Abstract Background: The sentinel lymph node (SLN) concept in breast cancer is an integral standard of care. However, several practical issues have been raised preventing proper identification of the sentinel node in certain clinical scenarios. Moreover, the criteria for definition of sentinel node have recently been questioned, including the value of dynamic information or the value of preoperative lymphoscintigraphy. The problems behind some of these questions rely on the impracticability to incorporate the preoperative imaging information into the operative procedure of sentinel lymph node biopsy (SLNB). Methods: For the freehand SPECT (fhSPECT, SurgicEye Germany) acquisition a gamma probe system and an infrared optical tracking system were combined in one system including a data processing unit in order to acquire the readouts of the probe and the position synchronously, process the readings into a 3D image and display them for the user. To date, 20 patients with invasive breast cancer undergoing SLNB were recruited and scanned intraoperatively using fhSPECT before excision of SLNs. The localization of SLNs with fhSPECT was compared to the position of SLNs detected using gamma probe and blue dye. FhSPECT was further used to prove the excision of the SLN postoperatively. Preoperative planar scintigraphy was used as a reference. Results: Preoperatively, 38 SLNs were mapped with conventional scintigraphy. In the pre-excision scan (performed in 19 of 20 patients) fhSPECT managed to map all but 3 SLNs in the identical position as compared to node location at planar scintigrams (34/37). Only in 1 patient fhSPECT did not find any SLN in the pre-excision scan. Gamma probe failed to detect any SLNs in 2 patients and mapped in total 30 of 38 nodes. 28 SLNs were resected and confirmed to be radioactive ex-vivo. fhSPECT detected 11 SLNs in 11 patients after primary SLN excision. In 4 cases additionally detected nodes were resected and confirmed to be radioactive, while in 7 cases harvesting of the additionally detected node was discarded as higher uptake SLNs had been removed already. In the remaining 9 patients no residual radioactivity was found in the axilla. Pre-excision fhSPECT acquisitions took approx. 3.1min (SD, 1.1min) while post-excision scans took 3.2min (SD, 0.9min). The surgical procedure was extended by 12min (6min before incision and 6min after excision of SLNs). Discussion: Intraoperative 3D imaging with fhSPECT for lymphatic mapping in breast cancer patients is feasible. Identification of the SLN can be repeated at any time during surgery to guide proper and precise resection of the sentinel node(s). For quality assurance the system can digitally document that all SLNs have been successfully removed. The freehand SPECT provides also new options concerning the above stated controversies in the principal sentinel lymph node concept. Controlled studies using 3D intraoperative imaging could help to clarify many of the discussed issues, including the role of lymphatic uptake on selecting the nodes for surgical resection. Also, the option to transfer dynamic information from preoperative imaging into the OR or the possibility to skip preoperative imaging bears additional advantages over the standard procedure. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-01-22.