Application of ACEA Quanteon Flow Cytometry in Detection of Circulating Tumor Cell CTCs

Circulating Tumor Cells (CTCs) refer to tumor cells that are spontaneously or detached from the primary tumor or metastases of the solid tumor into the peripheral blood circulation, and are the key factors for tumor recurrence and distant metastasis. The detection of CTCs contributes to the early diagnosis, efficacy monitoring, prognosis and drug resistance evaluation of malignant tumors , and its clinical application value has been widely recognized [1, 2] . Due to the rarity of CTCs, flow cytometry requires higher sensitivity for the identification of CTCs.

experimental design

CTCs content in the patient's blood is very rare, about every 10 6 to 10 7 leukocytes to only a few CTCs present, the sensitivity has been a major obstacle to testing and application. In many literature reports, tumor cell incorporation assays are often used to evaluate the sensitivity, specificity, accuracy, and repeatability of CTCs detection methods/systems.


In this study, colon cancer cell line (SW620) with high expression of EpCAM was selected as the incorporation cell (Fig. 1). In the first part, different numbers (0-256) of SW620 cells were mixed into 1×10 6 normal human PBMCs. The ACEA Quanteon flow cytometer detects the actual number of SW620 cells and determines the sensitivity of the instrument for detecting CTCs.


I. Identification of EpCAM on the surface of SW620 cells

Figure 1. Identification of EpCAM expression on SW620 cell surface. Fresh blood (2 mL) or fresh blood ( 1000/2 mL) spiked with SW620 cells were isolated from PBMCs with Ficoll-Paque PLUS lymphocyte separation (GE healthcare, 17-1440-02) , in the above samples and SW620 cells alone. The anti-CD45-FITC and anti-EpCAM-APC antibodies were separately incubated, and the isotype antibody control was set up and tested on the ACEA Quanteon flow cytometer. The data were analyzed by the same gate strategy. a. Results of staining of SW620 cells with IgG1 FITC and IgG2b APC isotype antibody; bd. Results of staining of SW620 cells, fresh blood, and fresh blood spiked with SW620 with CD45-FITC and EpCAM-APC antibodies.


Second, ACEA Quanteon can detect 1 tumor cell in 10 6 normal human leukocytes


Figure 2. Sensitivity analysis. Collect SW620 cells in logarithmic growth phase, label EpCAM-APC fluorescent antibody, set isotype antibody control, incubate on ice for 60 min, wash twice with D-PBS containing 0.5% BSA; press stained SW620 cells to 0, 2, 4,8,16,32,64,128,256,512 (20 μL in volume) were mixed into 2×10 6 PBMCs (180 μL in volume) and vortexed (theoretically, the number of SW620 cells per 100 μL) :0,1,2,4,8,16,32,64,128,256), at ACEA   100 μL was collected at a medium speed on a Quanteon flow cytometer, and the number of EpCAM+ cells was recorded . A. Set the door strategy. The F1 gate is set on the FSC-SSC scatter plot to remove debris and miscellaneous signals. Based on the P1 gate, the cells expressing EpCAM+ on the scatter plot are the SW620 cells incorporated; B. Test results on the ACEA Quanteon flow cytometer ;

 

in conclusion
The results of ACEA Quanteon flow cytometry showed that there was a good linear relationship between the number of cells actually detected and the theoretical number at different levels of cell incorporation. The sensitivity was 0.0001%, and 10 6 normal human leukocytes could be detected. One of the tumor cells demonstrated that the ACEA Quanteon flow cytometer has high sensitivity in detecting CTCs.

references

[1] Progress in detection and clinical application of circulating tumor cells. Zhao Qianwen, Situ Bo, Zheng Lei. Journal of Southern Medical University, 2017, 37(10): 1423-1426

[2] Research progress in circulating tumor cells. Wang Yiwen, Hu Kongwang. Journal of Anhui Medical University, 2017, 52(12): 1897-1900

* Article from Essen Bio

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