Establishment Of Capture And Identification Method For Breast And Colorectal Cancer Circulation Tumor Cell

ObjectiveThis research used the method of density gradient centrifugation and cytospin to enrich tumor cells in peripheral blood.Then,Giemsa staining was utilized to observe cell morphology combined with immunohistochemistry and immunofluorescence to identify whether the captured cells were tumor cells according to the specific molecular on cell surface.Hence,a more simple and convenient method of capture and detecting circulation tumor cells was likely to be established.Methods:1.Enrichment:The method of density gradient centrifugation and cytospin were used to enrich tumor cells in peripheral blood.2.Identification:Giemsa staining was used to observe cell morphology combined with immunohistochemistry and immunofluorescence to identify whether the captured cells were tumor cells according to the CK and EpCAM on cell surface.3.Recovery rate:Peripheral blood added with about 10,50 and 100 MCF-7or SW-620 turnor cells respectively to test recovery of the method.Experimental group,positive control group and negative control group were established to establish the new detecting method.1ml peripheral blood from breast or colorectal cancer patients was used.And the samples of positive control group and negative control group were from healthy persons,1ml peripheral blood added with given number of MCF-7 cells and 1ml peripheral blood respectively.Results:In positive control group,10 of 10 samples were identified to have tumor cells by Giemsa staining,immunofluorescence and immunohistochemistry.In negative control group,2/10 samples were identified to have large cells by Giemsa staining,lof 10 samples was detected anti-CK and anti-EpCAM positive cells by immunofluorescence,however,the size of detected cells was no larger than WBC.And no samples were detected positive by immunohistochemistry in negative control group.It meant high sensitivity and specificity.In the experimental group,large cells seemed like tumor cells can be seen in all of the patients.Anti-CK or anti-EpCAM positive cells can be seen in 10 of 10 screamed patients using immunofluorescence method.Strangely,anti-CK positive cell was seen only in one advanced colorectal cancer patient,but the size of positive cell was no more larger than white blood cell.The mean recovery rate was 88.8%in the tumor cell recovery test.ConclusionsIn this research,density gradient centrifugation and cytospin combined with Giemsa staining,immunofluorescence and immunohistochemistry was used to enrich and identify tumor cells in peripheral blood from healthy persons added with tumor cells and blood from breast and colorectal cancer patients.It showed satisfactory results in positive group,negative control group and recovery test which revealed high sensitivity and specificity of this detecting method.It deserved more research to perfect the method for applying to clinical use in the future.