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The NeoLSD™ MSMS kit is intended for the quantitative measurement of the activity of the enzymes acid-9glucocerebrosidase (ABG), acid-sphingomyelinase (GAA), 8 galactocerebrosidase (GALC), agalactosidase A (GLA) and a-L-iduronidase (IDUA) in dried blood spots (DBS) from newborn babies. The analysis of the enzymatic activity is intended as an aid in screening newborns for the following lysosomal storage disorders (LSD) respectively; Gaucher Disease, Niemann-Pick A/B Disease, Krabbe Disease, Fabry Disease, and Mucopolyaccharidosis Type I (MPS I) Disease.

The NeoLSD MSMS test system uses mass spectrometry to quantitatively measure the activity of six lysosomal enzymes simultaneously from a dried blood spot sample. The NeoLSD MSMS test system is comprised of: 1. NeoLSD MSMS kit, including substrates, internal standards, solutions and controls 2. The QSight Instrument is comprised of: QSight® 210 MD Mass Spectrometer O QSight HC Autosampler MD Instrument Software O QSight Binary Pump MD O Simplicity Instrument control software: O Simplicity Data Processing software (by sample): O O PerkinElmer MSMS Workstation Data Processing Software The NeoLSD MSMS kit evaluates enzyme activities by measuring the product generated when an enzyme reacts with a synthesized substrate to create a specific end product. The activities of the six lysosomal enzymes present in a 3.2 mm punch from a dried blood spot (DBS) are simultaneously measured by the NeoLSD MSMS kit. The punches are incubated with the assay reagent mixture which contains; - six substrates, one corresponding to each lysosomal enzyme - six stable-isotope mass-labeled internal standards (IS) each designed to chemically resemble each product generated - . a buffer to maintain the reaction pH, and to carry inhibitors to limit activity from competing enzymes if present and additives to enhance the targeted enzyme reactions.

The NeoLSD MSMS Kit is intended for the quantitative measurement of the activity of the enzymes acid-pglucocerebrosidase (ABG), acid-sphingomyelinase (ASM), acid-a-glucosidase (GAA), B-galactocerebrosidase (GALC), α-galactosidase A (GLA) and α-L-iduronidase (IDUA) in dried blood spots (DBS) from newborn babies. The analysis of the enzymatic activity is intended as an aid in screening newborns for the following lysosomal storage disorders (LSD) respectively; Gaucher Disease, Nieman-Pick A/B Disease, Pompe Disease, Fabry Disease, and MPS I Disease.

The NeoLSD MSMS test system uses mass spectrometry to quantitatively measure the activity of six lysosomal enzymes simultaneously from a dried blood spot sample. The NeoLSD MSMS test system is comprised of: 1. NeoLSD MSMS kit, including substrates, internal standards, solutions and controls 2. Waters TQD MSMS instrument comprised of, a. Waters 1525 sample pump b. Waters 2777c autosampler c. Waters MassLynx v4.1 firmware C. d. Power cables, tubing, syringes, connection cables 3. Waters NeoLynx v4.1 software and computer with monitor 4. PerkinElmer MSMS Workstation Software The NeoLSD MSMS kit evaluates enzyme activities by measuring the product generated when an enzyme reacts with a synthesized substrate to create a specific end product. The activities of the six lysosomal enzymes present in a 3.2 mm punch from a dried blood spot (DBS) are simultaneously measured by the NeoLSD MSMS kit. The punches are incubated with the assay reagent mixture which contains; . six substrates, one corresponding to each lysosomal enzyme . six stable-isotope mass-labeled internal standards (IS) each designed to chemically resemble each product generated . a buffer to maintain the reaction pH, and to carry inhibitors to limit activity from competing enzymes if present and additives to enhance the targeted enzyme reactions.

The SEEKER System, including the SEEKER Instrument and the SEEKER LSD Reagent Kit-IDUA|GAA|GBA|GLA for use on the SEEKER Instrument, is intended for quantitative measurement of the activity of a-L-iduronidase, a-D-glucosidase, Bglucocerebrosidase and a-D-galactosidase A from newborn dried blood spot specimens as an aid in screening newborns for Mucopolysaccharidosis Type I, Pompe, Gaucher and Fabry diseases. Reduced activity of these enzymes may be indicative of these lysosomal storage diseases. The enzymes measured using the SEEKER LSD Reagent Kit-IDUA|GAA|GBA|GLA and their associated lysosomal storage diseases are listed below. | Enzyme (abbreviation) | Disease | |----------------------------|--------------------------------------| | α-L-iduronidase (IDUA) | Mucopolysaccharidosis Type I (MPS I) | | α-D-glucosidase (GAA) | Pompe | | β-glucocerebrosidase (GBA) | Gaucher | | α-D-galactosidase A (GLA) | Fabry |

