Abstract: Objective ：To explore the screening method of circulating tumor cells (CTCs) staining with acridine orange fluorescent (AO-F) and its application in metastatic renal cell carcinoma (RCC) patients. Methods: Culture 769-P cell line and primary RCC cells，calculate the percentage of AO-F positive staining 769-P cell under 5 random sights after AO-F staining and evaluate the sensitivity of AO-F to cells. 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）respectively were mixed with 106 nucleated cells which were used to evaluate A0-F method’s specificity and reproducibility. 27 metastatic RCC patients were collected and use AO-F staining method to detect the expression of AO-F positive cells in their blood. Analyse the correlation between expression and clinical parameters. Results: The percentage of AO-F positive staining 769-P cell was 93%±3% under 5 random sights. 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. Zero case in controls was positive. 9 cases of 27 in test group were positive and the rate was 33.33%. There were no significant statistical difference between gender, ages, tumor sizes, pathological pattern, Furhman stages, metastasis of lung or not, presence of tumor or not and AO-F positive rate (P >0.05). Conclusion: It is confirmed that the method of CTCs staining with AO-F, which has high specificity and reproducibility, is feasible to detect CTC and worthy of being studied. There is a certain reference value to predict tumor recurrence and metastasis.

Key words: acridine orange,  microscopy, fluorescence, carcinoma, renal cell, circulating tumor cells