Evaluation of DiaSorin Molecular Enterovirus (EV) Primer Pair and Cytomegalovirus (CMV) Primer Pair in Laboratory Developed Test (LDT) Implementation on the Open, Random-Access NeuMoDx™ 288 Molecular System

AMP Corporate Workshop

Introducing the next generation of molecular diagnostics11/6/2019

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Facts and figures 2017

EMPLOYEESMore than 110 employees located in Ann Arbor, Michigan, USA110+

COMPANYNeuMoDx Molecular is a privately-held company which began operations in 20122012

SOLUTIONSA family of sample-to-result molecular (“MDx”) solutions for hospital and clinical reference laboratory customers. Superior clinical and operational performance achieved through integrating patented sample preparation chemistry, proprietary NeuDry reagents and patented multiplexed microfluidics and liquid handling robotics

2

CERTIFICATIONSISO13485:2016 certified company, cGMP compliant production facility & FDA cleared/registered and CE Certified products.

ISO

PATENTSStrong IP portfolio includes 20+ US patents, corresponding foreign patents & applications >20

4

– The ability to handle high and low volume tests

– Ability to continuously add samples

– Fast results for timely patient decisions

– STAT capability– Reflex capability– Quick to start with

minimal preparation– Low maintenance

– Fully integrated, including IT– Minimal hands on and long

walkaway– Visual prompts– Low training burden

Throughput

– Consolidating as many tests as possible on a single system

– CE-IVD– LDT– Scalable

– No batching of tests– No batching of sample types– No fixed criteria to make the

workflow efficient

– Cost constraints continue to drive decisions

– Laboratories understand they need to look at whole test costs

Laboratory demands driving platform choice in core labs

Turnaround Time Ease Of Use

Consolidation Random-access Total Cost of Ownership

5

Original design goals

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Key Customer Desires

Easy to Use

Full Automation/‘Sample-to-Result’

True Random Access

Fast Time to Result

Assays Stored On-Board

Low Cost per Test

High Throughput

Long In-Use Reagent Stability

Open System/LDT capability

Continuous Access

NeuMoDx Molecular Systems

NeuMoDx 96 Molecular System NeuMoDx 288 Molecular System

Medium-sizeCore/Commercial

Labs

LargeCore/Commercial

Labs

6(1) All info for 288 instrument. For 96 instrument, up to 96 specimen tubes, >4 hour walkaway window, up to 20 different tests stored on board, and up to 20 specimens per hour (2) Some assays currently between 15 and 30 days in-use life but expected to reach >60 days with additional testing

Highest PerformanceEasiest to Use Saves Most Time and Money

State-of-the-Art Sensitivity

Continuous Loading of Consumables, Reagents & Specimen Tubes

– Priority given to STAT samples

Quantitative and Qualitative Tests

Auto-Rerun and Auto-Reflex

All Common Specimen Types

‘Open’ System

– Multiple options to conduct LDTs through addition of liquid reagents, probe/primer only or all dry reagents

Extensive Multiplexing Capabilities

– Five color x High Resolution Melt(2) x multiple cartridge lanes

Fast Time to First Results

– 40-60 minutes for DNA

– 75-85 minutes for RNA

Low Cost per Result

– Competitive cost per test with low labor costs

Dry Format Assays (Load & Forget)

– Ambient storage shelf life >1 yr.

– >2 months(2) in-use life with no need to remove from system

High Throughput (1)

– Up to 42 specimens per hour

Minimized Specimen & Consumable Handling

– Lowest operator interaction of ANY MDx system

Walkaway System

– Load specimen tube carrier onto system and walkaway

– Up to 288 specimen tubes for >6 hour walkaway window (1)

True Random Continuous-Access

– Mix specimen types and tests; no batching required

Automated Consumable Loading and Disposal

Graphical User Interface

Up to 30 (1) Different Tests Stored in System for Immediate Use

– No need for operator to load desired test with each specimen

– Multiple tests from one specimen tube

The outcome

Implementing Lab Developed Tests on the NeuMoDx Molecular SystemMichelle MastronardiDirector of Systems IntegrationNeuMoDx

System Operation

1. Load primary specimen tube or secondary tube in Specimen Tube Carrier

2. Place Specimen Tube Carrier on Autoloader Shelf

3. Touch ‘load’ arrow on touchscreen user interface

4. Walk Away

When system has consumables loaded to complete the testing, the results will be available without further operator interaction.

