Background
Congenital hypothyroidism (CH) is a common, preventable cause of mental retardation, which is detected in many neonatal screening programs. Upon suspicion of CH, free thyroxine (FT4) and thyroid stimulating hormone (TSH) concentrations are measured. CH can be of thyroidal or central origin (CH-T and CH-C, respectively). While CH-T diagnosis is based on an elevated TSH with a low FT4, CH-C diagnosis is based on a low FT4 without a clearly elevated TSH. Currently, reliable neonatal reference intervals (RIs) for FT4 and TSH are lacking. Age-specific RIs would greatly improve the diagnostic process, especially for CH-C.

Objectives
To establish RIs for plasma FT4 and TSH in term neonates at day 3-7 (t=1) and day 13-15 of life (t=2; day of birth=0). The study was particularly designed to provide a reliable FT4 lower limit to facilitate CH-C diagnosis. In the Netherlands, the screening is performed at day 3-7, and neonates with an abnormal result suspect for CH-C are referred on average at day 14; time points were chosen accordingly.

Methods
Blood was collected from 120 neonates at two time points. For missed time points, additional participants were included (total number of participants>120). FT4 and TSH were measured with an electrochemiluminescence immunoassay (Cobas, Roche Diagnostics, Switzerland; adult RI for FT4 12-22 pmol/L, TSH 0.5-5.0 mU/L). RIs were calculated with MedCalc for Windows (version 18.5, Belgium). If data were not normally distributed, the non-parametric percentile method was used.

Results
From 146 participants (49% female) ≥1 measurement was available. 95% RIs for FT4 were 20.5-37.1 pmol/L (t=1) and 15.3-26.5 pmol/L (t=2). 95% RIs for TSH were 1.0-8.4 mU/L (t=1) and 1.4-8.6 mU/L (t=2).

Conclusion
FT4 concentrations differ significantly between the first and second week of life, with a FT4 lower limit of 20.5 pmol/L at day 3-7 and 15.3 pmol/L at day 13-15. These lower limits are considerably higher than the lower limit of the adult RI for the same assay.