Extracellular vesicles isolated from frozen whole blood show biomarker potential in small intestine neuroendocrine neoplasms.

Despite widespread storage of frozen whole blood (FWB) in biobanks worldwide, its suitability for extracellular vesicle (EV) research remains largely unassessed. Here, we developed a robust and practical workflow for EV-based cancer biomarker analysis from FWB by comparing differential ultracentrifugation (dUC), size-exclusion chromatography, and their combination. All methods successfully recovered vesicles within the expected 50-150 nm range, enriched for canonical EV markers, while reducing blood-derived EV contaminants, and displayed vesicle-like morphology. Among tested methods, dUC emerged as the most cost-effective and labor-efficient approach. Applying this pipeline to biobanked FWB samples from patients with small intestinal neuroendocrine neoplasms yielded an average of ∼1,000 proteins detected per sample using mass spectrometry-based proteomics, including known neuroendocrine markers and enrichment in synapse organization signaling pathways. These findings demonstrate the feasibility of EV isolation from FWB and biomarker-relevant downstream analysis, highlighting the untapped potential of EV-based biomarker discovery across diseases from existing biobanks.