Fail-Closed Validation of Lineage-Associated Transcript-Count Diversity After IFN-β Stimulation in a Public Human...

Shannon entropy of single-cell transcript counts reflects composition and sampling. We tested whether interferon-beta (IFN-β) stimulation was associated with donor-consistent diversity changes after employing technical controls. We reanalyzed GSE96583 peripheral blood mononuclear cells from eight donors with lupus, treating donors-not the 8949 target cells obtained from them-as inferential units. Analyses included sampling without replacement at 562 unique molecular identifiers, a 750-molecule sensitivity analysis, Miller-Madow correction, IFN-gene removal, and exact compositional decomposition. Mean paired-donor raw-count plug-in entropy increased by 0.138 bits in CD14+ monocytes and 0.329 bits in FCGR3A+ monocytes and decreased by 0.080 bits in natural killer cells. Directions persisted across prespecified robustness variants. Monocyte increases accompanied higher evenness and lower transcript dominance; natural killer cells showed the opposite pattern, without consistent richness changes. Adding IFN activity to donor-, condition-, and technical-covariate-adjusted models increased R² by at most 0.0108. Entropy contributions strongly overlapped pseudobulk expression changes. In GSE194122, the implemented binary chromatin-accessibility entropy was determined by open-peak count, precluding biological cross-modality coupling claims. These findings support a lineage-divergent association within this single lupus cohort; independent replication is required before broader biological or diagnostic interpretation.