Simplified Newborn Screening Protocol for ... - Semantic Scholar

Clinical Chemistry 57:9 1286–1294 (2011)

Automation and Analytical Techniques

Simplified Newborn Screening Protocol for Lysosomal Storage Disorders Thomas F. Metz,1,2† Thomas P. Mechtler,1,2† Joseph J. Orsini,3 Monica Martin,3 Bori Shushan,4 Joseph L. Herman,5 Rene Ratschmann,1 Chike B. Item,1,2 Berthold Streubel,6 Kurt R. Herkner,1,2 and David C. Kasper1,2*

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 highthroughput 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 solidphase 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

1

Department of Pediatrics and Adolescent Medicine and 2 Research Core Unit of Pediatric Biochemistry and Analytics, Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria; 3 Biggs Laboratory, Wadsworth Center, New York State Department of Health, Albany, NY; 4 Clinical Mass Spec Consultants, Toronto, ON, Canada; 5 Thermo Fisher Scientific, 101 Constitution Boulevard, Franklin MA; 6 Department of Pathology, Medical University of Vienna, Vienna, Austria † Thomas F. Metz and Thomas P. Mechtler contributed equally to the work, and both should be considered as first authors. * Address correspondence to this author at: Medical University of Vienna, Vienna, Austria, Department of Pediatrics and Adolescent Medicine, Wa¨hringer Gu¨rtel

1286

implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total analysis time to ⬍2 min per sample. © 2011 American Association for Clinical Chemistry

In recent years various approaches for highthroughput tandem mass spectrometry (MS/MS)7 screening of lysosomal storage disorders (LSDs) have been developed. The first assays were developed for the screening of Pompe, Gaucher, Niemann-Pick A/B, Fabry, and Krabbe disease in 2004 (1 ). The protocols have continuously evolved, and have been refined and optimized for high-throughput analysis (2, 3 ). The screening panel for LSDs was recently expanded and currently includes MS/MS assays for mucopolysaccharidosis (MPS) I, II, IVA, and VI (4 –7 ). However, newborn screening for LSDs is still a technological challenge. One of the major technical challenges in implementing MS/MS-based multiplex enzyme assays by using the currently available substrates is the complexity of sample preparation. Specimens collected from newborns are incubated with enzyme-specific buffers containing substrates for each lysosomal enzyme, followed by MS/MS detection of all respective enzymatic products and internal standards. The technology for screening several enzyme activities at once from more or less 1 single blood punch is complicated, time-consuming, and laborious, and includes the handling of hazardous reagents such as ethyl acetate (1, 3 ). Currently, routine newborn screening for LSDs has been introduced for Pompe disease in Taiwan (8 )

18-20, 1090 Vienna, Austria. Fax ⫹43-1-40400-3200; e-mail david.kasper@ meduniwien.ac.at. Received March 1, 2011; accepted June 30, 2011. Previously published online at DOI: 10.1373/clinchem.2011.164640 7 Nonstandard abbreviations: MS/MS, tandem mass spectrometry; LSD, lysosomal storage disorder; MPS, mucopolysaccharidosis; LLE, liquid-liquid extraction; SPE, solid-phase extraction; Turboflow, turbulent flow chromatography; UHPLC, ultra-HPLC; DBS, dried blood spots; NBS, newborn screening; ABG, acid ␤glucocerebrosidase; GLA, ␣-galactosidase; GAA, ␣-glucosidase; ASM, acid sphingomyelinase; GALC, galactocerebrosidase; DUA, iduronidase.