2

3

1

9

Uses ALL Room Temperature Stable Reagents & Consumables

LDT, Master Mix Test Strip NeuMoDx Extraction Plate NeuMoDx Lysis Buffer

NeuMoDx CartridgeLDT, Primer Probe Test Strip

• No additional user prep• Long Shelf Life and In-

Use Life• Barcoded for intelligent

tracking

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Principles of Operation

• Fully automated steps– Operator interacts with specimen and NeuMoDx Software – Instrument automates:

• Sample pickup from specimens• Combination with lysis buffer for lysis and binding step• Extraction and purification of nucleic acids• Real-Time PCR and results processing• On-board reagent and consumable inventory

• Sample process control (Internal Control) is co-extracted with every extraction– It is part of the NeuMoDx Extraction Plate– SPC1 for DNA and SPC2 for RNA specimens

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Running an LDT: Defining Assay Definition File (ADF)

Flexibility to….define extraction parameters:– Choose between 5 lysis buffers depending on specimen matrix– Choose specimen input volume up to 550 µL– Choose lysis time and temperature

define PCR cycling parameters:– Set PCR stages and steps, including temperatures and times – Detect on up to 5 channels, with one reserved for detection of the sample process control

define overall run settings:– Qualitative vs. Quantitative analysis– Define and/or Create Standard Curves for quantitative assays– Set up Standards, External Controls, and Calibrators

define stability settings for specimen and primer/probe (ASR) onboard life

Intuitive, User-Friendly Software

Running an LDT Test

• User loads Master Mix Test Strips• User loads LDT Test Strips containing assay specific primers/probes

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Once defined, the user can load specimens for automated sample processing.

User defines Assay Definition File User loads and maps test-specific Primer/Probe Mix to the LDT Test Strip

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LDT Workflow on NeuMoDx Molecular Systems

Easiest Workflow for LDT Implementation

Summary

• NeuMoDx Molecular Systems provide fully integrated, modular and scalable sample-to-insight solutions

• Easy to use, intuitive user interface• Very fast time to first result• True random access with up to 30 different assays onboard• Continuous loading & STAT capabilities for emergency

samples

• The NeuMoDx Systems allow seamless integration and automation of LDTs, using same workflow as IVD assays– Flexibility to run multiple IVDs and LDTs in parallel– Can automate commercially available ASRs or in-house

LDTs

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Evaluation of DNA and RNA LDT Implementation on the NeuMoDx Molecular System

Stephen Young, Ph.D., D(ABMM)Professor Emeritus of Pathology

University of New MexicoDirector of Research and Clinical Trials

TriCore Reference Laboratories Albuquerque, New Mexico 87102

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Background Previously we have reported implementation of two quantitative DNA Lab Developed Test onto the NeuMoDx Molecular System using Qiagen’s EBV and BKV ASRs based on Taqman technology.

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• Compared current manual EBV LDT process to NeuMoDx LDT

• System allowed IVD and LDT tests to be processed in parallel

• The time required to perform an LDT using the NeuMoDx 288 System vs. Current in-house method showed a reduction in both hands-on time and time to result

Workflow Advantages of NeuMoDx LDT Automation

AIMS of the Study

• Establish that Scorpion primer amplification chemistry could be used to develop LDTs on the NeuMoDx 288 System

• Develop two commercially available DiaSorin Molecular ASRs Scorpion primers into an LDT performed on the NeuMoDx 288 System

• Establish that it is possible to develop a quantitative DNA LDT and a qualitative RNA LDT.

• A quantitative CMV LDT using plasma as the matrix was selected.

• A qualitative Enterovirus assay using CSF as the matrix was selected.

