ABSTRACT: We analysed data collected as part of the Biomarkers of Acute Serious Illness in Children (BASIC) study. BASIC was a prospective cohort study that enrolled critically ill children admitted to four PICUs in London and Eastern England, UK, during emergency transport by the Children’s Acute Transport Service (CATS), the regional paediatric critical care retrieval service from 2014–2016. We aimed to identify, and validate, shared features of immune dysfunction (\"endotypes\") and assess association with clinically important outcomes (primary outcome: ventilator-free days at day 30). Whole blood was sampled into RNA-stabilising tubes (PAXgene, Qiagen, Germany) from recruited patients immediately during paediatric critical care retrieval. Samples were initially stored on ice packs (4ºC) prior to arrival to the admitting PICU, before storage at -80ºC and processing in batches using Illumina Human-HT-12 version 4 Expression BeadChips (Illumina, CA, USA). Statistical analysis was undertaken in R: A Language and Environment for Statistical Computing (version 4.4.1). Microarray data were compiled, log2 transformed and normalised. Of 384 samples assay, 382 met quality control criteria and went forwards for bioinformatics analysis. We used the top 15% (~5000) most variable transcript probes in the dataset before using k-means clustering (Hartigan and Wong algorithm) with two centres to determine BASIC endotype membership for individual children at retrieval to PICU. BASIC endotype membership was internally validated with a train/validation (2/3 training, 1/3 validation) partition of the dataset, and a nested cross-validation implementation of GLMNet. We identified two robust endotypes, BASIC endotype 1 (122, 31.9%, children), and BASIC endotype 2 (260, 68.1%, children), present in children with diverse illnesses and age groups. BASIC endotype 1 membership was associated with 4.1 (95% CI 2.0–6.2) days of increased length of mechanical ventilation and a non-significant association with mortality. BASIC endotype 1 membership was associated with increase naïve and resting memory CD4 T cell proportions, lower neutrophil proportions and lower gene expression sets associated with tumour necrosis factor (TNF)-α, interferon-γ, interferon-α, and interleukin-6/JAK/STAT pathways in comparison with BASIC endotype 2. These BASIC endotypes may enable stratified trials of treatment for immune dysfunction.