Simplified Screening for Lysosomal Storage Disorders

and by the state of New York for Krabbe disease (9 ). Other US states such as Washington (10 ) and countries such as Austria started the first pilot studies for multiplex MS/MS screening, in which a variety of LSDs are screened within a single assay (11–13 ). For future implementation of high-throughput LSD assays in routine clinical diagnostics, sample handling and MS analysis must be simplified; specifically, sample pretreatment, separation, and finally detection must become more integrated (14 ). The implementation of online multidimensional chromatography combining sample preparation with analysis in 1 protocol facilitates ease-of-use sample introduction and increases speed of analysis (15 ). Turbulent flow chromatography (Turboflow) involves the use of large-diameter particles (approximately 30 ␮m) with a high surface area packed into narrow-bore columns (0.5–1 mm i.d.) and available in a variety of stationary phases. Relatively crude samples such as dilute serum, cerebrospinal fluid, plasma, urine, blood-spot extracts, and whole blood can be injected directly into these columns under high mobilephase linear velocities, inducing turbulent-flow conditions. TurboFlow simultaneously eliminates matrix interferences from salts and proteins, the most common suppressors of MS/MS performance, and eliminates the need to perform liquid–liquid extraction (LLE) and solid-phase extraction (SPE) cleanup steps of previous methods (3, 16 ). The Turboflow cleanup is accomplished online in a matter of seconds and the analytes of interest, after cleanup of potential matrix interferences, are subsequently transferred to an analytical column for ultra-HPLC (UHPLC) separation before MS/MS analysis (17 ). We recently introduced a method for screening for LSDs that uses Turboflow chromatography technology (18 ). In the current study, we modified and optimized the system for high-throughput screening of 6 different LSDs in a single assay. To demonstrate the technical feasibility and robustness of this novel method, we applied it within a comprehensive pilot screening program that runs up to several thousand samples in a routine newborn screening laboratory, including samples from affected patients. Moreover, the protocol was expanded for the screening of MPS I, and parallel measurements were performed in collaboration with the newborn screening (NBS) laboratory in New York state, New York State Department of Health, Albany, NY, for evaluation of its use with their Krabbe assay (9 ). Material and Methods MATERIALS

Deionized water (18 M⍀) produced by a Millipore Milli-Q Reference A⫹ System. Methanol (#106035)

and isopropanol (#109634) were purchased from Merck Chemicals. Acetone (#650501), formic acid (#94318), and trifluoroacetic acid (#T62200) were purchased from Sigma Aldrich. Acetonitrile (#A/0627/17), Cyclone P 0.5 ⫻ 50 –mm Turbo Flow HTLC columns, and Hypersil Gold C8 2.1 ⫻ 50 –mm, 1.9-␮m particle size columns were purchased from Thermo Fisher Scientific. Microplates 96/U (#0030 601.203), microplates 96/F (#0030 601.106) and deep well plates (#0030 505.301) were purchased from Eppendorf. After we received informed consent from the parents, we collected dried blood spots (DBS) consecutively from 8586 newborns during the national routine Austrian NBS program. In Austria, NBS is centralized and conducted by one single laboratory located at the Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, for the entire population (approximately 78 000 births/year). In addition, NBS samples and 51 samples from patients with known LSDs were analyzed anonymously for 6 different lysosomal enzyme activities beyond the current screening panel for endocrine and metabolic disorders, including cystic fibrosis (19 ). The study was approved by the local ethics committee (EK 478/2009) and conducted in accordance with the institutional guidelines. The expanded screening for LSDs included the analysis of acid ␤-glucocerebrosidase (ABG), ␣-galactosidase (GLA), ␣-glucosidase (GAA), acid sphingomyelinase (ASM), galactocerebrosidase (GALC), and iduronidase (IDUA) enzyme activities in the anonymized DBS samples. SAMPLE PREPARATION

Sample preparation was performed according to a 2-day working protocol. On day 1, the protocol for the incubation of DBS with enzyme reagent cocktails was adapted from Zhang et al. (3 ) for ABG, ASM, GAA, and GLA and from Blanchard et al. (20 ) for IDUA. The GALC protocol was modified from Orsini and his coworkers (9 ). All protocols for day 1 and 2 are described in detail in the Supplementary Materials 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol57/ issue9. In brief, on day 1 aliquots of a single blood spot were incubated with enzyme-specific buffers containing substrates and internal standard for each lysosomal enzyme. On day 2, after an overnight incubation of 20 –22 h, the enzymatic reactions for ABG, ASM, GAA, GLA, and IDUA were quenched with 100 ␮L stop solution (80 mL acetonitrile/19.8 mL water/0.2 mL formic acid). After an overnight incubation of 19.5–21.5 h (30-min shorter than the previous incubation), the GALC enzyme reaction was quenched with 100 ␮L stop solution, sealed with silicone covers, and incubated again (30 min, 37 °C, 750 rpm) to maximize the product outcome. Finally, all 6 assays were transferred Clinical Chemistry 57:9 (2011) 1287