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Required Materials

• DiaSorin Molecular ASRs used in this study• CMV Primer Pair (Code MOL9002) • Enterovirus Primer Pair (Code MOL9020)

•NeuMoDx Reagents & Consumables Used • NeuMoDx Lysis Buffer 1, Recommended for DNA extraction from plasma/serum and

CSF• NeuMoDx Extraction Plate, containing nucleic acid binding NeuMagBeads, lytic

enzymes, and universal sample processing controls• NeuMoDx Wash and Release Solutions• NeuMoDx Microfluidic Cartridge• NeuMoDx LDT, DNA Master Mix Test Strip and NeuMoDx LDT, RNA Master Mix, which

contain all reagents necessary for amplification with the exception of the assay specific primers/probes

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Evaluation Study OutlineThe following protocols were executed to establish the LDT and subsequently determine the efficacy of implementation of the LDTs

Establishing the DNA/RNA LDT1. Limit of Detection 2. Limits of Quantitation and Linearity (Quant assay only)

Evaluating the DNA/RNA LDT 1. Interference Study2. Cross reactivity3. Within Lab Precision Study4. Method Correlation/comparison – using Verification

Panels

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Establishing the CMV Quantitative LDT with DiaSorin Molecular CMV ASR

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Implementation on NeuMoDx Molecular System• Prepared Primer/Probe Mix and added 4 µL per test into the empty Primer/Probe Strip

• Loading the ASR Primer/Probe Mix is the only extra step necessary to automate an LDT on the NeuMoDx System

• NeuMoDx Software used to define run parameters in the Assay Definition File for the assays• Sample matrix: plasma, 200 µL• Lysis Buffer selection: NeuMoDx Lysis Buffer 1, 900 µL• Lysis and binding: 60°C for 8 minutes• Rapid PCR cycling with detection of both target and sample process control 95°C denature for 6 seconds 60°C anneal for 30 seconds

• Positive result call criteria: Maximum Ct cutoff: 40Minimum End Point Fluorescence, 1000

CMV Volume Per Test (µL)

DiaSorin Molecular CMV Primer Pair 1

10 mM Tris-HCl, pH 8.0 3

Total 4

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Limit of Detection: CMV LDTThe Limit of Detection of the CMV LDT was determined using the 1st WHO CMV International Standard. Five levels of target with a negative were tested using NeuMoDx LDT, DNA Master Mix

The limit of detection of CMV was determined to be 195.97 IU/mL 95% based on Probit-style analysis with CI (147.35-315.66 IU/mL).

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LDT CMV, Limit of Detection

Conc (IU/mL) Avg Ct StDev #

samples Pos % Pos

600 34.88 0.53 48 48 100%

300 35.42 0.82 45 44 98%

150 36.38 1.23 48 44 92%

75 36.93 1.24 48 34 71%

37.5 37.81 1.75 43 18 42%

0 No Amp NA 48 0 0% 0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4

% P

ositi

veConcentration Log10 IU/mL

CMV LDT LoD, Probit Analysis

Sigmoid Fit

Expt. Data

Calculated LoD

Linearity and Master Standard Curve

• Master Calibration Curves were established using data generated across three lots of LDT DNA MM Test Strips

• Excellent Linearity with no lot-to-lot variation observed for CMV

• Upper Limit of Quantitation (ULoQ) determined to be 7.3 Log10 IU/mL

y = -3.2486x + 43.651R² = 0.9885

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Ct

Concen, Log10 IU/mL

CMV LDT Master Calibration Curve,

Conc.[Log10 IU/mL] Avg Ct SD Reported Conc.

