Measurement of immune cell-derived volatile organic compounds from ex vivo and in vitro cultures: a scoping review

BACKGROUND: Volatile organic compounds (VOCs) originate from cellular metabolic activity and disease-related biochemical processes and are emerging as non-invasive biomarkers. Although immune cells undergo marked metabolic and functional reprogramming during activation and differentiation, their contribution to the cellular volatile metabolome remains poorly characterised. AIM OF REVIEW: This scoping review aims to systematically map experimental studies reporting untargeted VOCs emitted from mammalian immune cell cultures, with particular emphasis on volatilomics workflows and confidence in metabolite identification. KEY SCIENTIFIC CONCEPTS OF REVIEW: A systematic literature search identified experimental studies analysing headspace VOCs from primary and immortalised immune cells. Data were extracted on cell models, stimulation conditions, headspace sampling strategies, analytical platforms and data processing workflows. Analytical quality and confidence in compound assignment were assessed using the Chemical Analysis Working Group-Metabolomics Standards Initiative (CAWG-MSI) criteria. Eleven studies met the inclusion criteria, employing a heterogeneous range of sampling and analytical approaches, including solid-phase microextraction, sorbent-based thermal desorption and secondary electrospray ionisation coupled to high-resolution mass spectrometry. Across studies, reported VOC profiles were able to distinguish immune cell type, activation state and external stimuli, supporting the biological plausibility of immune-derived volatilomic signatures. However, substantial methodological variability was evident. Only one study achieved CAWG-MSI level 1 identification, and most lacked key metadata, internal standards or validation procedures. Overall, immune cells appear to emit distinct VOC signatures linked to immunometabolic state, but current practices limit reproducibility, cross-study comparability and confident biological interpretation. This review identifies metabolomics-specific methodological priorities required to characterise immune-derived VOCs.