Table 1. Stepwise comparison of a protocol that includes liquid–liquid and solid-phase extraction and the new Turboflow chromatography online cleanup protocol (⬃400 newborns per day).

Day 1

Day 2

5-Plex protocola

6-Plex protocol (TLX2 system)

Gaucher, Niemann-Pick A/B, Pompe, Fabry, and Krabbe disease

Gaucher, Niemann-Pick A/B, Pompe, Fabry, Krabbe, and MPS I disease

Punching of DBS in duplicates (2 ⫻ 4 MTPs)b

Punching of DBS in duplicates (2 ⫻ 4 MTPs)

Reelute with 70 ␮L extraction buffer (first spot)

Reelute with 70 ␮L extraction buffer (first spot)

Incubate (1 h, 37 °C, 750 rpm)

Incubate (1 h, 37 °C, 750 rpm)

Incubation of 10-␮L DBS extraction aliquots from first spot with 15 ␮L reaction cocktail, separately for ABG, ASM, GAA, and GLA

Incubation of 10-␮L DBS extraction aliquots from first spot with 15 ␮L reaction cocktail, separately for ABG, ASM, GAA, GLA, and IDUA

Centrifuge all plates (1 min, 1000g)

Centrifuge all plates (1 min, 1000g)

Incubation of the second spot with 30 ␮L GALC reaction cocktail

Incubation of the second spot with 30 ␮L GALC reaction cocktail

Centrifuge (1 min, 1000g)

Centrifuge (1 min, 1000g)

Incubate (20–24 h, 37 °C, 250 rpm)

Incubate (20–24 h, 37 °C, 250 rpm)

Time: 2.7 h

Time: 2.7 h

⫹100 ␮L 1:1 methanol/ethyl acetate

⫹80 mL acetonitrile/19.8 mL water/0.2 mL FA to all GALC plates

Transfer all corresponding specimens into 1 deep well MTP

Shake GALC plate (10 min, 750 rpm)

⫹400 ␮L ethyl acetate

⫹100 ␮L 80:20 acetonitrile/water ⫹ 0.2% FA to all other plates

⫹400 ␮L HPLC Water

Transfer all corresponding specimens into 1 deep well MTP

Shake (5 min, ⬃500 rpm)

Repeat this procedure for all 4 plates

Centrifuge (⬃3000g, 10 min)

Centrifuge (⬃3000g, 15 min)

LLE; transfer into deep well MTP N2 dry (10–15 min) Reelute with 100 ␮L methanol/ethyl acetate SPE with silica gel N2 dry (20–25 min) Reelute with 50:50 acetonitrile/water

MS analysis

Repeat this procedure for all 4 plates MS analysis Total time: 4–5 h a b

Total time: 50 min

Adapted from Zhang et al. (2 ). MTP, 96-well microtiter plate; FA, formic acid; N2, nitrogen.

into 1 single deep well plate, sealed with aluminum foil, centrifuged for 15 min at 3320g, and measured with the TLX2 system by using online sample cleanup. Table 1 provides a short overview of the Zhang et al. protocol with the use of LLE and SPE compared with the new simplified protocol. Ultra-HPLC

