A novel method for detection of circulating tumor cells in peripheral blood of patients with lung cancer using immunomagnetic beads device

Abstract

Objective To establish a novel method for detecting circulating tumor cells (CTCs) in phripheral blood of lung cancer patients with high sensitivity and specificity. Methods Experimental study.42 cases of initial treatment patient who underwent resection and diagnosed to be non-small cell lung cancer by biopsy were studied,including 7 patients at stageⅠ,9 patients at stageⅡ,16 patients at stageⅢ and 10 patients at stageⅣ. As a control group,20 cases of healthy volunteers were selected.A series of experiments was conducted to determine the efficiency of tumor cells isolation,in which varied concentration (50,100,200,500,1000 cells) of A549 cells spiked into 2 ml peripheral blood drawn from healthy donors.The blood was removed of unwanted erythrocytes by lysis buffer,and made the rest of nucleated cells incubate with anti-EpCAM magnetic beads,then separated and enriched by a specific detector.All epithelia cells were retained on a slide because of a magnetic force and identified by H&E staining protocol.On the basis of cell recovery rate we calculated the sensitivity of tumor cells isolation.20 blood samples taken from healthy individuals were also detected to validate the specificity of this method.Samples of 42 patients with lung cancer were assayed for CTCs detection by above method.The correction of CTCs quantity with the patients′clinical features,for example,ages,gender,clinical stage,tumor size was analyzed in lung cancer patients by chi-square statistics.The correction of recovery cells with the spiked cells were assayed by linear correlation. Results The recovery rate was ranging from 68% to 82% by spiking varying numbers of A549 lung cancer cells into 2ml blood samples of healthy volunteers.Regression analysis of number of recovered vs. spiked A549 cells yielded a regression equation of Y=0.6419X+8.8875. The number of CTCs detected has signification correlate with the cells spiked (R2=0.9916,P 0.05),has signification with the clinical stage (P<0.05).None of the peripheral blood samples of the 20 healthy subjects analyzed was found to have CTCs. Conclusions This novel immunomagnetic separation technology is a sensitive and specific method,which provides a new tool allowing for feasible and specific detection of CTCs in lung cancer patients.The level of CTCs increases with the clinical stage and tumor size increased,which has important value to discover the early stage micrometastasis and redefine the clinical stage.But further multicenter and large sample clinical research are needed to confirm its clinical value.(Chin J Lab Med,2013,36:727-732) Key words: Lung neoplasms; Neoplasm circulating cells; Immunomagnetic separation; Antigens; neoplasm; Cell adhesion molecules