Construction Of Multichannel Membrane Enrichment Technology For Circulating Tumor Cells And Its Application In Breast Cancer

Objective: Based on the physical characteristics of Circulating tumor cells(CTC),a novel multichannel membrane enrichment technology for circulating tumor cells(MMET)was developed.And it was used to detect CTC in breast cancerMethods:(1)Establishment and optimization of MMET.Firstly,MCF-7 cells were selected to construct the CTC model of breast cancer patients.The effect of peristaltic pump flow rate on MMET cell recovery efficiency and leukocyte filtration rate was studied.Optimize staining method and specimen handling method.Then,the sensitivity and consistency of the device were studied,and the cells enriched on the membrane were analyzed and detected by pathological MGG staining and cell immunofluorescence.(2)Peripheral blood samples of clinical breast cancer patients were detected.92 patients with breast cancer as the research object were used to detect CTC on the filter membrane by MMET and immunofluorescence detection,count CTC,judge the type and immunophenotype of CTC,collect clinical data such as stage and tissue type,and conduct statistical analysis.The difference was considered statistically significant with P < 0.05.Results:(1)After optimization,the pressure pump flow rate was1ml/min,and the fixed concentration of methanol was 0.2%.The cell recovery was 86.00 ± 2.46%,and the filtration rate of leukocytes was96.54 ± 2.74%.(2)The MMET device can process 24 samples independently at the same time,with good consistency between channels.The detection range of the device was 0-200 cells /m L,and the detection sensitivity can reach 1 cell/ml.(3)CTC was detected in 39 of 92 patients,with a positive rate of 42.4%,including sCTC and CTC cluster.The positive rate of CTC,the number of sCTC and CTC clusters had no significant correlation with clinical stage,receptor status,pathological type,BI-RADS grade,mulifocality ang maximum diameter of mass,but had correlation with molecular typing of breast cancer.The composition of CTC was only correlated with molecular typing of breast cancer.The immunophenotypes of sCTC and CTC clusters were significantly correlated with clinical stage,receptor status,molecular typing,pathological type and BI-RADS grade.More E-sCTC and E-CTC clusters were detected in stage Ⅰ～Ⅱ,HR+HER2-,Luminal type and BI-RADS4.(4)Patients with CTC clusters tend to have more sCTC counts,and the difference is statistically significant.Patients with CTC-WBC tend to have more CTC cluster count and larger CTC cluster,with statistically significant differences,but no significant correlation with more sCTC count.Conclusions:(1)In this study,the MMET device was successfully constructed.The device is convenient to operate,has high efficiency and has certain capture efficiency and sensitivity,and can separate sCTC,CTC cluster and CTC-WBC.(2)The separation and enrichment of CTC based on MMET has high auxiliary diagnostic value for breast cancer.