The SEEKER System employs digital microfluidic technology to measure multiple lysosomal enzymatic activities quantitatively from newborn dried blood spot specimens. The following components are provided: - 1. SEEKER Instrument (including USB and power cables), Desktop PC with monitor, keyboard, mouse, and the Spot Logic software. - 2. SEEKER LSD Reagent Kit IDUA|GAA|GBA|GLA containing enzyme specific substrate reagents, dried blood spot extraction buffer, reaction stop buffer, 4 levels of calibrators and quality control dried blood spots containing 4 levels of quality control (OC) material. Each Reagent Kit contains sufficient consumables for 1440 tests. The contents of the kit are listed below: | Component | Contents | Quantity | |--------------------------------------|---------------------|------------| | Quality control<br>dried blood spots | QC-Base Pool (QCBP) | 1x15 spots | | | QC-Low (QCL) | 1x15 spots | | | QC-Medium (QCM) | 1x15 spots | | | QC-High (QCH) | 1x15 spots | | Enzyme substrates | IDUA | 9x100 µL | | | GAA | 9x100 µL | | | GBA | 9x100 µL | | | GLA | 9x100 µL | | Calibrators | Calibrant A (CALA) | 9x50 µL | | | Calibrant B (CALB) | 9x50 µL | | | Calibrant C (CALC) | 9x50 µL | | | Calibrant D (CALD) | 9x50 µL | Other components needed to run tests include the following: | Component | Contents | Quantity | |----------------|-------------------------|------------------| | Other Reagents | Extraction Buffer (EXT) | 9 x 30 mL | | | Filler Fluid | 9 x 10 mL | | Cartridge | SEEKER cartridge | 1 x 36 | The composition of the enzyme reagents and buffers are summarized below: | Reagent (Description) | Composition | |---------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------| | IDUA<br>(α-L-iduronidase substrate) | 2 mM 4-MU-α-L-iduronide sodium salt<br>3mM D-saccharolactone<br>0.04 M acetate buffer, pH 3.5<br>20 mM methyl β-cyclodextrin<br>0.01% Tween20 | | GAA<br>(α-D-glucosidase substrate) | 5 mM 4-MU-α-glucopyranoside<br>12 µM acarbose<br>0.04 M acetate buffer, pH 3.8<br>20 mM methyl β-cyclodextrin<br>0.01% Tween20 | | GBA<br>(β-glucocerebrosidase substrate) | 16 mM 4-MU-β-glucopyranoside<br>0.05 M/0.1M citrate phosphate buffer, pH 5.2<br>0.01% Tween20<br>1.5% sodium taurocholate | | GLA<br>(α-D-galactosidase A substrate) | 10 mM 4-MU-α-galactopyranoside<br>145 μM N-acetyl galactosamine<br>0.04 M acetate buffer, pH 4.6<br>20 mM methyl β-cyclodextrin<br>0.01% Tween20 | | Stop Buffer STB<br>(reaction stopping buffer) | 0.6 M NaHCO3, pH 11.0 in 0.04% Tween 20 | | Extraction Buffer EXT<br>(dried blood spot extraction buffer) | 0.1% Tween 20 in water | | Filler Fluid<br>(medium for droplet movement) | 0.1% Triton X-15 in 5cSt silicone oil | The Seeker Calibrators are supplied as part of the SEEKER LSD Reagent Kit -IDUA|GAA|GBA|GLA. The calibrators consist of 4 levels of aqueous preparation of 4methylumbelliferone sodium salt (4-MU) in 0.6M sodium bicarbonate buffer, pH 11.0 with 0.01% Tween 20. The concentration of 4-MU in each of the 4 calibrators is indicated in the table below: | Calibrator Level | Concentration of 4-MU | |---------------------|-----------------------| | Calibrant A (CAL A) | 0.0375 µM | | Calibrant B (CAL B) | 0.0750 µM | | Calibrant C (CAL C) | 0.1500 µM | | Calibrant D (CAL D) | 0.3000 µM | The quality control dried blood spots include 4 levels of control material: OC Low (QCL), QC Medium (QCM) and QC High (QCH). The composition of the 4 quality control dried blood spot (DBS) is summarized below. QCBP is used to fill empty wells on a cartridge. | Quality Control Level | Composition | |-----------------------|----------------------------------------------------------------------------------------------------------| | QCBP | Heat inactivated human serum, adjusted to ~50% hematocrit<br>using human red blood cells | | QCL | 5% cord blood and 95% heat inactivated serum, adjusted to<br>~50% hematocrit using human red blood cells | | QCM | 50% cord blood and 50% heat inactivated serum adjusted to<br>~50% hematocrit using human red blood cells | | QCH | Human umbilical cord blood, adjusted to ~50% hematocrit<br>using human red blood cells | The enzymatic activity values for the quality control DBS measured by the manufacturer are given on the lot specific quality control certificate included in each assay kit for all levels except QCBP. Each laboratory should establish its own mean and acceptable ranges for the quality control materials. ### 3. SEEKER Cartridges - 4. Finnipipette Novus 8-channel automatic pipette 1-10 uL - 5. Finnipipette Novus 1-channel automatic pipette 10-100 uL