The Negative Enrichment Method And Telomeraste Detecticn Of Tumor Cells In Malignant Pleural Effusions

ObjectiveThe purpose of this study was to explore a new way to detect tumor cells of pleuraleffusion with the negative enrichment by immunomagnetic beads and fluorescence in situhybridization (FISH) method, and to establish its standardization process, to improve thedetection rate of MPEs, and to provide a new approach for benign and malignant pleuraleffusion, further more, to clarify the influence of WBC on telomerase activity in pleuraleffusions.Methods1、Experiments specimens were collected from patients with benign or malignantpleural effusions, including17benign samples and36malignant samples.2、Tumor cells were enriched from malignant pleural effusion samples by means ofthe immunomagnetic beads negative enrichment method and density gradientcentrifugation method, followed by identification with cytology analysis (Wright’sGiemsa’s staining), immunofluorence staining (IF) and fluorescence in situ hybridization(FISH). Then we compared the sensitivity and specificity of tumor cells detection usingimmunomagnetic beads negative enrichment method and density gradient centrifugation.Five, ten, twenty, fifty and one hundred A549(lung adenocarcinoma) cells were labeledwith DAPI and added into20ml pleural effusions (containing1-10×106cells) from heartfailure patients, followed by the immunomagnetic negative enrichment method. Recoveredcancer cells were enumerated using a fluorescent microscope.3、TRAP-PCR-ELISA method was used to examine telomerase activity of tumor cellsenriched by the negative enrichment method and density gradient centrifugation method.Results1、Using negative enrichment method and density gradient centrifugation combinedwith cytology analysis, the positive rates of tumor cells in36malignant pleural effusionsamples were80.55%(29/36) and58.33%(21/36), respectively (2=4.00, P=0.039).Using negative enrichment method combined with IF, the positive rate was100%.Moreover, using negative enrichment method combined with FISH analysis, the positive rate of tumor cells was86.11%(31/36), it was much higher than that using density gradientcentrifugation combined with cytology analysis (2=5.818，P=0.012). In17cases ofbenign pleural effusions, using negative enrichment method combined with IF, the positiverate was100%. But we didn’t find cancer cells from benign pleural effusions using othermethods. After DAPI staining and mixing with right amount pleural effusions from heartfailure patients, the cell recovery rates of A549(lung adenocarcinoma) cells evaluatedunder fluorescence microscope were75%,78%,82%,85%,88%, the average recoveryrate was81.6%.2、The result of TRAP-PCR-ELISA test was shown below: In ten MPE samples,seven MPEs had telomerase activity after negative enrichment method enrichment, butthree MPEs had telomerase activity after density gradient centrifugation enrichment. Infive benign pleural effusion samples, they didn’t show telomerase activity after the twomethods enrichment. But there is no significant difference in two methods (2=2.25,P=0.125). However, using relative telomerase activities (RTA) calculation, the telomeraseactivity of MPEs after negative method enrichment is higher than they enriched bygradient centrifugation (P=0.007). In five benign pleural effusion samples, there is nosignificant difference between the two methods (P=0.08).ConclusionIt is practicable to enrich tumor cells from pleural effusions using negative enrichmentmethod by immunomagnetic beads. This method combined with cytology analysis or FISHobviously enhanced the sensitivity and specificity of tumor cells detection in pleuraleffusions. It is of great significance for distinguishing malignant from benign pleuraleffusions. But it is difficult to distinguish cancer cells from mesothelial cells usingimmunofluorence staining with CK, DAPI and CD45label. We should find more specificmarker to recognize tumor cells from pleural effusion.The detection of tumor cells telomerase activity after negative method enrichmentdepleted the influence of WBC to tumor cells’ telomerase activity, and improved thedetection sensitivity and specificity. This is beneficial to distinguish malignant pleuraleffusions from benign ones. The amount of WBC in pleural effusions will inhibittelomerase activity of tumor cells to a certain extent.