Detection And Clinical Significance Of Circulating Tumor Cells In Peripheral Blood Of Renal Cell Carcinona Patients Using Acridine Orange Fluorescent Method

ObjectiveExplore the screening method of circulating tumor cells(CTCs) staining with acridine orange florescence(AO-F) and its application in renal cell carcinoma(RCC) patients. Then evaluate its clinical application value of CTCs detection in patients with RCC. Methods1. Culture primary renal tumor cells and 769-P line and observe the difference of morphology and staining between them after AO-F staining. Then prepare two group cells of dead(75 ℃30min) and live(PBS 30min) and they were observed under fluorescence microscope after AO-F staining. Estimate the sensibility of AO-F dye with tumor cells by calculating AO-F positive staining cells in 100 dyed cells under 5 random microscope visions(×200).2. 5ml morning fasting venous blood of all controls was obtained to enrich nucleated cells. The CTCs model of renal cancer was established when known numbers of tumor cell(500、200、100、50、10/tube) respectively were mixed with 106 nucleated cells which were used to evaluate AO-F method’s sensitivity and reproducibility.3. 10 benign renal disease and 139 RCC patients(112 early renal cell carcinomaERCC and 27 metastasis renal cell carcinoma MRCC) had been chosen. Use AO-Fstaining method to detect the expression of positive cells. 10 blood samples takenfrom healthy individuals were also analyzed.4. Analyse the correlation between CTCs and patients’ clinical parameters. For example, gender, ages, tumor sizes, pathological pattern, T classification, Furhman stage, metastasis or not. Results1. The characteristics of primary renal tumor cells and 769-P cell line in morphology and staining had no difference after AO-F staining method. The nucleus of live group presented bright yellow, with cytoplasm presented flame-like orange. The dead group failed to emission specific fluorescence. The positive staining rate in live group was 93%±3.0% under five random microscope visions.2. The detection sensitivity was calculated as the rate of the number of AO-F positive staining cells to the number of total cells used in the system and the recovery rate(%) of four groups(500、200、100、50) was 10.2±3.8、9.2±2.3、10.8±2.6、10.5±1.9, respectively. No significant difference was found among four groups(F=1.001,P>0.05).The group(10) could find AO-F positive staining cells occasionally. The mean recovery rate of four groups was 10.16%±2.73%. The more tumor cells were added, the larger number of AO-F positive cells were detected. There was a positive correlation between them(Pearson=0.959,P=0.00).3. Zero case of 10 in control group and benign renal disease group was positive.19 cases of 119 were positive in RCC. The positive staining rate was 8.93%(10/112) for ERCC and 33.33%(9/27) for MRCC, which had a significant difference(P<0.05).4. There was no correlation between gender, ages, tumor sizes, pathological pattern and positive staining rate in 139 RCC patients(P>0.05). However,there was significant correlation between positive staining rate and metastasis. There was no significant statistical difference between clinical parameters and positive staining rate(P>0.05) in ERCC and MRCC.5. The AO-F positive staining rate has no significant difference in age, sex, pathological pattern, T 2/3 classification and Furhman grade between non-metastatic patients and metastatic patients, while a significant difference was found in T1. ConclusionIt is confirmed that the method of CTCs staining with AO-F, which has high sensitivity and repeatability, is feasible for predicting metastasis in RCC patients and worthy to being studied. There is a certain reference value to predict recurrence and metastasis.