Presentation on theme: "Next Generation Panel Oriented Diagnostics The FilmArray Pathogen Identification System Idaho Technology."— Presentation transcript:
1Next Generation Panel Oriented Diagnostics The FilmArray Pathogen Identification System Idaho Technology
2The FilmArrayToday we get to introduce you to a new molecular diagnostic platform that we are very excited about at Idaho Technology – The FilmArray. Today, we’ll be talking about:what the FilmArray ishow it worksour new respiratory paneland, most importantly, what it can do for youAdditionally, today, we are going to fire up the FilmArray and do a live demo so that you can see it action.
3User-Friendly Multiplex PCR The FilmArray is…User-Friendly Multiplex PCRAutomated ProtocolIntegrated Sample PrepAutomated Results AnalysisWe set out to make the FilmArray the most user-friendly multiplex PCR platform available. It has:Completely automated protocolIntegrated Sample PrepAutomated Results AnalysisSpeedMassive multiplex PCRLet me tell you a little more…Multiplex PCRSpeed
5Run Set-up Inject sample Load pouch in FilmArray Enter pouch and sample ID, user info, then press go!
6Automated ProtocolWe all know there is automated and then there’s automated. The FilmArray is automated.In a typical PCR reaction you start with a sample, take some reagents, some disposables and an DNA extraction robot and you extract the nucleic acids from the sample. You then transfer the nucleic acids, along with some additional reagents, to a thermal cycler instrument for PCR. If you are doing a deep multiplex analysis, you then transfer your amplified product, along with even more reagents, to analysis instrument which generates your results.With the FilmArray, you start with a sample add it to your sample buffer. You add your buffered sample and some rehydration solution to the FilmArray pouch, load the pouch on the FilmArray instrument, start the run and your done.
7Start the run & walk away Automated Protocol5 min of hands on timeStart the run &walk awayWith the FilmArray, you can set up a run in 5 minutes or less. This hands on labor involves simply injecting sample and rehydration buffer into a FilmArray pouch, loading the pouch into the FilmArray instrument, and starting the run. All subsequent sample manipulations, reagent transfers, and results analysis are completely automated – once you start the run you are free to walk away.The automated protocol includes integrated sample prep…
8Integrated Sample Prep One of the key aspects of our automated protocol that I want to emphasize is integrated sample prep. We have taken all the reagents, the instrument, and the disposables required to extract and purify nucleic acids from the patient specimen and integrated it all into the FilmArray. Integrated sample prep means less labor, less cost, less overhead, and less hassle for your lab.After the FilmArray extracts and purifies all the nucleic acids from the specimen, it automatically performs PCR analysis and reports the results.
9Multiplex PCR Simultaneous detection of dozens of targets from one patient sampleThe FilmArray performs multiplex PCR – simultaneously detecting dozens of DNA targets from one patient sample.This is the ideal approach for getting the most possible work done for least possible effort. In one run, with 5 minutes of labor, you get dozens of results.
10Automated Results Analysis Positive or negative call for each targetThe FilmArray analyzes real-time PCR curves, checks the results of the controls, and produces a positive or negative result for each target analyzed.
12Speed…. It’s really fast. Sample prep1 hourRT-PCRMultiplex PCRAnalysisResultsAnd it’s really fast.The FilmArrayExtracts and purifies nucleic acids from a patient specimenConverts and RNA into DNA via at Reverse Transcription stepPerforms a multiplex PCRAnalyzes all the dataAnd reports the resultsAll in less than 1 hour!This allows you to do what you do best – get results to your customers in a more timely fashion.And the icing on the cake is that you don’t get just one result in one hour but…
14Sample extraction & purification The FilmArray PouchReagent StorageChemical Circuit Board2 minutesThe FilmArray pouch is made up of two sections:A series of reagent storage chambersA chemical circuit boardAll the necessary reagents required for sample preparation, RT-PCR, PCR, and detection are stored in the reagent storage chambers.The chemical circuit board is made up of 2 layers of thin plastic film which contains various compartments for each of the steps I just mentioned as well as channels between each compartment.The ability to store and manipulate these reagents in this pouch allows us to completely automate the entire process downstream of sample loading.Sample extraction & purification1st stage multiplex PCR2nd stage PCR
15The FilmArray – How Does it Work? FilmArray Animation2 minutesTo further help you understand how the FilmArray works, here is an animated description.
