Tandem Mass Spectrometry Has a Larger Analytical Range than Fluorescence Assays of Lysosomal Enzymes: Application to Newborn Screening and Diagnosis of Mucopolysaccharidoses Types II, IVA, and VI

Abstract

BACKGROUND There is interest in newborn screening and diagnosis of lysosomal storage diseases because of the development of treatment options that improve clinical outcome. Assays of lysosomal enzymes with high analytical range (ratio of assay response from the enzymatic reaction divided by the assay response due to nonenzymatic processes) are desirable because they are predicted to lead to a lower rate of false positives in population screening and to more accurate diagnoses. METHODS We designed new tandem mass spectrometry (MS/MS) assays that give the largest analytical ranges reported to date for the use of dried blood spots (DBS) for detection of mucopolysaccharidoses type II (MPS-II), MPS-IVA, and MPS-VI. For comparison, we carried out fluorometric assays of 6 lysosomal enzymes using 4-methylumbelliferyl (4MU)-substrate conjugates. RESULTS The MS/MS assays for MPS-II, -IVA, and -VI displayed analytical ranges that are 1–2 orders of magnitude higher than those for the corresponding fluorometric assays. The relatively small analytical ranges of the 4MU assays are due to the intrinsic fluorescence of the 4MU substrates, which cause high background in the assay response. CONCLUSIONS These highly reproducible MS/MS assays for MPS-II, -IVA, and -VI can support multiplex newborn screening of these lysosomal storage diseases. MS/MS assays of lysosomal enzymes outperform 4MU fluorometric assays in terms of analytical range. Ongoing pilot studies will allow us to gauge the impact of the increased analytical range on newborn screening performance.

Liquid Chromatography–Tandem Mass Spectrometry Assay of Leukocyte Acid α-Glucosidase for Post-Newborn Screening Evaluation of Pompe Disease

“…The analytical range was defined as the enzyme-dependent assay response from the pooled normal control sample divided by the assay response for all GAA-independent processes (13). We anticipate that assays with a higher analytical range will be more accurate since the enzymatic activity values are spread over a larger range.…”

“…For MS/MS assays, the analytical range was calculated as: (GAA activity for normal control sample) − (GAA activity of the blank assay without leukocyte lysate)/(GAA activity of the assay without leukocyte lysate) (13). By using leukocyte lysates from non-Pompe pools, the analytical range for the LC-MS/MS assay of GAA was found to have a mean (SD) of 1135 (539) (see online Supplemental Table 3).…”

“…The classic GAA assay uses the fluorometric substrate 4-methylumbelliferyl- α -glucoside. Recent studies have shown that the analytical range of this assay, defined as the ratio of assay response measured with the normal control to that due to all GAA-independent processes, is relatively small compared to the corresponding value obtained with MS/MS assays (13), mainly due to the intrinsic fluorescence of the substrate itself (13). The low analytic range may be the reason that the fluorometric assay is unable to differentiate between IOPD vs LOPD in blood samples (14, 15).…”

“…The analytical range of an LC-MS/MS assay was recently shown to be nearly 2 orders of magnitude higher than that for the fluorometric enzyme assay (13). Here we describe a new LC-MS/MS method to accurately measure residual GAA activity in leukocytes isolated from venous blood of healthy patients, carriers, patients carrying 1 or more pseudodeficiency alleles, and Pompe patients with a range of severity.…”

Opening the toolbox of alternative sampling strategies in clinical routine: A key-role for (LC-)MS/MS

“…Direct screening examines endogenous substances, while indirect screening focusses on the conversion of substrates by specific enzymes 39 . Current NBS programs screen for up to over 50 disorders 40,41 .…”