Research paperFlow-based sorting of neonatal lymphocyte populations for transcriptomics analysis

•We report a method for sorting lymphocytes from small-volume infant blood samples.•Our gating strategy provided highly purified lymphocyte subpopulations.•Adherence to an SOP enabled reliable collection from two sites over time.•Sufficient cells were recovered from 2 to 3 ml of blood for transcriptomics analysis.

RationaleEmerging data suggest an important role for T lymphocytes in the pathogenesis of chronic lung disease in preterm infants. Comprehensive assessment of the lymphocyte transcriptome may identify biomarkers and mechanisms of disease.MethodsSmall volume peripheral blood samples were collected from premature infants enrolled with consent in the Prematurity and Respiratory Outcomes Program (PROP), at the time of discharge from the hospital. Blood samples were collected at two sites and shipped to a central laboratory for processing. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation and separated into individual lymphocyte cell types by fluorescence-activated cell sorting. Gating strategies were optimized to ensure reproducible recovery of highly purified lymphocyte populations over a multi-year recruitment period. RNA was isolated from sorted cells and characterized by high-throughput sequencing (RNASeq).ResultsBlood volumes averaged 2.5 ml, and sufficient PBMCs were collected from 165 of the 246 samples obtained (67%) from the 277 recruited subjects to complete sorting and RNASeq analysis on the resulting sorted cells. The number of total lymphocytes per ml of blood in the neonatal subjects was approximately 4 million/ml. Total lymphocyte frequencies recovered following sort varied widely among subjects, as did the frequency of individual lymphocyte and NK cell sub-populations. RNA yield from sorted cells varied according to cell type, but RNA of sufficient quantity and quality was recovered to enable RNASeq.SummaryOur results describe a validated procedure for the generation of genome-wide expression data from isolated lymphocyte sub-populations obtained from newborn blood.

Graphical abstractProcedure diagram. Summary of the critical steps involved in the standard operating procedure (SOP) developed for this study. A detailed SOP is included in the Supplement.Download full-size image