Homocysteine Measurement in Dried Blood Spot for Neonatal Detection of Homocystinurias

Abstract

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 mmol/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 mmol/L (n ¼ 99), and for the stored neonatal samples (stored dry, sealed in plastic at room temperature for 10 years) was 1.7-5.5 mmol/L, (n ¼ 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. Abbreviations AC

Performance of Expanded Newborn Screening in Norway Supported by Post-Analytical Bioinformatics Tools and Rapid Second-Tier DNA Analyses

“…Samples with a C5 concentration >1 µmol/L were therefore tested for the chromatographic separation of pivaloylcarnitine from C5 using an in-house developed LC-MS/MS method. A second-tier test to quantify allo-isoleucine was introduced in December 2017 to improve the screening for maple syrup urine disease (MSUD), based on the method of Alodaib et al [ 5 ]. The first-tier screening method does not differentiate between allo-isoleucine, leucine, isoleucine and hydroxyproline.…”

Guidelines for the diagnosis and management of cystathionine beta‐synthase deficiency

“…An alternative screening approach for determining tHcy is the analysis of DBS obtained by sampling capillary or venous blood on cards used in neonatal screening (McCann et al 2003; Bowron et al 2005; Turgeon et al 2010; Alodaib et al 2012; Bartl et al 2014). This is especially useful in clinical situations when pre-analytical and sample transport conditions cannot be met.…”

“…Low specificity of Met as the first tier analyte can be substantially improved by quantifying tHcy as a second-tier marker (Turgeon et al 2010; Mudd 2011; Alodaib et al 2012). This approach can reduce the false positive rate fivefold (Turgeon et al 2010) and data on tHcy in DBS as a secondary marker showed a good sensitivity with clear distinction between 31 CBS deficient patients (median tHcy 40 μmol/L) and controls (6 μmol/L).…”

A Rapid Screening Method for the Measurement of Neonatal Total Homocysteine in Dried Blood Spots by Liquid Chromatography-Tandem Mass Spectrometry

“…Methods with shorter retention times may produce less specific and inaccurate results that are caused by merging of neighboring interfering peaks with tHCY. Rapid LC-MS/MS methods using silica based and reversed phase columns for separation and quantitation of tHCY, as the free acid have been previously reported [5,7,28,29]. Our experience with chromatography of tHCY as both the free acid and as the butyl ester showed a significant improvement in peak symmetry and reproducibility of retention time and peak area for the latter: the butylation reduces the polarity and increases the interaction of the HCY with the column matrix.…”