Optimization Improved Iset(CTC-Biopsy) Detection System And Verify The Effect Of The Detection Of Circulating Tumor Cells In Cancer Patients

Background:Definition of circulating tumor cells(Circulating Tumor Cells, referred CTCs) for the invasion and metastasis of solid tumors or detached from the present in circulating tumor cells in a class, it is closely related to tumor recurrence and metastasis. Clinical studies have shown that peripheral blood circulation is closely related to the number of tumor cells in cancer patients with clinical stage of disease status. However, due to the number of rare CTCs, its research must first be enriched membrane filtration separation technology(Isolating by size of epithelial tumor cells, ISET) technology uses tumor cells than normal blood volume is usually large, as well as the principle of differential cell deformability, the large size of cells trapped in the filter membrane surface.Objective:Earlier our research group Improvement of ISET Technology, and the development of automated testing equipment CTC-Biopsy. The purpose of this experiment is to improve and optimize the CTC-Biopsy detection system, and to verify the effect of CTC in the peripheral blood of tumor patients, and to explore the value of clinical application.Methods: Department of tumor cell A549, Fa Du, MB-MDK-468, PC3, PAN-2, MGC803, SMMC7721, respectively, using Millipore filters and CTC-Biopsy membrane and optimizes the CTC-Biopsy detection system from the following 4 aspects:1)Study on the effect of the application of a variety of tumor cell line cells by CTC-Biopsy detection system;2)Study on the effect of cell staining by using a variety of tumor cell lines in the simulation of CTC-Biopsy system;3)Using a variety of tumor cell lines to simulate the detection of tumor cells in the peripheral blood by CTC-Biopsy detection system.4)Using a variety of tumor cell lines to simulate the stability and reproducibility of tumor cells in the detection of tumor cells by using CTC-Biopsy detection system. 2 the effects of high speed centrifugation on CTM detection results were studied using a single and group CTC-Biopsy(SMMC7721) tumor cell line. 3 the accuracy of CTC and CTM in the peripheral blood of tumor patients was detected by CTC-Biopsy assay. 4. According to China’s State Food and Drug Administration(CFDA, China’s State food and Drug Administration) clinical trial management specification(GCP, good clinical practice) requirements using CTC-Biopsy detection system of 156 cases of tumor patients and 22 healthy volunteers in clinical trials.Result: 1.1) after the tumor cell line was detected by CTC-Biopsy staining, the nucleus and cytoplasm of the tumor cells were obvious, and the structure of the cell nucleus was clear, and the pathological diagnosis was reached.Respectively, using Millipore and CTC-Biopsy, compared with the, it is found that the CTC-Biopsy filter is more clear, and the transmittance is better than the Millipore filter. 2) CTC-Biopsy detection system of respectively using millipore and CTC-Biopsy membrane experimental simulation results showed that the tumor cells into the blood after after staining with CTC-Biopsy membrane filtration background of clear, membrane surface and only a small number of lymphocytes residue, does not affect the judgement of the results; Tumor cell nucleus, slurry apparent contrast, nucleus structure clear, reach the cell pathology diagnosis standard, CTC-Biopsy membrane compared to Millipore filters has higher transmittance, convenient microscopic observation, membrane background more clearly. 3) immunocytochemistry was used to calculate draw the that CTC-Biopsy detection system using CTC-Biopsy filter to detect the amount of different types of tumor cell lines cell retention rate were above 85%, and the application of Millipore filters obtained interception rate was more than 70%. 4) CTC-Biopsy detection applications CTC-Biopsy membrane detection for a number of different tumor cell retention rate of 66.7-90.4% higher than when using the Millipore filters 58.3%-78.5%, CV value 4.8-12.4% 2.9-10.8% below using Millipore filters. 2. In the simulation environment of human blood, cells of a single tumor after CTC-Biopsy detection system after blood sample pretreatment process, do not gather produce clusters of tumor cells; combined with high speed centrifugal CTC-Biopsy detection system for detecting steps lead to simulation experiment conclusion CTM scattered: clusters of tumor cells in centrifugal pretreatment process was scattered.The reasons may be: cultured into clusters of tumor cells relative adhesion is loose and the actual CTM has a certain gap;(2) high speed centrifugal may cell clusters scattered; 3 tumor cells are not fixed three or five cell as a group,so in the count may exist certain errors. But this also deduce that if application CTC-Biopsy detection system in tumor patients blood samples to check into clusters of tumor cells that CTM, it can be concluded that the adhesion between these cells are very close, with characteristics of tumor thrombus, or has the potential to become cancerous thrombus. We infer that detected in the peripheral blood of patients with tumor of CTM is not because CTC-Biopsy detection system produced, but patients themselves in the process of occurrence and development of tumor tumor cell aggregation adhesion arising. 3. The experiment results show that cells intercepted by CTC-Biopsy detection system. According to the morphological judgment criteria of CTC, CTM, have received immunocytochemistry confirmed that cell morphology judgment standard can be used for the judgment of the CTC, CTM; CTC-Biopsy detection system staining and immunofluorescence staining can detect the occurrence of EMT CTC, CTM. 4. clinical trials were detected in a sample of 178 cases, the real effective samples for 172 cases, including 22 cases of healthy subjects, tumor subjects 150 cases, 133 cases were male, 45 cases of women, age range 25-81 years, including 11 cases of lung cancer, esophagus carcinoma in 28 cases, 44 cases of gastric cancer, colorectal cancer in 46 cases, 9 cases of liver cancer, renal cell carcinoma in 12 cases. CTC-Biopsy detection system detection sensitivity of disease of malignant diseases: 51.33%, specificity: 100%, false positive rate: 0.00%, false negative rate: 48.67%, the positive predictive value: 100% and negative predictive value of 23.16%, Youden index: 0.513. Regardless of diseases in stage IV patients with stage I- III stage were detected rate had no significant difference; in addition to the outside of the stomach the disease the stage between the detection rate was no significant difference; in patients with stage IV gastric cancer detection rate was significantly higher than that of the patients with I- III stage(P = 0.002). The detection rate was 51.33% in this clinical trial, and the detection rate was 27.91% higher than that of the optimized detection procedure(gradient centrifugal pretreatment blood sample). CTC-Biopsy detection system test specimens were stained with clean background, clear cell morphology, cell nucleus and cytoplasm clear boundaries; except for abnormal cells, sample only residual small amounts of mononuclear cells. The 95%CI detection rate of CTC detection system is 43.33%, which is higher than that of the ISET 43.06%.Conclusion: 1 CTC-Biopsy detection system of the cell staining effect to achieve the requirements of cell pathology diagnosis, recovery rate reached the international level of similar detection system, and the stability is good, the repeatability is strong. 2.CTC-Biopsy detection system was not produced in the process of CTM; tumor patients in the peripheral blood CTC and CTM part of the cells in the presence of EMT phenomenon. 3 the nature of CTC-Biopsy and CTC in the peripheral blood of tumor patients was detected by immunofluorescence assay, and the correctness of the criterion of the morphology was confirmed by CTM assay; 4. The GCP clinic trail confirmed CTC-Biopsy detection system for detecting cancer patients peripheral blood in CTC sensitivity, specificity and other indicators have reached the international similar detection system level; the detection system is easy to operate and convenient, with efficient, stable and economical advantages, with good clinical value.