title: Simplified newborn screening protocol for lysosomal storage disorders.
abstract: 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.
authors.affiliation: Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Metz
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Mechtler
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Orsini
authors.forename: Monica
authors.initials: M
authors.lastname: Martin
authors.forename: Bori
authors.initials: B
authors.lastname: Shushan
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Herman
authors.forename: Rene
authors.initials: R
authors.lastname: Ratschmann
authors.forename: Chike B
authors.initials: CB
authors.lastname: Item
authors.forename: Berthold
authors.initials: B
authors.lastname: Streubel
authors.forename: Kurt R
authors.initials: KR
authors.lastname: Herkner
authors.forename: David C
authors.initials: DC
authors.lastname: Kasper
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002985:Clinical Protocols; D000795:Fabry Disease / Q000175:diagnosis; D005776:Gaucher Disease / Q000175:diagnosis; D006009:Glycogen Storage Disease Type II / Q000175:diagnosis; D006801:Humans; D007231:Infant, Newborn; D007965:Leukodystrophy, Globoid Cell / Q000175:diagnosis; D016464:Lysosomal Storage Diseases / Q000175:diagnosis*; D013058:Mass Spectrometry; D008059:Mucopolysaccharidosis I / Q000175:diagnosis; D015997:Neonatal Screening / Q000379:methods*; D052536:Niemann-Pick Disease, Type A / Q000175:diagnosis; D052537:Niemann-Pick Disease, Type B / Q000175:diagnosis; D010865:Pilot Projects; D012680:Sensitivity and Specificity