Background: Sentinel lymph node (SLN) analysis is conventionally analysed using HES and CK19 immunohistochemistry. In case of SLN involvement, a second surgery is required for axillary lymph node (ALN) resection thus delaying the initiation of adjuvant therapies. Methods: 381 pts with invasive breast cancer were considered in this retrospective study. SLN were detected using combined radio-isotope and color detection. SLN involvement was analysed using OSNA for CK19 mRNA, in 100 pts (group 1) and compared to conventional histopathology in 281 pts (reference population, group 2). In all cases, control cytology was performed. Results: No significant difference was found between group 1 and 2 regarding patients characteristics, tumor localization, size, grade, steroid receptors and HER2 expression and the mean number of SLN analysed per pt, 2.4 (range 1-7) and 2.5 (range 1-8), respectively. Considering positive SLN as “++” (CK19 mRNA copy number>5000), “+” (250 < CK19 mRNA copy number < 5000) and positive by inhibition in group 1 and macro-, micrometastases and isolated tumor cells in group 2, no difference in lymph node involvement rate was found between the two groups with 29.0 and 29.9% of positive SLN, respectively. Only one discordance case was observed with negative OSNA analysis and positive cytology with isolated tumor cells. Using OSNA intraoperatively, the mean time to process SLN was 42 min (range 10-104) allowing immediate ALN resection. Significant (P<.01) reduction of re-intervention rate was observed when OSNA was used (9 vs 39%), including margins insufficiency (5 vs 13%), ALN resection (3 vs 15%) or both (1 vs 11%). Delay before adjuvant therapy was also significantly (P <.01) reduced when OSNA was used with 43 (range 20-82) vs 59 (range 14-212) days. Conclusions: Results achieved with OSNA are fully consistent with those achieved using conventional immunohistochemistry analysis. SLN analysis using OSNA avoids a second operation in most patients for ALN resection and shortens the delay for adjuvant therapy.