Optical Fluidic Flow Cytometry For The Separation And Analysis Of Circulating Tumor Cells And Typing And Counting Detection

Liquid biopsy based on circulating tumor cells has become one of the hot topic and frontier fields in noninvasive diagnosis and real-time monitoring of tumors in recent years due to its advantages such as comprehensive molecular information,convenient sampling,low invasion,no radioactive contamination and low cost.CTCs is a collective name for all types of tumor cells in the peripheral blood,which escape from the tumor site and infiltrate the surrounding tissues and enter the blood,and reach other organs and tissues through blood circulation.CTCs is an important way of tumor metastasis.Studies have shown that CTCs appear before the formation of visible solid tumors,and their type and number are closely related to the progression of tumors.Therefore,at different stages of the disease,CTCs phenotypic count detection is helpful for cancer typing diagnosis,prognosis evaluation and efficacy monitoring.However,CTCs are rare,1 mL of peripheral blood may contain only a few to a few dozen CTCs,but there are about 1 million white blood cells and 5 billion red blood cells,making many traditional cell analysis methods no longer suitable.For example,flow cytometry(FCM)can take more than 24 hours to detect a sample,while automated digital microscopy(ADM)is difficult to screen for the effective number of such rare cells.Currently,most of the developed CTCs detection methods capture CTCs using epithelial cell adhesion molecule(EpCAM)antibodies coupled to magnetic beads or microfluidic chips,and then use immunofluorescence imaging or reverse transcription polymerase chain reaction(RT-PCR)to characterize CTCs.Some methods according to the tumor cells and normal cells in terms of size,density and other physical properties difference,including microporous filter,density gradient centrifugation,and the microstructure filtering based on microfluidic chip,the migration of inertia,deterministic methods of lateral migration to separatio n of CTCs,qualitative and then to collect CTCs to count.Although these approaches represent important advances in this area,technical bottlenecks remain for CTCs,which are highly biologically and physically heterogeneous.First,currently,single antibody capture dependent on epithelial marker EpCAM results in CTCs with low or no expression of EpCAM antigen being unable to becaptured,resulting in missed detection.Secondly,although the separation method based on physical properties is relatively simple,low cost and independent of cell surface markers,due to the heterogeneity of CTCs in physical properties,such methods tend to lose small CTCs,resulting in incomplete separation and low purity.More importantly,the current CTCs detection method with independent separation and analysis technologies is not only low in automation,tedious and time-consuming,but also easy to cause sample loss and pollution in cell transfer,container replacement and other links,leading to large systematic errors.Therefore,the development of sensitive,rapid,and can provide CTCs separation analysis and type counting and other functions of the new detection technology,CTCs detection research is an important topic.In this paper,microfluidic,optical,electronic and data processing technologies are integrated to develop a optofluidic flow cytometry,which automatically and continuously realizes the separation of CTCs in blood samples,3D focusing,real-time single-cell analysis and typing counting and other operations as well as sensitive and high-throughput detection.The paper consists of the following three parts:The first chapter,introduction,first introduces the significance of circulating tumor cell detection,then introduces the progress of circulating tumor cell detection and photofluidic flow cytometry,and puts forward the key scientific and technical problems.The second chapter,based on the current lack of sensitivity,high throughput analysis and classification was isolated from peripheral blood count CTCs instrument,build give a kind of integrated multiplex injection pump,multistage microfluidic chip,multi-parameter optical detection,data processing and system control module,such as optical flow control,flow cytometry instrument(Optofluidic Flow Cytometer,OFCM),numerical simulation and fluorescent microsphere mixture and added to the healthy blood cells test experiment,inspected the OFCM performance.This has laid a solid foundation for the subsequent applied research of clinical samples.The third chapter,using OFCM,CTCs isolated from blood samples of 15 patients with metastatic breast cancer and 5 healthy volunteers were analyzed and phenotypic counting,and has carried on the comparison with the CellSearch method.The resultsshowed that the method based on OFCM has the advantages of sensitivity and high throughput,which may provide a new device and method for CTCs liquid biopsy.