Homocysteine measurement in dried blood spot for neonatal detection of homocystinurias.

Expanded newborn screening (NBS) leads to an increased number of false positive results, causing parental anxiety, greater follow-up costs, and the need for further metabolic investigations. We developed and validated a second-tier approach for NBS of homocystinurias by measuring the total homocysteine (tHcy) on the initial dried blood spot (DBS) samples to reduce the need for further investigation, and investigated newborn DBS homocysteine values in patients with homocystinuria. Total DBS homocysteine was measured in normal newborns, and retrospectively in newborns with established disorders, using liquid chromatography tandem mass spectrometry (LC-MS/MS) with stable isotope-labelled internal standards for homocysteine. Analytes were separated using reverse phase chromatography with a total run time of 3 min. The method was linear over the range of 10-100 μmol/L of tHcy and showed excellent precision; intra-batch CV was 4% and inter-batch precision 6.5%. Comparison of 59 plasma values with DBS for tHcy taken at the same time showed excellent correlation, (r (2)>0.97). The reference range for current neonatal samples was 5.4-10.7 μmol/L (n=99), and for the stored neonatal samples (stored dry, sealed in plastic at room temperature for 10 years) was 1.7-5.5 μmol/L, (n=50), both being normally distributed. The clinical utility of this method was checked by retrospective analysis of stored NBS samples from patients with different forms of homocystinuria, including four different remethylating disorders. All had clear elevations of tHcy.