1. FilmArray™ - Nested Multiplex PCR for Multi-pathogen Screening
Beth Lingenfelter
Regulatory Affairs Manager
Kirk Ririe
CEO, FilmArray Inventor
Mark Poritz, PhD
Senior Scientist
Film Array Program Lead
Todd Ritter
Corporate Development Officer
Marshall McCarty
Director QA/RA

2. Meeting Objectives and Agenda
Start a dialogue about FilmArray
Obtain input for product design
Gain insights concerning regulatory issues
Agenda
Introductions and background– Beth Lingenfelter
Principle of operation – Kirk Ririe
Test system evaluation data – Kirk Ririe
Controls – Mark Poritz
Questions – OIVD?

3. Idaho Technology Inc – A PCR company
1990 – Rapid Air Thermocyclers
1996 – ITI launches LightCycler
1997 – Roche licenses LC from ITI
1999 – R.A.P.I.D., military version of LC
2003 – Awarded JBAIDS contract
2005 – Cleared JBAIDS Anthrax Detection Kit
2007 – Submitted JBAIDS Plague and Tularemia Detection Kits

4. How does it work? Nested Multiplex PCR (nmPCR)
High-sensitivity assays for large panels of biological agents.
Chemical Circuits in a Pouch –
Closed system for complex chemistry
Bead Beating to liberate nucleic acids
Magnetic bead nucleic acid extraction
Reverse transcription for RNA targets
Nested Multiplex PCR
High Resolution DNA melting curve analysis
Instrument Automation

5. Nested PCR Pathogen Genome RNA or DNA Outer primers,
Reverse transcription,
1st Stage PCR
200bp
Dilution Step,
Inner primers, 2nd Stage PCR
90bp

6. Primary Multiplex RT-PCR
Nested Multiplex PCR
2nd Stage PCR
LCGreen or Taqman
Dilute 100 fold
Between 1st & 2nd stage reactions 1F 1R 2R 3R 5R 6R 3F 2F 6F 4F 5F 4R
Primary Multiplex RT-PCR

7. Robust Multiplex PCR 1 - 5000 x LOD C. burnetti
x LOD C. burnetti + 0.1x LOD B. anthracis (100 ul of a 100 CFU/ml BA sample)

8. 20 Organism Pool

9. Chemical Circuit-boards
Freeze dried reagents for sample preparation and nested multiplex PCR (nmPCR) (A)
Reagents delivered into working blisters pneumatically (B)
1st Stage PCR (Blisters F and G)
High-density array of 120, 1 ul 2nd stage nmPCR reactions (I)

10. Instrument Automation – Electro-pneumatic Integration
Silicone bladders inflate over blisters to move liquids
Pistons close channels to select flow path
All components integrated in a single core module
Air Channels
Valve
Window - 2nd Stage PCR
Pneumatic Bladders

11. Prototype FilmArray™ Instrument
Fully automated
1 hour to result
System weight 14 lbs + laptop
Five prototype systems in operation

12. The Instrument Generates a Complex Data Set

13. MetaAnalysis Summary

14. Analysis for each organism

15. Analyze the results for each assay

16. System Evaluation Data
Demonstration for US Air Force EOS program,
Lackland AFB,
San Antonio, TX
URI pre-clinical test
Primary Children’s Medical Center (PCMC)
Salt Lake City, UT
IRB approved protocol

17. Lackland AFB Demo – Jul 07 3 Pre-production FilmArray™ Instruments
Pooled ATCC samples and frozen de-identified clinical specimens

18. Lackland Results
Blinded test demonstrated very reproducible multiplex results across three instruments

19. PCMC Test System Evaluation
100 frozen samples tested at ITI, (collected February-March 2007)
50 Samples tested at PCMC (October – November 2007)
Triplicate results are compared to DFA, discordant results are being followed up with sequencing

20. Detection Rates for FilmArray and DFA

21. PCMC Study Results To-date

22. Triple Infection
Bocavirus – black
PIV2 – blue
Human Rhinovirus - green
Bocavirus – black, PIV2 – blue, Human Rhinovirus - green