Methodology Of Colorectal Exfoliated Cells In Feces In The Colorectal Cancer Screening

Background and Objective:Colorectal cancer is one of the most commonmalignancies in the world. The morbidity of colorectal cancer ranks second only togastric cancer and esophageal cancer among gastrointestinal malignancies. Significantclinical symptoms always could be found not in the early but the advanced stages ofcolorectal cancers. Removal of the early stage tumors could usually lead to betterprognosis. Therefore, it is crucial of early dectection, diagnosis and treatment toimprove the survival rates of the population with colorectal cancers.Currently, themethods of colorectal cancer screening mainly include fecal occult blood test (FOBT),barium enema, and colonoscopy, etc. FOBT is the most commonly used method despiteits low sensitivity and specificity, as well as high rates of false-positive andfalse-negative. Colonoscopy is the gold standard method in the screening and diagnosisof colorectal cancers but the invasiveness and high expenses impede it to be thepreferred screening method.Compared with these conventional methods, thescreening/detection of colorectal exfoliated cells in stool can be recognized as a novelmethod in）diagnosis of the colorectal cancers for its high specificity, high sensitivityand non-invasiveness. In order to improve the extraction rate of exfoliated cells fromstool and simplify the extraction process, the research aims to improve the originalexfoliated cells extraction process, emphasizing the theory and its application process inthe tri-layer screening method and the method of nano-magnetic beads enrichment ofexfoliated cells. This thesis also evaluates the feasibility and diagnosis efficiency ofthese methods, hoping to increase their practical applicability and expand in real clinicalapplication. Methods:1.The evaluation and significance of tri-layer method in extracting exfoliated cells.131patients were enrolled randomly with51colorectal cancers,43colorectaladenomas,16ulcerative colititises and21controllers (with negative colonoscopy)according to the colonoscopy results. Three stool examples were taken from eachsubject and were given different treatments. Through the improved centrifugal washingmethod and tri-layer method, the exfoliated cells were extracted and given HE staining.Immunol FOBT was done in each subjects.the observation under the HE stain.2.The evaluation and significance of nano-magnetic beads enrichment in extractingexfoliated cells.2.1The detection of the magnetic beads enrichment efficiency in the colorectalcancer cell cultured in vitro.Preferably a nano bead enrichment were cultured in vitro colon cancer cellsHCT116, and repeatedly test validation to find the maximum enrichment of cells, theoptimal reaction conditions.2.2The nanobeads enrichment verification experiment of colorectal cancer inpatients with fecal shedding cells38routine colonoscopy and confirmed by pathology of colorectal cancer patientsand18healthy controls were collected for each patient will naturally and bowel willnano-bead enrichment off the cell extraction of exfoliated cells, HE staining and lightmicroscopyunder observation; and each patient immunoassay fecal occult blood test.Results:1.The results of improved centrifugal washing method and tri-layer screeningmethod in extracting exfoliated cells.In51stool examples taken from51colorectal cancer patients, malignance cellswere detected in35examples through tri-layer screening method, with a positive rate of68.63%(35/51). Malignance cells were detected in39examples through improvedcentrifugal washing method and the positive rate was76.47%(39/51). The positive ratewas80.39%（41/51）through FOBT.(X~2=8.956,X~2=2.981,P <0.05).2.The evaluation and significance of nano-magnetic beads enrichment in extractingexfoliated cells.2.1The detection of the magnetic beads enrichment efficiency through culturingthe colorectal cancer cell in vitro.Comprehensive enrichment cell number and HE staining read the piece, the optimal reaction conditions as follows: take100ul colorectal cancer cells HCT116suspension (10×105/ml), by adding40ul coated nanobeads③high sugar compounds,37℃water bath and incubated for60min, and will be equipped with the incubation,beads-a centrifuge tube and magnet of the cell suspension adherent reaction time was15min.2.2The nanobeads enrichment verification experiment of colorectal cancer inpatients with fecal shedding cells38cases of colorectal cancer patients, using nano-bead enrichment method in anatural stool in colorectal cancer detection sensitivity of65.79%(25/38), the site of theatypical cells positive rate of colorectal cancer tissue related(P <0.05), Dukes stage andhistological differentiation were unrelated (P>0.05). In the clear intestinal colorectalcancer detection sensitivity of71.05%(27/38).18cases of healthy people group, fall offthe cytology of colorectal malignancies and specificity of100%.No significant difference could be found between the nano-magnetic beads andtri-layer method and FOBT test in the sensitivity of CRC detection (X~2=3.167,X~2=0.457,P>0.05). however, significance could be found between the two methods inthe specificity of CRC detection (X~2=6.278,X~2=8.418,P﹤0.05).With regard to the HE staining, the exfoliated cells gathered by nano-magneticbeads enrichment method were evenly dispersed and the background was clear withminimal amount of stool, which did not affect the observation of cells.Conclusions:1. There are two improvements in extracting exfoliated cells in stool.(1) Theapplication of tri-layer screening method in extracting exfoliated cells in stool couldsimply the extraction process and could be used in large scale clinical application.(2)The establishment of nano-magnetic beads enrichment of exfoliated cells which is, tosome extent, creative and valuable for its high recovery rate of cells, high specificity,and clear smear background.2.The application of tri-layer screening method and nano-magnetic beadsenrichment method of exfoliated cell is easyly to handle and dependable, whose couldgreatly improve the detection rate of colorectal cancers.