Transcend UHPLC systems (part of the TLX-2) with Allegro quaternary pumps were used (ThermoFisher Scientific). The mobile phases were A: 0.1 mL formic 1288 Clinical Chemistry 57:9 (2011)

acid/0.01 mL trifluoroacetic acid/99.89 mL water; B: 0.1 mL formic acid/0.01 mL trifluoroacetic acid/99.89 mL acetonitrile; C: 45 mL isopropanol/45 mL acetonitrile/10 mL acetone; D: 80 mL acetonitrile/19.8 mL water/0.2 mL formic acid. Mobile phase C was used as a wash solvent and mobile phase D was used to prevent air bubbles in the pump system. The injection volume was 10 ␮L. The Transcend system employs 2 HPLCs and two 6-port valves per channel, which were configured in the standard focus mode (17 ). The online sample cleanup and separation performed by use of the


Fig. 1. Chromatogram including the retention time and peaks for substrates, products, and internal standards for all 6 lysosomal enzymes.

TLX-Turboflow System was recently described by Kasper et al. (18 ). The cleanup and chromatographic sequence are described in detail in the online Supplementary Materials 1.

and blood spot volume, according to the method reported by Li et al. (16 ). Blank values (obtained with extracts of 3.2-mm blood-free filter disks) were subtracted to give the final values of lysosomal enzyme activity.

MULTIPLEXED SAMPLE INTRODUCTION

The total time for the multidimensional TurboFlow UHPLC experiment was 4 min. The analytes of interest emerged from the analytical column at around 2.15 min in a window that was 1.30-min wide. MS/MS data acquisition started 2.15 min after injection and continued for 1.3 min until all analyte signals were recorded (see Fig. 1). In the current experimental setup, a dualchannel TLX-2 system was used (2 TurboFlow and 2 analytical columns). We used staggered injections, so that while one sample was being cleaned up on the Turboflow column another was being chromatographed into the MS/MS analyzer. Hence, the effective analysis time for each sample was ⬍2 min instead of the 4 min required if samples were run serially. MASS SPECTROMETRY

MS was performed on a Thermo Scientific TSQ Quantum Ultra with an HESI-2 heated electrospray probe. We monitored single-reaction–monitoring transitions for the 6 products and their respective internal standards. A chromatogram that includes the retention and peaks for substrates, products, and internal standards for all 6 lysosomal enzymes is shown in Fig. 1. The MS settings for each compound of interest are provided in Table 2. In the final step, the amount of product was calculated from the ion abundance ratio of the product to the internal standard for a sample multiplied by the amount of added internal standard, time of incubation,

Results DEVELOPMENT OF A HIGH-THROUGHPUT HEXAPLEX SCREENING ASSAY FOR LSDs

The multiplexed, multidimensional UHPLC MS/MS screening method was optimized (Table 2) without the need for time-consuming offline sample preparation such as LLE and SPE (see online Supplementary Material 1). The assays for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and MPS I disease were less laborious, which led to time savings of 4 –5 h per 400 samples on day 2 (Table 1). In a first pilot study, all 6 lysosomal enzyme activities for 8586 newborns and 51 patients with known LSDs were analyzed (Table 3). The cutoff values were chosen as ⱕ3.2 ␮mol 䡠 L⫺1 䡠 h⫺1 for Gaucher, ⱕ0.9 ␮mol 䡠 L⫺1 䡠 h⫺1 for Niemann Pick A/B, ⱕ3.3 ␮mol 䡠 L⫺1 䡠 h⫺1 for Pompe, ⱕ2.9 ␮mol 䡠 L⫺1 䡠 h⫺1 for Fabry, and ⱕ0.7 ␮mol 䡠 L⫺1 䡠 h⫺1 for MPS I disease, respectively, according to the 0.1 percentile of the normal population. The cutoff for Krabbe disease was below 20% of the daily mean activity according to Orsini et al. (9 ). We did not observe any statistical difference in lysosomal enzyme activities between single and multiplex analysis with the use of the combined online sample cleanup and the separation of substrates and products with an analytical column (see online Supplemental Fig. 1). Clinical Chemistry 57:9 (2011) 1289

