Assessment of inter-operator variability in peripheral monocyte subset gating strategy using flow cytometry in patients with suspected acute stroke

Background: Innovative tools to reliably identify patients with acute stroke are needed. Peripheral monocyte subsets, that is, classical-Mon1, intermediate-Mon2, and non-classical-Mon3, with their activation marker expression analyzed using flow-cytometry (FCM) could be interesting cell biomarker candidates. Aim: To assess the inter-operator variability in a new peripheral monocyte subset gating strategy using FCM in patients with suspected acute stroke. Methods: In BOOST-study ("Biomarkers-algOrithm-for-strOke-diagnoSis-and Treatment-resistance-prediction," NCT04726839), patients >= 18 years with symptoms suggesting acute stroke within the last 24 h were included. Blood was collected upon admission to emergency unit. FCM analysis was performed using the FACS-CANTO-II (R) flow-cytometer and Flow-Jo (TM)-software. Analyzed markers were CD45/CD91/CD14/CD16 (monocyte backbone) and CD62L/CD11b/HLA-DR/CD86/CCR2/ICAM-1/CX3CR1/TF (activation markers). Inter-operator agreement (starting from raw-data files) was quantified by the measure distribution and, for each patient, the coefficient of variation (CV). Results: Three operators analyzed 20 patient blood samples. Median inter-operator CVs were below the pre-specified tolerance limits (10% [for Mon1 counts], 20% [Mon2, Mon3 counts], 15% [activation marker median-fluorescence-intensities]). We observed a slight, but systematic, inter-operator effect. Overall, absolute inter-operator differences in fractions of monocyte subsets were <0.03. Conclusion: Our gating strategy allowed monocyte subset gating with an acceptable inter-operator variability. Although low, the inter-operator effect should be considered in monocyte data analysis of BOOST-patients.