X. Kate Zhang

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.

Development and Evaluation of Quality Control Dried Blood Spot Materials in Newborn Screening for Lysosomal Storage Disorders

BACKGROUND Lysosomal storage disorders (LSDs) comprise more than 40 genetic diseases that result in the accumulation of products that would normally be degraded by lysosomal enzymes. A tandem mass spectrometry (MS/MS)-based method is available for newborn screening for 5 LSDs, and many laboratories are initiating pilot studies to evaluate the incorporation of this method into their screening panels. We developed and evaluated dried blood spot (DBS) QC materials for LSDs and used the MS/MS method to investigate their suitability for LSD QC monitoring. METHODS We incubated 3.2-mm punches from DBS controls for 20-24 h with assay cocktails containing substrate and internal standard. Using MS/MS, we quantified the resulting product and internal standard. Samples were run in triplicate for 3 consecutive days, and results were reported as product-to-internal standard ratios and enzyme activity units (micromol/L/h). RESULTS Enzyme activity interday imprecision (CV) for the high, medium, and low series were 3.4%-14.3% for galactocerebroside alpha-galactosidase, 6.8%-24.6% for acid alpha-galactosidase A, 7.36%-22.1% for acid sphingomyelinase, 6.2%-26.2% for acid alpha-glucocerebrosidase, and 7.0%-24.8% for lysosomal acid alpha-glucosidase (n = 9). In addition, DBS stored at -20 degrees and 4 degrees C showed minimal enzyme activity loss over a 187-d period. DBS stored at 37 degrees and 45 degrees C had lower activity values over the 187-day evaluation time. CONCLUSIONS Suitable QC materials for newborn screening of LSDs were developed for laboratories performing DBS LSD screening. Good material linearity was observed, with goodness-of-fit values of 0.953 and higher. The QC materials may be used by screening laboratories that perform LSD analysis by MS and/or more conventional fluorescence-based screening methods.

How well does urinary lyso-Gb3 function as a biomarker in Fabry disease?

BACKGROUND Fabry disease is characterized by accumulation of glycosphingolipids, such as globotriaosylceramide (Gb(3)), in many tissues and body fluids. A novel plasma biomarker, globotriaosylsphingosine (lyso-Gb(3)), is increased in patients with the disease. Until now, lyso-Gb(3) was not detectable in urine, possibly because of the presence of interfering compounds. METHODS We undertook to: 1) characterize lyso-Gb(3) in urine; 2) develop a method to quantitate urinary lyso-Gb(3) by mass spectrometry; 3) evaluate urinary lyso-Gb(3) as a potential biomarker for Fabry disease; and 4) determine whether lyso-Gb(3) is an inhibitor of α-galactosidase A activity. We analyzed urinary lyso-Gb(3) from 83 Fabry patients and 77 healthy age-matched controls. RESULTS The intraday and interday bias and precision of the method were <15%. Increases in lyso-Gb(3)/creatinine correlated with the concentrations of Gb(3) (r(2)=0.43), type of mutations (p=0.0006), gender (p<0.0001) and enzyme replacement therapy status (p=0.0012). Urine from healthy controls contained no detectable lyso-Gb(3). Lyso-Gb(3) did not inhibit GLA activity in dried blood spots. Increased urinary excretion of lyso-Gb(3) of Fabry patients correlated well with a number of indicators of disease severity. CONCLUSION Lyso-Gb(3) is a reliable independent biomarker for clinically important characteristics of Fabry disease.

Newborn Screening for Pompe Disease by Measuring Acid α-Glucosidase Activity Using Tandem Mass Spectrometry

