Preserving neutrophils in single cell sequencing — our experience with a multi-center cohort 2309567

Introduction 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. To address this gap, we generated a high-quality single-cell neutrophil atlas from patients with ANCA-associated vasculitis and related rheumatic diseases. Methods 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. 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 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. 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 Immature neutrophil substates, which are largely absent in healthy donors, are essential for understanding autoimmune pathology. 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.