Breast-conserving surgery has become the preferred treatment method for breast cancer. Surgical margin assessment is performed during surgery, as it can reduce local recurrence in the preserved breast. Development of reliable and lower-cost ex vivo cancer detection methods would offer several benefits for patient care. Here, a practical and quantitative evaluation method for the ex vivo fluorescent diagnosis of breast lesions was developed and confirmed through a three-step clinical study. Gamma-glutamyl-hydroxymethyl rhodamine green (gGlu-HMRG) has been reported to generate fluorescence in breast lesions. Using this probe, we constructed a reliable and reproducible procedure for the quantitative evaluation of fluorescence levels. We evaluated the reliability of the method by considering reproducibility, temperature sensitivity, and the effects of other clinicopathological factors. The results suggest that the fluorescence increase of gGlu-HMRG is a good indicator of the malignancy of breast lesions. However, the distributions overlapped. A 5 min reaction with this probe could be used to distinguish at least part of the normal breast tissue. This method did not affect the final pathological examination. In summary, our results indicate that the methods developed in this study may serve as a feasible intraoperative negative-margin assessment tool during breast-conserving surgery.
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