BACKGROUND Pompe disease, caused by the deficiency of acid alpha-glucosidase (GAA), is a lysosomal storage disorder that manifests itself in its most severe form within the first months of life. Early detection by newborn screening is warranted, since prompt initiation of enzyme replacement therapy may improve morbidity and mortality. We evaluated a tandem mass spectrometry (MS/MS) method to measure GAA activity for newborn screening for Pompe disease. METHODS We incubated 3.2-mm punches from dried blood spots (DBS) for 22 h with the substrate [7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenylcarbamoyl]- ethyl}-carbamic acid tert-butyl ester] and internal standard [7-d(5)-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic acid tertbutyl ester]. We quantified the resulting product and internal standard using MS/MS. We assessed inter- and intrarun imprecision, carryover, stability, and correlation between enzyme activities and hematocrit and punch location and generated a Pompe disease-specific cutoff value using routine newborn screening samples. RESULTS GAA activities in DBS from 29 known Pompe patients were <2 micromol/h/L. GAA activities in routine newborn screening samples were [mean (SD)] 14.7 (7.2) micromol/h/L (n = 10,279, median 13.3, 95% CI 14.46-14.74 micromol/h/L) and in normal adult samples 9.3 (3.3) micromol/h/L (n = 229, median 9, 95% CI 8.88-9.72 micromol/h/L). GAA activity was stable for 28 days between 37 degrees C and -80 degrees C. Carryover could not be observed, whereas intrarun and interrun imprecision were <10%. The limit of detection was 0.26 micromol/h/L and limit of quantification 0.35 micromol/h/L. CONCLUSIONS The measurement of GAA activities in dry blood spots using MS/MS is suitable for high-throughput analysis and newborn screening for Pompe disease.

Newborn screening for Fabry disease by measuring GLA activity using tandem mass spectrometry

BACKGROUND Fabry disease (FD) is an X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A (GLA). We evaluated a tandem mass spectrometry method to measure GLA activity. METHODS One 3.2mm punch from a dried blood spot sample (DBS) was incubated with substrate and internal standard in the reaction buffer for 22 h. The resulting product was quantified against internal standard using MS/MS. RESULTS The median GLA activity of male newborn DBS (N=5025) was 9.85 + or - 6.4 micromol/h/l (CI 95% is 9.67-10.02 micromol/h/l); The median GLA activity of female newborns (N=4677) was 10.2 + or - 6.3 micromol/h/l (CI 95% is 10.02-10.38 micromol/h/l). The difference between the two subgroups is within assay analytical variation. The GLA activities in the DBS samples from 9 juvenile and adult males with previously identified FD were below 1.64 micromol/h/l. The GLA activities from 32 juvenile and adult females with confirmed FD were below 4.73 micromol/h/l. In 5 (16%) females GLA activities were above the 0.5th percentile of lower limit of CI 95% at 3.18 micromol/h/l. CONCLUSIONS The MS/MS method for Fabry disease newborn screening is robust and can be readily multiplexed with other lysosomal disorders such as Pompe, Gaucher, Niemann-Pick, and Krabbe diseases.

Implementation of newborn screening for Krabbe disease: Population study and cutoff determination

OBJECTIVE The aim of this study was to develop a newborn screening algorithm for Krabbe disease. DESIGN AND METHODS We measured the galactocerebrosidase activity of 139,074 anonymous newborns, 56 known carriers, and 16 Krabbe patients using a tandem mass spectrometry method. The activities were converted to percentages of daily mean activity (%DMA), and the results from diseased and normal populations were used to establish cutoffs. RESULTS The absolute activities for the newborns ranged from 0.17 to 355 micromol/L h (N=139,074) and activities for Krabbe-positive controls ranged from 0.08 to 0.48 micromol/L h (N=16, n=91 measurements) while activities for carriers ranged from 0.28 to 2.71 micromol/L h (N=56, n=72 measurements). Cutoffs were set based on results from Krabbe-positive and carrier controls and the newborn population distribution. CONCLUSIONS The algorithm and cutoffs we propose provided 100% detection of all positive controls with 60/100,000 screen positive results predicted. In the course of this study, one anonymous newborn was predicted to have Krabbe disease based on enzyme activity and subsequent DNA analysis.

