Aim: The overall goal will be to derive synergistic evidence from the randomized PRIMAL Clinical Study on whether blood biomarker can be identified that indicate favorable and adverse postnatal immune maturation in VPIs and MPIs in terms of the occurrence of sepsis in the short term and the burden of infectious diseases and need of antibiotic treatments in the long term. Specifically, we want to dissect the role of the microbiome quality early in life and the influence of probiotics and antibiotics on the development of blood immunoprofiles of VPIs and MPIs.

Design: Every blood sample collected from the clinical sites of the PRIMAL consortium will be assessed by Chipcytometry with a predefined marker panel aligned with the PRIMAL consortium. Chipcytometry is a platform for single-cell immunophenotyping that uses small microfluidic devices to capture cells on activated surfaces enabling long-term storage of the precious clinical samples from preterm infants. Chipcytometry allows fluorescence-based sequential marker staining cycles in a virtually unlimited manner and reassessment at later time points. Detailed phenotype information on blood immune cells based on cell surface and intracellular protein expression including rare events like transcription factors will be generated. Fluorescence data will be quality controlled, steadily processed into analog raw data and transferred to the computational pipelines.

Expected outcome: We will identify

• which cellular marker profiles including cross-correlating combinations thereof are linked to sepsis and
• which profiles are linked to a persistent high susceptibility to infectious diseases by training a classifier using the development of sepsis and the amount of infectious diseases in the first year of life (high/low) as binary categories. After technical filtering and feature selection steps, ranking of the contribution of different predictors allows to identify variables of particular importance for the predictive power and, hence, potential biomarkers.
• Integration of intestinal microbiota data and information on probiotics/antibiotics intake will suggest favorable as well as adverse impacts thereof on the development of non-resident immunity. The data we generate will be crucial for developing novel strategies to preventive interventions targeting long-term health costs.