[Log10 IU/mL] Bias SD TAE %Pos

7.30 19.65 0.61 7.39 0.09 0.19 0.46 100%6.30 23.13 0.46 6.32 0.02 0.14 0.30 100%5.30 26.84 0.32 5.17 -0.13 0.10 0.06 100%4.30 30.00 0.39 4.20 -0.10 0.12 0.13 100%3.30 32.65 0.41 3.39 0.09 0.13 0.34 100%2.30 36.50 0.92 2.34 0.04 0.28 0.60 100%

y = 1x - 5E-05R² = 0.9885

0.0000

1.0000

2.0000

3.0000

4.0000

5.0000

6.0000

7.0000

8.0000

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Repo

rted

Con

c, L

og10

IU/m

L

Expected Conc, Log10 IU/mL

CMV LDT Linearity

Excellent correlation between CMV LDT reported concentrations and the expected concentrations with slope of correlation plot at 1 and intercept (bias) ~ 0.00005.

Lower Limit of Quantification – CMV LDT• Limit of Quantitation was defined as the concentration of target detected consistently (≥95%

sensitivity) with a total analytic error =1.0.• Here the Total Analytic Error (TAE) is defined as

𝑻𝑻𝑻𝑻𝑻𝑻 = 𝑻𝑻𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨 𝑩𝑩𝑩𝑩𝑩𝑩𝑨𝑨 + 𝟐𝟐 ∗ 𝑺𝑺𝑨𝑨𝑩𝑩𝑺𝑺𝑺𝑺𝑩𝑩𝑺𝑺𝑺𝑺 𝑫𝑫𝑨𝑨𝑫𝑫𝑩𝑩𝑩𝑩𝑨𝑨𝑩𝑩𝑨𝑨𝑺𝑺

• As seen from data above, TAE=1.0 for a value 150 IU/mL and 75 IU/mL (both below LoD levels)

• However, since the LLoQ cannot be less than LoD, the LLoQ was determined to be 2.3 Log10 IU/mL, or 200 IU/mL.

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LLoQ

Conc [IU/mL]

Expected Conc[Log10 IU/mL]

Actual Conc [Log10 IU/mL] N SD Bias TAE % POS

600 2.78 2.70 48 0.16 -0.08 0.25 100

300 2.48 2.53 45 0.25 0.06 0.56 98

200 2.30 2.34 20 0.28 0.04 0.60 100

150 2.18 2.24 48 0.38 0.06 0.82 92

75 1.88 2.07 48 0.38 0.19 0.96 71

37.5 1.57 1.80 43 0.54 0.22 1.30 42

LDT Assay Definition Finalized• At this point, the following parameters were determined for the CMV Quantitative LDT

• Limit Of Detection (LoD)• Lower Limit of Quantitation (LLoQ)• Linearity & Master Calibration Curve• Upper Limit of Quantitation (ULoQ)

• Final results processing parameters were defined with special attention to cutoff criteria for establishing valid amplification

• A Quantitative Assay Definition File was finalized for further evaluation

• Prior to performing further testing, two Calibrators (High and Low) were processed in triplicate to establish the Test Strip Lot-specific Calibration

• Calibration Validity was automatically evaluated by NeuMoDx Software

• Once established, the calibration is valid until the Lot of the Test Strip is changed or the ADF is modified

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Evaluation of Established CMV Quantitative LDT

Interference Study• Potential Interference of CMV LDT, with representative endogenous moieties typically encountered in

plasma matrix, was evaluated to determine the robustness of the test.

• Interference panels were prepared by spiking CMV at 600 IU/mL, into negative plasma samples containing high concentrations of the potential endogenous interference moieties listed in the table below.

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Interference Moiety

Avg Ct(N=3) StDev % Pos Exp Conc

[Log10 IU/mL]Conc

[Log10 IU/mL] Bias

Albumin 120 mg/mL 34.93 0.56 100 2.79 2.67 -0.12

Bilirubin0.025 mg/mL 34.45 0.54 100 2.79 2.82 0.03

Hemoglobin 1 mg/mL 34.66 0.51 100 2.79 2.76 -0.03

Triglycerides 2.5 mg/mL 34.69 0.66 100 2.79 2.75 -0.04

Pos Ctrl 34.94 0.61 100 2.79 2.67 -0.12

Neg Ctrl NA NA 0 NA NA NA

No interference was observed in the presence of any of the four interfering moieties tested

Within Lab Precision Study• Within Lab Precision was assessed by using a 4-member panel for CMV• Testing was performed across 6 rounds of 2 runs, spanning multiple days

