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Multiplex Enzyme Assay Screening of Dried Blood Spots for Lysosomal Storage Disorders by Using Tandem Mass Spectrometry

Genzyme Corporation, Framingham, MA

^aaddress correspondence to this author at: Five Mountain Road, Framingham, MA 01701. Fax 508-872-9080; e-mail kate.zhang{at}genzyme.com.

Abstract

Background: Reports of the use of multiplex enzyme assay screening for Pompe disease, Fabry disease, Gaucher disease, Niemann-Pick disease types A and B, and Krabbe disease have engendered interest in the use of this assay in newborn screening. We modified the assay for high-throughput use in screening laboratories.

Methods: We optimized enzyme reaction conditions and procedures for the assay, including the concentrations of substrate (S) and internal standard (IS), assay cocktail compositions, sample clean-up procedures, and mass spectrometer operation. The S and IS for each enzyme were premixed and bottled at an optimized molar ratio to simplify assay cocktail preparation. Using the new S:IS ratio, we validated the modified assay according to CLSI guidelines. Stability of the S, IS, and assay cocktails were investigated. Dried blood spots from 149 healthy adults, 100 newborns, and 60 patients with a lysosomal storage disorder (LSD) were tested using the modified assay.

Results: In our study, the median enzyme activity measured in adults was generally increased 2–3–fold compared to the original method, results indicating higher precision. In the multiplex format, each of the 5 modified enzyme assays enabled unambiguous differentiation between samples from healthy individuals (adults and newborns) and the corresponding disease-specific samples.

Conclusions: The modified multiplex enzyme assay with premixed S and IS is appropriate for use in high-throughput screening laboratories.

The following articles in journals at HighWire Press have cited this article:

				D. S. Millington Rapid and Effective Screening for Lysosomal Storage Disease: How Close Are We? Clin. Chem., October 1, 2008; 54(10): 1592 - 1594. [Full Text] [PDF]

				A. Dajnoki, A. Muhl, G. Fekete, J. Keutzer, J. Orsini, V. DeJesus, X. K. Zhang, and O. A. Bodamer Newborn Screening for Pompe Disease by Measuring Acid {alpha}-Glucosidase Activity Using Tandem Mass Spectrometry Clin. Chem., October 1, 2008; 54(10): 1624 - 1629. [Abstract] [Full Text] [PDF]