ObjectiveNewborn screening (NBS) for cystic fibrosis (CF) was introduced in Switzerland in 2011 based on an immunoreactive trypsinogen (IRT)-DNA-IRT protocol. CF diagnosis was confirmed by sweat test and/or genetics but remained inconclusive for some newborns (cystic fibrosis transmembrane conductance regulator related metabolic syndrome (CRMS)/CF screen positive, inconclusive diagnosis (CFSPID)). We aimed to (1) Describe IRT levels in healthy newborns in the first year of life and by gestational age (GA), and (2) Compare IRT at two time points between healthy newborns and newborns with CF and CRMS/CFSPID.DesignRetrospective study.SettingNational NBS database.PatientsAll children with an IRT measurement by heel prick test from 2011 to 2019.InterventionsNone.Main outcome measuresIRT values were extracted from the National NBS Laboratory, and clinical characteristics of positively screened children from the CF-NBS database. Second IRT assessment in positively screened children was usually performed after 18–24 days. We calculated internal IRT Z-Scores and multiples of the median to compare our results across different laboratory tools.ResultsAmong 815 899 children; 232 were diagnosed with CF, of whom 36 had meconium ileus (MI); 27 had CRMS/CFSPID. Among all samples analysed, mean IRT Z-Scores were higher for newborns with GA <33 weeks and ≥43 weeks (all Z-Scores >0.11) compared with term babies (all Z-Scores ≤0.06). Repeated IRT Z-Scores after a median (IQR) of 19 (17–22) days remained high for infants with CF with or without MI but decreased for infants with CRMS/CFSPID.ConclusionsMeasurement of a second IRT value can help distinguish between children with CRMS/CFSPID and CF, early in life.