Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening

Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards containing dried blood spots as the enzyme source.

For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newborn-screening centers.

We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid alpha-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed.

We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann-Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5-9 heterozygous carriers were approximately one-half those measured with 15-32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals.