Background: Although sentinel lymph node biopsy (SLNB) is a common procedure in treatment of early stage breast cancer, SLN examination protocols have not been standardized. OSNA assay is a semi-automated lymph node examination method using molecular biological technique and has been valued as a tool for the intraoperative SLN examination with potential for standardization. Also, OSNA assay is quite unique in providing semi-quantitative result (++/+) reflecting volume of metastatic foci by measuring the amount of CK19mRNA in a lymph node. This study evaluated clinical utility of OSNA assay in the clinical setting at 10 institutes in Japan. Methods: 415 clinically node negative breast cancer patients scheduled for SLNB were enrolled. Each surgically-obtained SLN was applied to both postoperative pathological examination and OSNA assay; a central slice of each SLN with 1 mm thickness was applied to pathological examination for one section H&E staining, and the residual portion was applied to OSNA assay. For patients who received axillary dissection (ALND), non-SLN status was examined by routine method at each site. Results: OSNA showed significantly high number of positive patients over pathological examination (94 patients by OSNA assay vs. 67 patients by pathological examination). Among 35 patients who were judged as SLN positive only by OSNA assay, 32 patients received ALND and 6 (18.8%) of these patients had metastases in non-SLN. Among those who received ALND, 50 patients showed OSNA ++ and 34 patients OSNA +, with metastases in non-SLN in 22 patients and 6 patients respectively. Positive predictive value of OSNA ++ for non-SLN metastasis (22/50 = 44.0%) was significantly higher than that of OSNA + (6/34 = 17.6%) (p=0.01). Conclusions: These results suggest that OSNA assay can be more sensitive for detection of SLN metastases than pathological examination using one section H&E staining and semi-quantitative result of OSNA (++/+) defined by the amount of CK19mRNA can be a strong predictive factor of additional non-SLN involvement.