Detecting multiple lysosomal enzymes in dried blood spots by tandem mass spectrometry

Objective Establish a method for measuring the activities of Galactocerebrosidase (GALC), α-Glucosidase(GAA), α-Galactosidase (GLA) and α-L-Iduronidase (IDUA) in dried blood spots specimen by tandem mass spectrometry (MS/MS). Methods A total of 2175 dried blood spot samples forinborn errors of metabolism in neonatalscreening center of Shanghai Xinhua hospital were collected in July and November, 2013.And twenty dried blood spot samples from patients withlysosomalstorage disorders(LSDs)of Shanghai Xin Hua Hospital were collected from September 2012 to January 2014. The extraction of DBS was incubated with enzyme substrates and internal standards.After liquid-liquid and solid-phase extraction, the extraction solution was dried under nitrogen and reconstituted. Then enzyme reaction products and internal standards were analyzed by MS/MS. Linearity, precision, accuracy and the limit of detection were evaluated. 2175 dried blood spot samples were detected to establish the normal reference range for the activities of four enzymes according to 0.5th to 99.5th percentiles. 20 specimens from patients withLSDs were detected to verify the reference range inclinical judgment. Results The intraassay and interassay precisions ranged from 1.7% to 11.8%, and the intraassay and interassay accuracies ranged from 85% to 115%. The linear coefficients for measured concentration of enzyme products/internal standards and theoretical concentration were 0.997-0.999. The limits of detection forGALC, GAA, GLA and GLA were 0.03 μmol/(L·h), 0.09 μmol/(L·h), 0.12 μmol/(L·h) and 0.16 μmol/(L·h). The normal reference values for GALC, GAA, GLA and GLAwere 0.51-8.51 μmol(L·h), 1.99-22.22 μmol/(L·h), 1.68-41.59 μmol/(L·h) and 2.36-19.21 μmol/(L·h). The enzymes of 20 patients with LSDs were remarkably decreased compared to the normal range. The Krabbe, Pompe, Fabry, MPSⅠ patients can be effectively detected by this MS/MS method. Conclusions A MS/MS method for measuring GALC, GAA, GLA and IDUA enzyme activities in DBShas been established.(Chin J Lab Med, 2016, 39: 761-765) Key words: Lysosomal storage diseases; Tandem mass spectrometry; Dried blood spot testing; Galactosylceramidase; Alpha-Glucosidases; Alpha-Galactosidase; Iduronidase