Research Scientist Evotec International GmbH Goettingen, Niedersachsen, Germany
Disclosure(s):
Melinda Barkhuizen, PhD: No relevant disclosure to display
Introduction/Rationale: o Neutrophils are the most abundant innate immune cells, yet they remain underrepresented in single-cell studies because they are not captured in PBMC preparations and rapidly deteriorate in whole blood. o To address this gap, we generated a high-quality single-cell neutrophil atlas from patients with ANCA-associated vasculitis and related rheumatic diseases.
Methods: o Fresh whole blood was collected from participants enrolled in three ethics-approved observational studies at collaborating university hospitals. Single-cell profiling was performed using the standard 10x Genomics workflow optimized for unfixed material. o To define quality control parameters, we conducted a time-series analysis of neutrophil retention (2–28 hours post-collection) and compared fresh versus frozen aliquots from matched samples.
Results: o Frozen samples showed marked neutrophil loss. Beyond 6 hours post-collection, cells exhibited reduced viability, increased autoreactivity, and transcriptional alterations, including ICAM-1 upregulation indicative of artifactual activation. After optimizing processing parameters, we generated a robust neutrophil atlas comprising 168 samples and 1.2 million cells, capturing disease-relevant states in ANCA-associated vasculitides. o We here provide first evidence for early developmental stages (promyelocytes: DEFA3, MPO; myelocytes: RTN, LCN2, CYBB), immature neutrophils (MME), activated subsets (S100A8, S100A9, MMP9), interferon-responsive populations, and exhausted phenotypes related to disease phenotype and severity of ANCA-associated vasculitides.
Conclusion: o Immature neutrophil substates, which are largely absent in healthy donors, are essential for understanding autoimmune pathology. o Processing fresh whole blood within 6 hours is critical to preserve native transcriptomic states and prevent activation artifacts such as ICAM-1 induction, underscoring processing time as a key variable in neutrophil single-cell studies.