Table 2. MS parameters. MS parameters

Scan type

Positive SRMa

Ion spray voltage

3500 V

Vaporizer temperature

350 °C

Sheath gas pressure

60 arb

Ion sweep gas pressure

2.0 arb

Auxiliary gas pressure

35 arb

Capillary temperature

250 °C

Skimmer offset

0V

Collision gas pressure

0.4 mTorr

Scan width

0.05 m/z

Peak width (FWHM) 0.3 m/z

Q1

0.7 m/z

Q3 Section 1 Enzymeb

a b

0.0–0.35 min Precursorion, m/z

Scan time 0.05 s/channel Production, m/z

Collision energy, V

Tube lens, V

IDUA

391.1

291.0

7

80

IDUA IS

377.2

277.1

7

80

GLA

484.3

233.3

28

103

GLA IS

489.3

238.3

25

104

GAA

498.3

247.3

25

96

GAA IS

503.3

252.3

26

97

Section 2

0.35–1.3 min

Enzyme

Precursorion, m/z

Scan time 0.05 s/channel Production, m/z

Collision energy, V

Tube lens, V

ABG

482.3

264.3

25

96

ABG IS

510.4

264.3

24

95

ASM

398.3

264.3

18

103

ASM IS

370.1

264.3

18

51

GALC

426.2

264.3

20

55

GALC IS

454.3

264.3

21

100

SRM, single-reaction monitoring; IS, internal standard; FWHM, full width at half maximum; arb, arbitrary units. Enzyme names are representative for reaction products and corresponding internal standards, respectively.

DETECTION OF AFFECTED PATIENTS WITH LSDs

For method evaluation, we analyzed a total of 51 patients with known LSDs (12 patients with Pompe, 13 with Gaucher, 22 with Fabry, 1 with Niemann-Pick A/B, 1 with Krabbe, and 2 with MPS I disease). All disease control patients had diminished enzyme activities that showed little or no overlap with enzyme activity values obtained from the normal reference population (Table 3; also see online Supplemental Table 3). METHOD COMPARISON FOR THE KRABBE DISEASE ASSAY

The GALC enzyme activities for 176 newborns were measured in parallel by the New York screening laboratory to evaluate the Turboflow assay (Table 4). The 1290 Clinical Chemistry 57:9 (2011)

coefficient of determination (R2) between both methods was 0.79. The mean activity measured with the Turboflow assay was lower, but SDs and CVs were similar (Table 4). Both assays clearly differentiated the affected and nonaffected newborns. Discussion LSDs result in the accumulation of macromolecular substrates that would normally be degraded by lysosomal enzymes. The combined incidence has been estimated at 1 per 7700 live births for whites (21, 22 ). The relatively high combined incidence, availability of effective therapies for some of these diseases, and dire

0.4

0.2 0.2

44.1 0.08

0.08 0.02

16.91 2.2

0.3 1.5

91.0 1.4

0.2 1.3

86.8 0.4

0.4 0.3

62.6 Maximum

0.2 2.6

93.9

Minimum

2.2

0.4

0.4 28.7

20.4 0.08

0.08 16.35

7.7 2.1

2.2 73.0

38.8 1.4

1.4 75.9

55.7 0.4

0.4 43.2

23.8

76.5 Percentile 99.9%

2.2

41.8 Percentile 99%

2.1

0.3

0.4 10.0

7.4 0.08

0.08 1.53

0.78 0.8

1.6 11.9

8.0 0.6

0.8 27.7

20.9 0.4

0.4 11.0

8.0 0.4

1.4

15.6

20.5 Percentile 75%

11.7

Median

0.2

0.2 5.2 0.08 0.43 0.5 5.5 0.4 15.3 0.4 5.8

0.2

Percentile 25%

0.2

1.5

0.7 0.08

0.08 0.14

0.04 0.3

0.3 3.0

2.9 0.2

0.2 6.2

3.3 0.4

0.4 2.5

0.9

4.6 Percentile 1.0%

0.2

3.2 Percentile 0.1%

0.2

0.3

5.1 —

7.9 0.08

6.31 —

1.27 1.0

22.0 —

10 0.5

6.2 —

22.5 0.4

4.5 —

8.9 0.8

— % Maximum activity of affected patients/mean of newborns activity

13.1

16.8 Mean

Affected patients (n ⴝ 2) Newborns (n ⴝ 8586) Affected patients (n ⴝ 1) Newborns (n ⴝ 8586)