Expected Conc[Log10 IU/mL] N N (POS) % POS Avg Conc

[Log10 IU/mL] Abs Bias Within Round SD

Within Run SD

5.82 36 36 100% 5.71 0.11 0.42 0.41

4.82 34 34 100% 4.77 0.05 0.12 0.08

3.82 36 36 100% 3.82 0.00 0.14 0.14

2.82 35 35 100% 2.78 0.04 0.20 0.18

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Verification Panel Testing

Good correlation between CMV LDT reported concentrations and the expected concentrations of the verification panel.Slope of correlation plot is 0.93 with intercept (bias) < 0.09

• The quantification of CMV using the CMV LDT was confirmed using a commercially available CMV verification panel from Exact Diagnostics.

• Exact Verification Panels consist of 6 panel members spanning the dynamic range of the CMV LDT.

Exact CMV Verification Panel (CMVP200)

Panel [Log10 IU/mL]

N N (POS) % POS

Reported Conc.

[Log10 IU/mL]SD Bias

[Log10 IU/mL]

6.60 5 5 100% 6.32 0.26 -0.28

5.60 5 5 100% 5.32 0.29 -0.28

4.60 4 4 100% 4.17 0.27 -0.43

3.60 4 4 100% 3.19 0.30 -0.41

2.60 5 5 100% 2.42 0.31 -0.18

2.30 5 4 80% 2.51 0.73 0.21

y = 0.9251x + 0.0853R² = 0.9847

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00Re

port

ed C

onc.

Log

10 IU

/mL

Expected conc. Log10 IU/mL

CMV Verification Panel

Method CorrelationThe concordance of DiaSorin Molecular CMV LDT with an FDA cleared CMV quant assay, cobas6800/8800 TaqMan® CMV Test, was evaluated with a contrived 5 member CMV panel spanning the dynamic range of the CMV LDT.

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Panel [Log10 IU/mL]

% Pos (N=4)

Avg[Log10 IU/mL]

Bias from Expected Bias from COBAS®

6.48 100% 6.41 -0.07 -0.055.48 100% 5.32 -0.16 -0.064.48 100% 4.31 -0.17 -0.013.48 100% 3.45 -0.03 0.152.48 100% 2.63 0.15 0.24

NeuMoDxN288

Panel [Log10 IU/mL]

Conc[IU/mL]

Volume Adjusment*

Conc[Log10 IU/mL]

Bias from Expected

6.48 5.0E+06 2.9E+06 6.46 -0.025.48 4.2E+05 2.4E+05 5.38 -0.104.48 3.7E+04 2.1E+04 4.32 -0.163.48 3.5E+03 2.0E+03 3.30 -0.182.48 4.3E+02 2.5E+02 2.39 -0.09

Roche cobas 6800

* The lowest volume on COBAS is 350 µL. The quantification has been equalized to 200 µL for the comparison purpose.

Method Correlation

y = 1.0807x - 0.4127R² = 0.9988

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

LDT

Conc

en. [

Log1

0 IU

/mL]

cobas 6800 conc. [Log10 IU/mL]

CMV LDT vs Roche cobas 6800

• Excellent correlation between CMV LDT reported concentrations and the expected concentrations of the verification panel with slope of correlation plot, 0.94 and intercept (bias) ~ 0.2.

• Excellent concordance between CMV LDT and Roche COBAS CMV Quant with slope of correlation plot, 1 and intercept (bias) < 0.45.

y = 0.9426x + 0.2029R² = 0.9962

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Repo

rted

Con

c. [L

og10

IU/m

L]

Expected Conc. [Log10 IU/mL]

CMV LDT, Exp vs Reported

Establishing the EV RNA LDT Using DiaSorin Molecular EV ASR

33

Implementation Of DiaSorin Molecular EV LDT on NeuMoDx Molecular System

• Prepared Primer/Probe Mix and added 4 µL per test into the empty Primer/Probe Strip

• Loading the ASR Primer/Probe Mix is the only extra step necessary to automate an LDT on the NeuMoDx System

