BackgroundSentinel node (SN) biopsy in penile cancer (PeCa) is typically performed using 99mTc-nanocolloid and blue dye. Recent reports suggested that the hybrid (radioactive and fluorescent) tracer indocyanine green (ICG)-99mTc-nanocolloid may improve intraoperative optical SN identification. ObjectiveThe current study aimed to confirm the reliability of ICG-99mTc-nanocolloid and to assess whether blue dye is still of added value. Design, setting, and participantsA total of 400 ≥T1G2N0 PeCa patients were staged with SN biopsy at a single European centre. SNs were preoperatively identified with lymphoscintigraphy and single-photon emission computed tomography. Intraoperatively, SNs were detected via gamma tracing, blue staining, and fluorescence imaging. Outcome measurements and statistical analysisAll patients (n=400, 740 groins) received ICG-99mTc-nanocolloid. Intraoperative SN identification rates were retrospectively evaluated. In those patients who received ICG-99mTc-nanocolloid and blue dye (n=266, 492 groins), SN visualisation rates were compared using the McNemar test. Results and limitationsIn total, 740 groins were assessed. No tracer-related (allergic) reactions were reported. All preoperatively defined SNs (n=1163) were localised intraoperatively. Of all excised SNs, 98% were detectable with gamma probe and 96% were visible with fluorescence imaging. In the analysis of the patients who received ICG-99mTc-nanocolloid and blue dye, fluorescence imaging yielded a 39% higher SN detection rate than blue dye (95% confidence interval 36–43%, p<0.001). Of the SNs that were tumour positive, 100% were intraoperatively visualised by fluorescence imaging, whereas merely 84% of the positive nodes stained blue. ConclusionsThis study confirms that ICG-99mTc-nanocolloid is a reliable SN tracer for PeCa that significantly improves optical SN detection over blue dye. Patient summaryHybrid indocyanine green (ICG)-99mTc-nanocolloid is a safe and reliable sentinel node (SN) tracer, as established in this large series of 400 penile cancer patients (740 groins). It enables accurate pre- and intraoperative SN identification and significantly improves SN detection rate compared with blue dye, without staining the surgical field or the need for an additional injection.