent should be consistent with that of the antibody; 　——Tumor cells and normal cells should have nuclear staining at the same time. Positive marker immune characteristics can be divided into three levels: weak positive (+), moderate positive (++), and strong positive (+++). Immunoenzyme markers are manifested as light yellow fine particles, brownish yellow particles, and brownish yellow coarse particles. The latter are dazzling and easy to see. Immunofluorescence rules are manifested as light green fluorescence, obvious green fluorescence, and bright green dazzling fluorescence. 10.3 Fluorescence in situ hybridization method 10.3.1 The validity of the test results is as follows: 　——Positive control: abnormalities appear at any two or more sites (including two) of the cell chromosome or two or more (including two) abnormalities appear at one site (gene deletion/amplification, gene fusion or gene rearrangement); 　——Negative control: no abnormal signal of the cell chromosome. 10.3.2 Cells with abnormal chromosomes and positive nuclear staining are tumor cells. The chromosome abnormalities include gene deletion/amplification, gene fusion or gene rearrangement. The determination method is as follows: 　——Gene amplification: single-color probes usually count the absolute amount of each tumor cell signal, while dual-color probes calculate the ratio of the target gene site signal of each tumor cell to the reference chromosome signal; 　——Chromosome translocation: It can occur within or between chromosomes. In a few cases, translocation can also result in the loss of one of the separated probe signals; Note 1: If it occurs within a chromosome, the translocation is determined by the spacing between the separated probe signals reaching 1 to 2 times the signal diameter. Note 2: If it occurs between chromosomes, there should be clear independent signals. ——Gene deletion: When using a single-color probe (i.e., without a reference chromosome probe), the number of counted cells needs to be increased to avoid interpreting a positive result due to a lack of signal caused by the slide; multicolor probes above dual-color probes may have different threshold requirements for each signal. 10.4 Reverse transcription polymerase chain reaction (RT-PCR) 10.4.1 The validity of the test results is as follows: 　——Positive control: a typical S-type amplification curve (confirming that the amplification system is effective); 　——Negative control: a straight line or a slight oblique line, without an exponential growth period (excluding contamination of the amplification system); 　——PCR melting curve is a single-peak curve; 　——In the exponential amplification area of the amplification curve, the standard deviation of the CT value between duplicate wells should be <0.2; 　——The threshold setting is reasonable (set in the exponential growth area); 　——Amplification efficiency is between 90% and 110%, and the standard curve R>0.99, R2>0.98; the standard deviation should be <0.2. The Ct of the positive treatment result is about 15 to 30; the Ct of the negative control result is >35. 10.4.2 Result determination: 　——Negative: F≤A; 　——Positive: F＞A. F=2-?? Ct, ?? Ct=(Ct test group gene 1-Ct test group gene 2)-(Ct control group gene 1-Ct control group gene 2), where gene 1 is the test gene for EpCAM, CK19, gene 2 is the internal reference gene such as ?-Actin, GAPDH, etc., and Ct is the threshold of the test sample; 　——A is the critical value of the relative gene quantity of a specific gene (such as EpCAM, CK19, etc.) between negative samples and positive samples. 10.5 Gene sequencing method 10.5.1 The validity of the determination result is as follows: 　——The sequencing result is a single peak and there is little overlap with other peaks, indicating that the sequencing result is valid; 　——If multiple peaks appear at one site of a single peak, it indicates that the sequencing result is invalid. 10.5.2 Result determination: 　——Positive: Specific gene mutations are detected; the positive judgment value is that the mutation ratio is not less than 0.4%, the effective sequencing depth is not less than 500×, the absolute copy number of mutations is not less than 2, and the chain balance is between 0.1 and 0.9 (fusion is not applicable); 　——Negative: No specific gene mutations are detected. Example: Specific gene mutations, such as: EGFR gene/exon 20 p.T790 missense mutation/c.2369C>T/P.Thr790Met, mutation abundance is 1.22%. 11 Test results 11.1 Test result expression 11.1.1 Cytopathological identification method 11.1.1.1 The detection of a single CTC, CTC surrounded by a group of white blood cells, or a cluster of CTCs in 2 mL of blood samples is positive. When there are no less than 5 CTCs in a malignant solid tumor, it indicates a poor clinical prognosis. 11.1.1.2 If no single CTC, CTC surrounded by leukocytes, or CTC clusters are detected in 2 mL of blood, the test is negative, indicating that the test sample does not contain CTCs. 11.1.1.3 The results should be used in conjunction with information from diagnostic tests (i.e., imaging, laboratory tests), physical examination, and complete medical history in accordance with appropriate patient management procedures. 11.1.2 Immunocytochemistry and Immunofluorescence 11.1.2.1 Detection of single CTC, CTC surrounded by leukocytes, or CTC clusters with brown-yellow staining or green fluorescence in 2 mL of blood. 11.1.2.2 No single CTC, CTC surrounded by leukocytes, or CTC clusters are detected in 2 mL of blood. 11.1.3 Fluorescence in situ hybridization 11.1.3.1 Detect single CTCs, CTCs surrounded by white blood cells, or CTC clusters with chromosomal abnormalities (gene deletion/amplification, gene fusion, or gene rearrangement) in 2 mL of blood sample. 11.1.3.2 No single CTCs, CTCs surrounded by white blood cells, or CTC clusters with chromosomal abnormalities (gene deletion/amplification, gene fusion, or gene rearrangement) were detected in 2 mL of blood sample. 11.1.4 Reverse transcription polymerase chain reaction (RT-PCR) 11.1.4.1 Detect CTCs with abnormal amplification/expression of specific biomarkers (such as EpCAM, CK19, etc.) in 2 mL of blood sample. 11.1.4.2 No CTCs with abnormal amplification/expression of specific biomarkers (such as EpCAM, CK19, etc.) were detected in 2 mL of blood sample. 11.1.5 Gene sequencing method 11.1.5.1 CTCs with specific gene mutations (such as EGFR, Her2, etc.) were detected in 2 mL of blood samples. 11.1.5.2 CTCs with specific gene mutations (such as EGFR, Her2, etc.) were not detected in 2 mL of blood samples. 11.2 Use of test results 11.2.1 Both the fluorescence in situ hybridization method and the reverse transcription polymerase chain reaction method (RT-PCR) should be met in the same experiment, otherwise the experiment is invalid and should be repeated. 11.2.2 The final results obtained by the 11.1 method should be used in combination with all clinical information from diagnostic tests (i.e. imaging, laboratory tests), physical examinations and complete medical history in accordance with appropriate sampler management procedures. 12 Precautions 12.1 Safety management Laboratory safety management and staff behavior comply with the provisions of GB19489. Staff should wear latex disposable gloves, masks and laboratory caps for all operations handling blood. 12.2 Anti-pollution of reagents and materials The laboratory should be divided into functional areas. The work clothes, laboratory tools and laboratory record books in each area should be distinguished and marked and should not be mixed. Pay attention to the pollution caused by droplets and dust caused by conversation or other activities. If necessary, operate in a clean bench. 12.3 Waste treatment The operation area should have special solid and liquid waste collection containers, and the waste should be treated harmlessly.

T/CSBM 0044-2023 history

2023 T/CSBM 0044-2023 Non-antibody-dependent magnetic separation detection method of circulating tumor cells in peripheral blood

T/CSBM 0044-2023Non-antibody-dependent magnetic separation detection method of circulating tumor cells in peripheral blood (English Version)

T/CSBM 0044-2023 history

T/CSBM 0044-2023
Non-antibody-dependent magnetic separation detection method of circulating tumor cells in peripheral blood (English Version)