Establishment Of Micronucleus Test And Its Application In Cancer Diagnosis

Objective:1.To establish a routine culture method for micronucleus detection.To establish the dose-response curve of micronucleus induced by X-ray irradiation in human peripheral blood lymphocytes in vitro.2.To establish an effective and simple anti centromere antibody staining staining method for the detection of aneuploidy agents.3.Micronucleus test was applied to clinical detection of cancer patients,observation and analysis of gastric cancer,thyroid cancer,colon cancer and total cancer patients peripheral blood lymphocyte micronucleus rate,and analyze the correlations between micronucleus rate and CEA,CA19-9 expression in gastric cancer group and colon cancer group,as well as the correlations between micronucleus rate and FT3,FT4,TSH levels in thyroid cancer group.4.To meta-analyze the diagnostic performance of micronucleus rate in conforming cancers.Methods:1.Different dose(0.00-5.00Gy)of X-ray was utilized to irradiate human peripheral venous blood.The micronucleus rate of peripheral blood lymphocytes was detected by routine culture of whole blood,and the dose-response curve of micronucleus rate and irradiation dose was plotted.2.The micronucleus of peripheral blood lymphocytes induced by vincristine(0.01μg/ml,0.02μg/ml,0.04μg/m and 0.08μg/ml)was detected by the modified crest staining method.3.The micronucleus rate of peripheral blood micronucleus cells(‰)was observed and analyzed by micronucleus test in patients with newly diagnosed thyroid cancer,colon cancer and stomach cancer,and normal healthy people were included as the control group.The correlations between micronucleus rates of gastric cancer group and colon cancer group and CEA,or CA19-9,as well as the correlations between micronucleus rate of thyroid cancer and FT3,FT4 or TSH was analyzed by using SPSS 25.0 software.4.The online Medline/Pub Med and other databases were extensively searched to obtain the studies that documented the diagnostic value of micronucleus rate in cancers.Stata 12.0 and meta-cisc1.4 were used to analyze the extracted data.The combined indexes included sensitivity,specificity,positive likelihood ratio(PLR),negative likelihood ratio(NLR),diagnostic ratio(DOR)and area under receiver operating characteristic curve(ROC),etc.Spearman correlation coefficient,Cochran-Q test and I2 test were used to evaluate the heterogeneity among studies;sensitivity and meta-regression analyses were used to trace the potential sources of heterogeneity;Deeks funnel plot was used to evaluate the potential publication bias among the pooled effects.Results:1.Under five different doses of 0.00,0.50,1.00,3.00 and 5.00Gy,the micronucleus rate of peripheral blood lymphocytes increase with the increase of dose.The best fitting equation of 0.00-5.00Gy is as follows:y=3.00+36.786D~2.2.The micronucleus rate of peripheral blood lymphocytes increase with the increase of VCR concentrations(0.00μg/ml,0.01μg/ml,0.02μg/ml,0.04μg/ml and 0.08μg/ml respectively).The percentage of centromere positive’s micronuclei induced by VCR was about 70-75%by CREST method.3.The micronucleus rates of lymphocytes in peripheral blood of cancer group,colon cancer group,gastric cancer group and thyroid cancer group were significantly different from those of healthy control groups(all with P<0.01).The micronucleus rate of cancer patients group was higher than that of healthy control group.The AUC of the area under the ROC curve of colon cancer group,gastric cancer group,thyroid cancer group and total cancer group were 0.78,0.70,0.84 and 1.0,respectively.In the correlation study,the correlation coefficients between micronucleus rate and CEA and CA19-9 in the colon cancer group were 0.558 and 0.578,respectively,showing a moderately positive correlation.The correlation coefficients between the micronucleus rate and CEA and CA19-9 in the gastric cancer group were 0.781 and 0.314,respectively.That is,the micronucleus rate of patients with gastric cancer was highly positively correlated with CEA,but not with CA19-9.The micronucleus rate in the thyroid cancer group was not correlated with FT3,FT4,and TSH.4.There were 5 articles about the diagnostic value of micronucleus in cancer.The pooled sensitivity,specificity,PLR,NLR,DOR and AUC of micronucleus in conforming cancers were 0.71,0.80,3.70,0.48,9.45and 0.8096,respectively.Conclusion:1.The micronucleus was detected by the whole blood micro routine culture method,and the dose-response curve of micronucleus of human peripheral blood lymphocytes in vitro induced by X-ray was established,and the curve has a good fit.2.By detecting the micronucleus induced by vincristine,an effective and simple CREST staining method for the detection of aneuploidy agent was established.3.Cancer(including colon cancer,stomach cancer and thyroid cancer)is closely related to the increase of micronucleus.There is a certain correlation between the micronucleus rate of colon cancer and the tumor markers CEA and CA19-9,and there is a high correlation between the micronucleus rate of gastric cancer and the tumor markers CEA.The combined detection of micronucleus and tumor marker can be used as a new biological monitoring method for cancer.4.The meta-analysis for the first time showed that micronucleus has a high diagnostic value in the diagnosis of cancer,which can be used as a new biomarker for the diagnosis and prediction of cancers.