Intraoperative Flow Cytometry for the Differentiation of Glioblastomas from Metastatic Brain Tumours: A Preliminary Prospective Study

Background: Glioblastoma (GBM) and metastatic brain tumours are the two malignant intracranial lesions most frequently encountered in adult neurosurgical practice, and their intraoperative separation may be difficult when imaging is atypical, when no extracranial primary tumour is known, or when frozen-section examination is deferred or inconclusive. Intraoperative flow cytometry (iFC) is an emerging adjunct that provides a rapid, quantitative assessment of tumour DNA content and cell cycle distribution, with a preliminary result available within approximately 5–6 min of tissue receipt. Aim: The aim was to determine, in a preliminary prospective cohort, whether iFC-derived DNA ploidy and cell cycle parameters differ between GBM and brain metastases, and to quantify their discriminatory performance. Methods: iFC was applied to 52 patients operated on for intracranial tumours, including 40 glioblastomas and 12 brain metastases. Tumour samples were analysed for DNA index, G0/G1-phase fraction, and Tumour Index (S + G2/M phases) using the “Ioannina-protocol”. Groups were compared with the Mann–Whitney U test. Discrimination was assessed by receiver operating characteristic (ROC) analysis. Results: Metastases showed a higher DNA index (median 1.05, interquartile range [IQR] 1.00–1.32 versus 1.00, IQR 1.00–1.00; p = 0.004) and a higher Tumour Index (24.5%, IQR 19.0–36.0 versus 12.0%, IQR 8.0–19.0; p = 0.034), whereas GBM showed a higher G0/G1-phase fraction (88.0%, IQR 81.0–92.0 versus 75.5%, IQR 64.0–81.0; p = 0.034). Aneuploidy (DNA index outside 0.9–1.1) was present in 6/12 (50.0%) metastases versus 5/40 (12.5%) GBM (p = 0.011). ROC analysis of the Tumour Index yielded an area under the curve of 0.70 (95% CI 0.51–0.88); at the Youden-derived cut-off of 22 the sensitivity was 75.0% (95% CI 42.8–94.5), specificity 75.0% (95% CI 58.8–87.3), positive predictive value 47.4%, negative predictive value 90.9% and overall accuracy 75.0% (95% CI 61.1–86.0). Conclusions: In this small, single-centre preliminary study of 40 glioblastomas and only 12 metastases, iFC detected reproducible differences in DNA ploidy and cell cycle distribution at the group level. Overall discrimination was moderate, so that these parameters cannot at present support an individual-patient intraoperative diagnosis. DNA-based iFC should be regarded as a potential complement to standard intraoperative neuropathological assessment, and requires validation in larger cohorts.