Detection Of Circulating Tumor Cells With Cell Filtration-Laser Scanning Cytometry

Objective:To establish a kind of method of cell filtration(isolated by the size of epithelial tumor cells,ISET)conbined with laser scanning cytometry(LSC)to detect circulating tumor cells(CTC)in patients with cancer.Methods: (1) ISET (Filtrated the sample with the polycarbonate membrane containing cylindrical pores with 8 um in diameter)was used to separate the tumor cells, to react with the mixed liquor of Cytokeratin 8(CK8) and leukocyte common antigen(LCA)CD45 for 45 minutes, and then to be dyed with 4,6-diamidino-2- phenylindole(DAPI),and finally to be scanned and analyzed by LSC, selecting the cells of CK8+ and CD45-, and relocating and confirming tumor cells visually.(2) Tumor cells(MCF-7) were mixed into the blood sample from healthy volunteers in different proportions ,detected to ascertain the recovery, the sensitivity and the specificity.(3)To examine 50 advanced(stageⅣ) tumor patients(including 25 advanced breast cancer patients) with metastasis,22 early (stageⅠ-Ⅲ) tumor patients (including 20 breast cancers)who can be operated on and 30 healthy volunteers for control, the correlation was analyzed between the number of CTCs and clinical factors such as clinical stage, the size of tumor and lymphatic metastasis ect .Results: (1)It was confirmed that the polycarbonate filtration membrane with pores of diameter of 8um can filter 99% normal leukocytes while keeping 97% tumor cells. The CK8 antibody was specific to tumor cells and CD45 to leukocytes(both exceeding 99%).We could discriminate initially the positive cells after scanning and analyzing by LSC and finally confirm the tumor cells according to their morphological properties.(2) The repeated sensitive experiments confirmed that the recovery rate of mixed tumor cells with this method remained about 10.34±1.15 and the result kept stable when the number of the mixed cells was between 60 and 600000(P>0.05), We could detect 1 tumor cell in 1 ml peripheral blood while no positive cells were detected in healthy control, showing the high sensitivity and specificity of the method.(3)In the clinical specimen, 39 samples(78%) in 50 patients of stageⅣand 11 samples(50%) in 22 patients of stageⅠ-Ⅲwere detected with the CTCs . However, CTCs were not(0.00%)detected in all 30 healthy samples by control. The detection rate of CTCs in tumor patients was higher than that in 30 healthy volunteers (P<0.05), and the detection rate of CTCs in 50 patients of stageⅣwas higher than that in 22 tumor patients of stageⅠ-Ⅲ(P<0.05).(4)For 45 breast cancers, we found the more advanced the stage was ,the bigger the number of CTCs. The number of CTCs(8.60±1.39)of 25 breast cancers in stageⅣwas bigger than that(3.45±1.08) of the patients in stageⅠ-Ⅲ(P<0.05), and the number of CTCs(8.44±1.33) of the patients in stageⅢ-Ⅳwas bigger than that(3.11±1.05) of the patients in stageⅠ-Ⅱ(P<0.05).Also ,the number of CTCs(7.63±1.26) of patients with lymphatic metastasis was bigger than that(2.80±1.46) of the patients with no lymphatic metastasis, but the number of CTCs had no significant difference respectively between the patients with different primary tumor size (T) , the different states of ER and PR, and the different express levels of HER-2(all of P>0.05).Conclusions:(1)The method to detect CTCs with cell filtration- laser scanning cytometry was convenient with high sensitivity and good specificity.(2)We could detect CTCs not only in most advanced tumor patients and but also in tumor patients with no metastasis ,indicating the method could be used to detect CTCs and evaluate the treatment (chemotherapy for example) in advanced tumor patients and even the detection of CTCs and treatment evaluation for early stage tumor patients.(3)The correlation between the number of CTCs and the clinical stage of breast cancers and lymphatic metastasis suggested that number of CTCs could be used as a judgment criterion of prognosis for breast cancers.