• NeuMoDx Software used to define run parameters in the Assay Definition File for the assays• Sample Matrix: CSF, 200 µL. Minimum sample volume: 350 µL using Low Volume Carrier• Lysis Buffer selection: NeuMoDx Lysis Buffer 1, 900 µL• Lysis and binding: 45°C for 10 minutes• Rapid PCR cycling with detection of both target and sample process control: Reverse transcription: 50°C for 15 min Inactivation: 95°C denature for 4 min 95°C denature for 6 seconds 60°C anneal for 30 seconds

• Positive result call criteria: Maximum Ct cutoff: 40 Minimum End Point Fluorescence, 1000

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EV Volume Per Test (µL)

DiaSorin Molecular EV Primer Pair 0.8

10 mM Tris-HCl, pH 8.0 3.2

Total 4

Limit of Detection: EV LDTThe Limit of Detection of the EV LDT was determined using Exact EV control material.Five levels of target and a negatives were tested using one lot of NeuMoDx LDT, RNA Master Mix

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EV Concen.

EV LoD in CSF

Avg Ct StDev # samples POS % Pos

160 c/mL 35.16 1.71 48 47 98%

80 c/mL 36.30 2.46 42 39 93%

40 c/mL 37.52 3.34 44 36 82%

20 c/mL 40.80 3.86 47 24 51%

10 c/mL 38.13 4.09 47 15 32%

Neg na na 47 0 0%

The limit of detection of EV was determined to be 97.7 cp/mL, 95% based on Probit-style analysis with CI ( 69.8, 166.0).

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4

% P

ositi

ve

Concen Log10 cp/mL

EV Limit of Detection, Probit Analysis

Probit Fit Series2

Expt Data Calculated LoD

LDT Assay Definition Finalized

• At this point the Qualitative Assay Definition File was finalized for EV for use in further evaluation

• Final results processing parameters were defined with special attention to cutoff criteria for establishing valid amplification

• Prior to performing further testing, user-defined positive and negative external controls were processed to qualify the System.

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No interference was observed in the presence of any of the four interfering moieties tested

Interference Study

EV LDT – Interference Study Results

Interfering Moiety Concentration % Pos (N=3) Ct SD

Albumin 120 mg/mL 100% 34.89 0.77Hemoglobin 3 mg/mL 100% 34.56 0.58Triglycerides 5 mg/mL 100% 34.91 0.09

Blood 5% 100% 33.4 0.15

Control, Pos NA 100% 34.59 0.10

Control, Neg NA 0 NA NA

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• Potential Interference of EV LDT, with representative endogenous moieties typically encountered in CSF matrix, was evaluated to determine the robustness of the test.

• Interference panels were prepared by spiking EV into negative CSF samples containing abnormally high concentrations of endogenous moieties, listed in the table.

Within Lab Precision Study• Within Lab Precision was assessed by using a 4-member panel for both EV RNA LDT• Testing was performed across 6 rounds of 2 runs, across multiple days• Testing was performed using one lot of LDT, Master Mix reagent

Panel Conc [cp/mL] n n (POS) % POS Avg Ct SD Ct Within

Round SDWithin Run

SD

Medium Positive

1100 (~ 10x LoD) 24 24 100% 32.53 0.54 0.48 0.35

Low Positive 330 (~3X LoD) 24 24 100% 34.47 0.88 0.78 0.64

Low Negative 3.3 (~1/100 LoD) 23 2 8.7% 39.46 6.42 na na

True Negative 0 23 0 0% na na na na

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Verification Panel Testing: EV LDTThe detection of EV by EV LDT was determined using EV LoD verification panel (Exact)The panel contain 6 members with Cox A9 concentrations from 31.3 to 1000 cp/mL.