Affected patients (n ⴝ 13)

Newborns (n ⴝ 8586)







IDUA GALC GLA GAA ASM ABG

Table 3. Enzymatic activities (␮mol 䡠 Lⴚ1 䡠 hⴚ1) in affected patients and healthy controls determined by the Turboflow chromatography method.


clinical outcomes for lack of timely treatment justify implementation of LSDs into routine NBS protocols. Substantial medical challenges still remain for the development of successful therapies for many of the other LSDs, as well as challenges regarding the availability of those therapies that are successful (23 ). The screening for several LSDs simultaneously in a single sample can be time-consuming, expensive, and work-intensive without the use of highly automated online systems. Current limitations include the requirement to perform LLE and SPE cleanup, and the use of hazardous reagents such as ethyl acetate (1–3 ). The introduction of online sample cleanup that uses multidimensional chromatography essentially eliminates the use of such organic compounds from the screening of LSDs by MS/MS (Table 1), and reduces the need for large amounts of consumables (see online Supplemental Material 2). The use of an analytical column in conjunction with an online cleanup column improves a multiplex approach by separating several enzymatic products from residual substrates. The first such protocol was described in a report by la Marca et al. (24 ), in which they suggested that online multidimensional HPLC sample preparation eliminated the need for laborious preanalytical steps with the use of a homebuilt multidimensional LC method in which a POROS cleanup column (Applied Biosystems) and an analytical C18 column were used. A similar protocol that included the use of a Turboflow cleanup column was recently reported by our group (18 ); this protocol was expanded to a hexaplex assay for the screening of MPS I in the present work. A serious problem with some published LSD assays is the potential for interference of the enzyme product signal from excess substrate due to insource fragmentation (14, 15 ). In these previous studies interference was minimized by either detuning the ion source, i.e., purposefully making the instrument less sensitive, or adding off-line cleanup steps such as SPE. We found that the use of an online analytical column in conjunction with the Turboflow online sample cleanup column, and the higher resolving powers of UHPLC, completely eliminated such interference while keeping the total analysis time to ⬍2 min per sample and facilitated the expansion of the screening panel. This expansion has practical value because new substrates for the screening for MPS II, IVA, and VI are on the horizon (4 –7 ). To illustrate this point, we integrated the screening of GALC activity with our published pentaplex assay (18 ) and evaluated the resulting GALC activities in collaboration with the New York screening laboratory by performing parallel measurements. Although our GALC enzyme activities were lower than those measured by the New York laboratory, we could clearly differentiate Clinical Chemistry 57:9 (2011) 1291

Table 4. Parallel measurement results for Krabbe disease in 176 newborns by 2 screening laboratories. Laboratorya

1

2

1

2

Specimen type

Newborns

Newborns

Affected patientsb

Affected patients

Mean ratios of product/internal standard

0.74

0.22

0.12

0.02

Mean activity, ␮mol 䡠 L⫺1 h⫺1

2.66

0.77

0.23

0.08

0.05

0.01

SD activity, ␮mol 䡠 L⫺1 h⫺1 CV

21.74

12.34

Minimum activity, ␮mol 䡠 L⫺1 h⫺1

0.69

0.17

0.19

0.07

Maxaximum activity, ␮mol 䡠 L⫺1 h⫺1

5.02

2.29

0.27

0.09

5.50

6.31

% Daily mean activity according to Orsini et al. (9 ) a b

Laboratory 1: Albany, New York; laboratory 2: Austrian Newborn Screening Laboratory, Vienna, Austria. 4 Replicates.

