Simplified newborn screening protocol for lysosomal storage disorders

BACKGROUND: Interest in lysosomal storage disorders, a collection of more than 40 inherited metabolic disorders, has increased because of new therapy options such as enzyme replacement, stem cell transplantation, and substrate reduction therapy. We developed a high-throughput protocol that simplifies analytical challenges such as complex sample preparation and potential interference from excess residual substrate associated with previously reported assays.

METHODS:

After overnight incubation (16-20 h) of dried blood spots with a cassette of substrates and deuterated internal standards, we used a TLX-2 system to quantify 6 lysosomal enzyme activities for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and mucopolysaccharidosis I disease. This multiplexed, multidimensional ultra-HPLC-tandem mass spectrometry assay included Cyclone P Turbo Flow and Hypersil Gold C8 columns. The method did not require offline sample preparation such as liquid-liquid and solid-phase extraction, or hazardous reagents such as ethyl acetate.

RESULTS:

Obviating the offline sample preparation steps led to substantial savings in analytical time (approximately 70%) and reagent costs (approximately 50%). In a pilot study, lysosomal enzyme activities of 8586 newborns were measured, including 51 positive controls, and the results demonstrated 100% diagnostic sensitivity and high specificity. The results for Krabbe disease were validated with parallel measurements by the New York State Screening Laboratory.

CONCLUSIONS:

Turboflow online sample cleanup and the use of an additional analytical column enabled the implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total analysis time to <2 min per sample.

Published in Clinical Chemistry. Read the online article here. 

Authors / corresponding author* 

Thomas F. Metz*,1,2; Thomas P. Mechtler*,1,2;  Joseph J. Orsini, 3;  Monica Martin, 3;  Bori Shushan, 4; Joseph L. Herman, 5; Rene Ratschmann, 1; Chike B. Item, 1,2; Berthold Streubel,6; Kurt R. Herkner,1,2; David C. Kasper,1,2

1 Department of Pediatrics and Adolescent Medicine; Medical University of Vienna
2 Research Core Unit of Pediatric Biochemistry and Analytics, Department of Pediatrics and Adolescent; Medical University of Vienna
3 Biggs Laboratory, Wadsworth Center, New York State Department of Health, Albany, NY
4 Clinical Mass Spec Consultants, Toronto, ON, Canada
5 Thermo Fisher Scientific, 101 Constitution Boulevard, Franklin MA
6 Department of Pathology, Medical University of Vienna, Vienna, Austria

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