Table 1.

First-tier biochemical marker, second-tier genotyping markers and target conditions.

First-Tier Biochemical		Second-Tier Genetic		Target Condition	Historical Biochemical Cutoff for Recall without 2nd Tier (μmol/L)
Biochemical Marker	Cutoff (μmol/L)	Genotyping Marker	Cutoff
Free carnitine-C0	5.0–6.5 n/c *	Sequence variant in SLC22A5 gene	≥one P/LP variant	Carnitine uptake defect (CUD, MIM #212140, also known as systemic primary carnitine deficiency)	≤6.2 c
Citrulline	25–50 n/c ^	Sequence variant and target screening for IVS16ins3kb in SLC25A13 gene $	1. ≥one P/LP variant and citrulline ≥ 35 μmol/L, or 2. ≥two P/LP variant and citrulline ≥25 and <35 μmol/L @	Citrin deficiency (CD, MIM #605814, also known as citrullinemia type II)	≥35 c
C5DC-carnitine	>0.17 n >0.35 c	Sequence variant in GCDH gene	≥one P/LP variant	Glutaric acidemia type I (MIM #231670)	≥0.35 c
C5OH-carnitine	>0.9 n >0.7 c	Sequence variant in ACAT1, HLCS and HMGCL gene	≥one P/LP variant	Beta-ketothiolase deficiency (#203750) Holocarboxylase synthetase deficiency (MIM #253270) 3-Hydroxy-3-methylglutaryl-CoA lyase deficiency (MIM #246450).	≥0.78 c

Notes: “n”: NeoBase2 cutoff. “c”: MassChrom cutoff. “n/c”: same cutoff for NeoBase 2 and MassChrom. “P”: pathogenic. “LP”: likely pathogenic. “*”: NBS cases with free carnitine C0 <5.0 μmol/L are recalled immediately as positive and excluded from second-tier genetic screening test. “^”: NBS cases with citrulline >50 μmol/L are recalled immediately as positive and excluded from second-tier genetic screening test. “$”: Target screening for IVS16ins3kb in SLC25A13 gene was implemented since May 2023; all cases positive for citrulline elevation before May 2023 were retrospectively reviewed. “@”: During 1–30 September 2021, cases with citrulline ≥25 and <35 μmol/L and one P or LP variant would be recalled as screening positive; the second-tier genetic screening cutoff was later refined to “≥two P or LP variant and citrulline ≥25 and <35 μmol/L” to prevent excessive recalls of CD carrier.