the affected Krabbe patient and healthy newborns [patient⬘s GALC activity was below the cutoff for retesting of ⬍12% according to Orsini et al. (9 )]. The lower GALC activity might be due to slightly different sample workup. These preliminary results demonstrated that both assays are comparable, but further studies with more positive controls are required. We minimized a potential source of ion suppression and residual substrate interference due to insource fragmentation by using the analytical column to separate the substrate from the product. Other sources of ion suppression such as salt, proteins, and phospholipids were eliminated by using Turboflow chromatography (18 ). The purchase of a commercial 2-channel online sample cleanup system (TLX-2) is approximately €150 000, and screening for LSDs requires one additional mass spectrometer for the high-throughput analysis of approximately 100 000 samples per year. A detailed overview of cost calculations for both LLE and SPE screening and a Turboflow assay is provided in the online Supplementary Materials 2. The latter protocol has lower run-time costs (⬍€0.80) and fewer personnel requirements, and does not require the purchase of liquid-handling stations for automated processing of SPE. One limitation of the current protocol is the separate incubations on day 1, which require several additional microtiter plates and sample handling. However, Gelb et al. recently demonstrated that these preanalytical steps can be simplified by combining incubation buffers for Pompe, Fabry, and MPS I diseases; hence additional time and expenses for consumables can be saved (2 ). With a biochemical assay it is not possible to discriminate between pseudodeficiency genes that result in low enzyme activities and genes that lead to a severe phenotype. Prospective screening for LSDs has to be accompanied by genetic confirmatory testing [e.g., 1292 Clinical Chemistry 57:9 (2011)

Krabbe screening in New York state (9 )]. Results of this and other studies have demonstrated that enzymatic assays that use MS are robust and accurate for the detection of known symptomatic LSD patients (2, 11, 12 ). The rarity of some of these diseases will, however, necessitate more extensive population-based studies to accurately evaluate the true frequency of both false-negative and false-positive results that occur with the use of this biochemical screening approach. Quality of screening is an important issue, and hence certified substrates and QCs must be produced under good manufacturing practices with regulatory approvals. Quality assurance programs, e.g., the Newborn Screening Quality Assurance Program at the CDC (Atlanta, GA) provides QC standards that are essential to maintain a high quality of screening (25 ). Worldwide collaborative projects such as the Region 4 Stork website (http://www.region4genetics.org) are necessary to ensure safety and consistency of screening between states and even within countries (26 ). The availability of suitable treatments for some of these disorders has resulted in increased efforts to develop new, reliable, and robust methods that can be used to perform high-throughput population screening through established NBS programs worldwide. New protocols and technologies are now available that permit higher efficiency (2, 27, 28 ). Nonetheless, the current experience of nationwide screening for LSDs is limited. Several small pilot projects have been started, but whole-population LSD NBS has been implemented on a routine basis in only 2 jurisdictions. Screening for Pompe disease was introduced in Taiwan because of the high population prevalence of this disease (8 ), and screening for Krabbe disease was started in 2006 in New York state (29 ) because hematopoietic stem cell transplantation during the newborn stage demonstrated improved outcomes (30 ).


We are faced with rapidly advancing technical possibilities and industry-driven development of enzyme replacement therapies that are still limited to just a small number of LSDs, a situation that is raising controversial discussions worldwide. These disorders were low-priority targets in the American College of Medical Genetics expert panel, in part because detection was not considered feasible, cost-effective, and simple (31 ). This viewpoint has changed owing to recent developments regarding high-throughput screening technologies (3, 27, 28 ) that allow simplified, less laborious, and more cost-effective (⬍€1 per sample) multiplex screening of several LSDs from a single blood spot.

or revising the article for intellectual content; and (c) final approval of the published article.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting

Acknowledgments: We are grateful to Joseph DiBussolo and Jeffrey Zonderman of Thermo Scientific for support and technical assistance with mass spectrometry, Hui Zhui and Victor de Jesus of the CDC, and Joan Keutzer of Genzyme.

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: B. Shushan, Thermo Scientific. Stock Ownership: None declared. Honoraria: None declared. Research Funding: Grant from the Austrian Ministry of Health (GZ 20.501/40-IV/A/2007). Expert Testimony: None declared. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

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