Lysosomal storage disorder 4 + 1 multiplex assay for newborn screening using tandem mass spectrometry: Application to a small-scale population study for five lysosomal storage disorders

BACKGROUND We sought to modify a previously published tandem mass spectrometry method of screening for 5 lysosomal storage disorders (LSDs) in order to make it better suited for high-throughput newborn screening. METHODS Two 3-mm dried blood spot (DBS) punches were incubated, each with a different assay solution. The quadruplex solution was used for screening for Gaucher, Pompe, Krabbe and Fabry diseases, while a separate solution was used for Niemann-Pick A/B disease. RESULTS The mean activities of acid-β-glucocerebrosidase (ABG), acid sphingomyelinase (ASM), acid glucosidase (GAA), galactocerebroside-β-galactosidase (GALC) and acid-galactosidase A (GLA) were measured on 5055 unidentified newborns. The mean activities (compared with their disease controls) were, 15.1 (0.35), 22.2 (1.34), 16.8 (0.51), 3.61 (0.23), and 20.7 (1.43) (μmol/L/h), respectively. The number of specimens that fell below our retest level cutoff of <20% daily mean activity (DMA) for each analyte is: ABG (6), ASM (0), GAA (5), GALC (17), and GLA (2). CONCLUSIONS This method provides a simplified and reliable assay for screening for five LSDs with clear distinction between activities from normal and disease samples. Advantages of this new method include significant decreases in processing time and the number of required assay solutions and overall decreased complexity.

Determination of psychosine concentration in dried blood spots from newborns that were identified via newborn screening to be at risk for Krabbe disease

BACKGROUND New York State has screened over 1.2 million newborns for Krabbe disease, and we identified 4 newborns with infantile Krabbe disease. In addition, 6 other newborns were identified with very low galactosylcerebrosidase (GALC) activity. Because these patients remain asymptomatic, we investigated whether psychosine levels could be a useful marker for disease. METHODS HPLC-MS/MS methodology was used to determine the psychosine concentrations in dried blood spots (DBS) collected from the following cohorts: known Krabbe patients, screened babies that were determined to have infantile Krabbe disease, asymptomatic infants with low GALC activity, and normal controls. RESULTS The psychosine concentrations from the known Krabbe patients ranged from 7 to 50 ng/ml. Newborns identified by screening who were confirmed with infantile Krabbe disease ranged from 23 to 73 ng/ml. Asymptomatic individuals with low GALC activity had concentrations ranging from 1.7 to 5.7 ng/ml. Concentrations in newborns with normal GALC activity were all <3 ng/ml. CONCLUSIONS The psychosine concentrations in DBS from confirmed infantile patients are at least four times higher than the asymptomatic newborns and nearly an order of magnitude greater than normal newborns. Further studies are needed to determine if psychosine can be used as a predictor of disease status/progression in screen positive newborns.

Lyso-sphingomyelin is elevated in dried blood spots of Niemann–Pick B patients

Niemann-Pick disease type B (NPD-B) is caused by a partial deficiency of acid sphingomyelinase activity and results in the accumulation of lysosomal sphingomyelin (SPM) predominantly in macrophages. Notably, SPM is not significantly elevated in the plasma, whole blood, or urine of NPD-B patients. Here, we show that the de-acylated form of sphingomyelin, lyso-SPM, is elevated approximately 5-fold in dried blood spots (DBS) from NPD-B patients and has no overlap with normal controls, making it a potentially useful biomarker.

Effect of sample collection on α-galactosidase A enzyme activity measurements in dried blood spots on filter paper

BACKGROUND Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha galactosidase A (AGAL, EC 3.2.1.22). Despite increasing utilization of dried blood spot (DBS) as samples for AGAL enzyme assays, the effects of blood sample collection techniques on enzyme activity have not been studied. METHODS DBS samples were prepared by spotting blood collected into an ethylenediaminetetraacetic acid (EDTA) tube and by direct application of blood from a finger prick or a venipuncture syringe. AGAL activity was measured quantitatively by detecting the fluorescence of 4-methylumbelliferone (4-MU) generated using the substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUGal) in an acidic pH for 20 h. N-acetyl-D-galactosamine (GalNAc) was used to inhibit alpha-galactosidase B (EC 3.2.1.49). RESULTS We studied 88 previously diagnosed Fabry disease patients and 690 healthy controls. Average AGAL activity in DBS samples prepared using EDTA tubes was higher compared to those spotted directly irrespective of disease status. CONCLUSIONS The study confirms the need for collection method-specific reference ranges using DBS samples.