Simple SummaryIntraoperative Flow Cytometry (iFC) is a new technique that can help assess the malignancy grade, diagnose tumor type, and evaluate resection margins during solid tumor surgery. This study focuses on the role of iFC in grading gliomas and evaluating resection margins. iFC can analyze tissue samples within 5–6 min and was utilized to evaluate samples from patients with gliomas who underwent surgery over an 8-year period. The study found that high-grade gliomas had a significantly higher tumor index than low-grade gliomas. A cut-off value of 17% in the tumor index was identified as being able to accurately differentiate low- from high-grade gliomas. All low-grade gliomas were diploid, while 22 high-grade gliomas were aneuploid. iFC was also able to verify the presence of malignant tissue in every case when evaluating glioma margins. The study concludes that iFC is a promising intraoperative technique for glioma grading and resection margin assessment.AbstractBackground: Intraoperative Flow Cytometry (iFC) is a novel technique for the assessment of the grade of malignancy and the diagnosis of tumor type and resection margins during solid tumor surgery. Herein, we set out to analyze the role of iFC in the grading of gliomas and the evaluation of resection margins. Material and Methods: iFC uses a fast cell cycle analysis protocol (Ioannina Protocol) that permits the analysis of tissue samples within 5–6 min. Cell cycle analysis evaluated the G0/G1 phase, S-phase, mitosis, and tumor index (S + mitosis phase fraction) and ploidy status. In the current study, we evaluated tumor samples and samples from the peripheral borders from patients with gliomas who underwent surgery over an 8-year period. Results: Eighty-one patients were included in the study. There were sixty-eight glioblastoma cases, five anaplastic astrocytomas, two anaplastic oligodendrogliomas, one pilocytic astrocytoma, three oligodendrogliomas and two diffuse astrocytomas. High-grade gliomas had a significantly higher tumor index than low grade gliomas (median value 22 vs. 7.5, respectively,p= 0.002). Using ROC curve analysis, a cut-off value of 17% in the tumor index could differentiate low- from high-grade gliomas with a 61.4% sensitivity and 100% specificity. All low-grade gliomas were diploid. From the high-grade gliomas, 22 tumors were aneuploid. In glioblastomas, aneuploid tumors had a significantly higher tumor index (p= 0.0018). Twenty-three samples from glioma margins were evaluated. iFC verified the presence of malignant tissue in every case, using histology as the gold standard. Conclusion: iFC constitutes a promising intraoperative technique for glioma grading and resection margin assessment. Comparative studies with additional intraoperative adjuncts are necessary.