39

EV Verification Panel

Concen [cp/mL]

N (valid results) N (POS) %POS AVG Ct SD Ct

1000 12 12 100% 31.39 1.53

500 11 11 100% 31.86 1.46

250 11 10 91% 33.39 1.13

125 12 8 67% 33.27 3.75

62.5 12 8 67% 34.53 2.95

31.3 13 6 46% 33.54 2.74

Strain Coverage The inclusivity of EV LDT was demonstrated with 22 strains of EV cultures (1:10 dilution in UVT)

EV Strains Results Ct EV Strains Results Ct

Cox A9 Positive 14.35 Echo 6 Positive 14.84

Cox B1 Positive 14.50 Echo 9 Positive 13.52

Cox B2 Positive 14.70 Echo 11 Positive 16.76

Cox B3 Positive 14.31 Echo 13 Positive 14.04

Cox B4 Positive 15.08 Echo 17 Positive 16.06

Cox B5 Positive 14.34 Echo 18 Positive 15.40

Cox B6 Positive 13.79 Echo 20 Positive 16.30

Echo 1 Positive 14.73 Echo 30 Positive 13.73

Echo 2 Positive 14.13 Enterov68 Positive 17.84

Echo 3 Positive 13.97 Enterov70 Positive 14.44

Echo 4 Positive 14.79 Enterov71 Positive 14.85

EV LDT detected all the 22 strains tested.

Cross Reactivity/Interference Ten organisms that have been detected in CSF were spiked into CSF at concentrations > 1E6 CFU, cp/mL or TCID50/mL. These organisms did not cross react in the EV LDT. The potential interference of these organisms to EV LDT was also tested in the presence of EV at near LoDlevel, 330 cp/mL. No interference by these 10 organisms was observed.

OrganismsNo EV (N=3) With EV at 330 cp/mL (N=3)

OrganismsNo EV (N=3) With EV at 330 cp/mL (N=3)

% Pos Ct % Pos Avg Ct StDev % Pos Ct % Pos Avg Ct StDev

HSV1, 1E6 cp/mL 0 NA 100 34.8 1.21

Neisseria meningitidis A,

6E6 CFU/mL0 NA 100 33.08 0.09

HSV2,1E6 cp/mL 0 NA 100 35 0.46

Neisseria meningitidis D,

6E6 CFU/mL0 NA 100 33.09 0.44

Enterobacter aerogenes,

6E6 CFU/mL 0 NA 100 32.9 0.75 E.coli,

6E6 CFU/mL 0 NA 100 31.30 0.11

CMV,1E6 cp/mL 0 NA 100 34.7 0.81

Streptococcus agalactiae,

6E6 CFU/mL0 NA 100 34.14 0.26

Zika,6E6 TCID50/mL 0 NA 100 34.2 0.54

Staphylococcus aureus,

6E6 CFU/mL0 NA 100 32.90 0.38

Pos Ctrl NA NA 100 35.4 1.38 Pos Ctrl 0 NA 100 33.60 1.15

Neg Ctrl 0 NA 0 NA NA Neg Ctrl 0 NA 0 NA NA

Summary• One quantitative DNA and one qualitative RNA LDTs using commercially available

ASRs from DiaSorin Molecular were implemented on the NeuMoDx Molecular System providing a fully automated Sample-to-Result solution for LDTs

• Excellent analytical performance was demonstrated for both LDTs implemented in the study

• General purpose reagents provided by NeuMoDx were utilized in the study with no further modification/optimization

• Implementation of the LDTs with different ASRs or PCR chemistry on the NeuMoDx Molecular System was extremely straightforward; the NeuMoDx Software is intuitive and easy to use

• The NeuMoDx Molecular System provide a truly random access LDT platform. Both DNA and RNA LDT, quantitative and qualitative LDT can be run at the same time with any batch testing

• Onboard & room temperature stable reagents provided in unitized format significantly simplify the entire process and minimize waste

42

AcknowledgementsTriCore Reference Laboratories

Aaron Wagner

NeuMoDx MolecularHui Lin LeeMichelle MastronardiCatherine CouturePatrick MateasBetty WuSundu Brahmasandra

DiaSorin MolecularBojana Rodic-PolicMichelle Tabb

We would like to express our gratitude to DiaSorin Molecular for providing CMV and EV ASR Primer Pairs for this study.