Aim:To establish a standardized protocol for the isolation and enumeration of circulating tumor cells(CTCs)from peripheral blood of patients with metastatic breast cancer.Methods:The protocol used tumor cells spiked i...Aim:To establish a standardized protocol for the isolation and enumeration of circulating tumor cells(CTCs)from peripheral blood of patients with metastatic breast cancer.Methods:The protocol used tumor cells spiked in a lymphoid cell line with detection by flow cytometry and quantitative reverse transcription polymerase chain reaction(QRT-PCR).Cells of the human mammary cancer subtypes were spiked into Jurkat cells,which served as the lymphocyte designate in numbers from 10 to 500 per 105 Jurkat cells.This mixed population was probed for CD45,EpCAM,and pancytokeratin acquired from flow cytometry and characterized by microscopy.QRT-PCR was done for CK-19,MUC-1,EpCAM,and GAPDH.Validation was attained with blood samples from 22 patients with metastatic breast cancer and 20 healthy individuals.Results:Flow cytometry could detect 1 breast cancer cell per 100,000 Jurkat cells,with similar detection levels in the breast cancer subtypes.Samples from patients with breast cancer showed a range of CTCs from 1-85 per 10 mL of blood.Quantitation of expression for EpCAM,CK-19,Muc-1,and Her2neu confirmed the presence of CTCs in 76%of samples.Conclusion:Density gradient and immunomagnetic enrichment accomplished isolation of CTCs and quantitation was achieved using flow cytometry.Combined QRT-PCR and imaging further validated these findings,rendering a robust methodology.展开更多
文摘Aim:To establish a standardized protocol for the isolation and enumeration of circulating tumor cells(CTCs)from peripheral blood of patients with metastatic breast cancer.Methods:The protocol used tumor cells spiked in a lymphoid cell line with detection by flow cytometry and quantitative reverse transcription polymerase chain reaction(QRT-PCR).Cells of the human mammary cancer subtypes were spiked into Jurkat cells,which served as the lymphocyte designate in numbers from 10 to 500 per 105 Jurkat cells.This mixed population was probed for CD45,EpCAM,and pancytokeratin acquired from flow cytometry and characterized by microscopy.QRT-PCR was done for CK-19,MUC-1,EpCAM,and GAPDH.Validation was attained with blood samples from 22 patients with metastatic breast cancer and 20 healthy individuals.Results:Flow cytometry could detect 1 breast cancer cell per 100,000 Jurkat cells,with similar detection levels in the breast cancer subtypes.Samples from patients with breast cancer showed a range of CTCs from 1-85 per 10 mL of blood.Quantitation of expression for EpCAM,CK-19,Muc-1,and Her2neu confirmed the presence of CTCs in 76%of samples.Conclusion:Density gradient and immunomagnetic enrichment accomplished isolation of CTCs and quantitation was achieved using flow cytometry.Combined QRT-PCR and imaging further validated these findings,rendering a robust methodology.
