Multiplex enzyme assay screening of dried blood spots for lysosomal storage disorders by using tandem mass spectrometry.
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.
Zhang
X Kate
XK
Genzyme Corporation, Framingham, MA 01701, USA. kate.zhang@genzyme.com
Elbin
Carole S
CS
Chuang
Wei-Lien
WL
Cooper
Samantha K
SK
Marashio
Carla A
CA
Beauregard
Christa
C
Keutzer
Joan M
JM
D000328:Adult; D016022:Case-Control Studies; D006801:Humans; D007231:Infant, Newborn; D016464:Lysosomal Storage Diseases / Q000097:blood / Q000175:diagnosis*; D053719:Tandem Mass Spectrometry / Q000379:methods*