A robust pipeline for high-content, high-throughput immunophenotyping reveals age- and genetics-dependent changes in blood leukocytes

• DOI: 10.1016/j.crmeth.2023.100619
• Publication Date: 2023-10-25T11:25:04Z
• Authors: Thomas Liechti; Sofie Van Gassen; Margaret Beddall; Reid Ballard; Yaser Iftikhar; Renguang Du; Thiagarajan Venka...; David Novak; Massimo Mangino; Stephen Perfetto; H. Benjamin Larman; Tim Spector; Yvan Saeys; Mario Roederer
• Keywords: QUALITY-ASSURANCE; Science; B-Lymphocyte Subsets; QD415-436; Biochemistry; Article; Immunophenotyping; Medicine and Health Sciences; Leukocytes; Humans; ASSAY; OPTIMIZATION; MUTATION; CP: Systems biology; FLOW-CYTOMETRY; Q; HUMAN IMMUNE-SYSTEM; Biology and Life Sciences; Reproducibility of Results; CP: Immunology; PERFORMANCE; Flow Cytometry; 3. Good health; CD45 GENE; VISUALIZATION; TP248.13-248.65; CELL RESPONSES; Biotechnology

High-dimensional flow cytometry is the gold standard to study the human immune system in large cohorts. However, large sample sizes increase inter-experimental variation because of technical and experimental inaccuracies introduced by batch variability. Our high-throughput sample processing pipeline in combination with 28-color flow cytometry focuses on increased throughput (192 samples/experiment) and high reproducibility. We implemented quality control checkpoints to reduce technical and experimental variation. Finally, we integrated FlowSOM clustering to facilitate automated data analysis and demonstrate the reproducibility of our pipeline in a study with 3,357 samples. We reveal age-associated immune dynamics in 2,300 individuals, signified by decreasing T and B cell subsets with age. In addition, by combining genetic analyses, our approach revealed unique immune signatures associated with a single nucleotide polymorphism (SNP) that abrogates CD45 isoform splicing. In summary, we provide a versatile and reliable high-throughput, flow cytometry-based pipeline for immune discovery and exploration in large cohorts.