16Automated Protocol Bladders inflate over blisters to push liquid Air ChannelsPistons close channelsPneumatically driven plungers in the FilmArray instrument are driven into the reagent storage chambers and delivers the appropriate reagents at the right time and the right place.The chemical circuit board is manipulated by pneumatically opening and closing the various reaction blisters as well as the channels between the reaction blisters. In this way, the FilmArray instrument is able to drive reagents left and right through the chemical circuit board.All of this allows us to provide you with a completely automated protocol. The FilmArray mixes all the appropriate reagents at the appropriate place and at the appropriate time – so that you don’t have to.Pneumatic Bladders
17The FilmArray: Video analysis 2 minutesTo further help you understand how the FilmArray works, here is a video showing how the reagents are moved around within the pouch.This is time-compressed actual footage of a FIlmArray run.
18Integrated Sample Prep Once the patient specimen is injected into the pouch and run began, the specimen is bead beat to release all nucleic acids. Bead beating has been shown to be an effective and robust method for nucleic acid extraction for a variety of sample types. It’s like a micro-blender for cells and viruses.After the nucleic acids have been released they are bound by magnetic beads and then moved from the sample lysis blister to the purification blisters. Here the nucleic acids are washed 3 times to remove any remaining cellular and viral debris.The last sample prep step is to elute the nucleic acids from off the magnetic beads and move them to the PCR I blister.++=
19Nested MultiPlex PCR 3 minutes Next, the purified nucleic acids are moved to the PCR I blister. Here any RNA is converted into DNA via a reverse transcription step. Then a massive multiplex PCR reaction is performed – but only for a limited number of cycles. During the early cycles of PCR, reagents are plentiful and do not become a limiting factor until the later PCR cycles. So after a limited number of PCR cycles, before competitive effects become problematic, the FilmArray halts the PCR reaction.The products from PCR I are then diluted to functionally remove any remaining PCR I primers. The diluted PCR I products are then combined with a fresh primer free master mix. Aliquots of this solution are then distributed to each well of the array in the PCR II blister. Individual primer sets are pre-spotted in each well of this array. Each of these primer sets are nested primers designed to detect and amplify one of the PCR products generated in PCR I. Thus PCR II is performed in traditional single-plex fashion – so during the detection phase the later cycles of PCR, we eliminate the competitive effects common to multiplex PCR.
20Automated Results Analysis 102 individual 2nd stage PCR wellsEach well contains one reactionFluorescent data is generated for each wellDuring the PCR II phase, the FilmArray generates real time PCR curves for each well in the array.
21Automated Results Analysis All targets tested in triplicateWith so much real-estate in this array we are able to test all targets in duplicates or triplicates.
22Automated Results Analysis For some organisms we target multiple genes – each tested in triplicateSoftware generates an automated call for each organismAnd some organisms, we look at multiple genes – each in triplicate.At the completion of PCR II, the FilmArray software analyzes the data from each well – including several controls. The software performs a complex meta analysis of the data for each replicate of each target for each organism. Based on this analysis the FilmArray reports a positive or negative result for each organism.
23The FilmArray Respiratory Panel ViralAdenovirusBocavirusCoronavirus 229ECoronavirus HKU1Coronavirus OC43Coronavirus NL63Entero VirusInfluenza AInfluenza A H1Influenza A H1 2009Influenza A H3BacterialBordetella pertussisChlamydophila pneumoniaeMycoplasma pneumoniaeInfluenza BMetapneumovirusParainfluenza 1Parainfluenza 2Parainfluenza 3Parainfluenza 4Respiratory Syncytial VirusRhinovirus
27Triple Infection Bocavirus – black PIV2 – blue Human Rhinovirus - greenBocavirus – blackPIV2 – blueHuman Rhinovirus - green27
28Simultaneous Detection of 12 Organisms Flu A H1Flu A H3Flu BRSVPara Flu 1Para Flu 2Para Flu 3AdenoHuman Meta PneumoRhinoBocaS. pneumoniae1 minuteThis experiment was run using our Bio-Threat panel. In this experiment 5 low-copy targets were spiked with 1 high copy target into one sample. As you can see the FilmArray was able to detect all 5 low-copy targets at their limit of detection.
30Internal pouch Controls Controls currently in the pouch:RNA Process mRNA from freeze-dried S. pombePouch pass/fail is set on this control1st Stage PCR synthetic oligos in PCR12nd Stage PCR synthetic oligos in PCR2Useful for understanding failure modesHuman mRNA assay for human mRNA in sampleWe are collecting data
31Location of the controls PCR2 control(spotted with PCR2 primers)2 minutesThe FilmArray pouch is made up of two sections:A series of reagent storage chambersA chemical circuit boardAll the necessary reagents required for sample preparation, RT-PCR, PCR, and detection are stored in the reagent storage chambers.The chemical circuit board is made up of 2 layers of thin plastic film which contains various compartments for each of the steps I just mentioned as well as channels between each compartment.The ability to store and manipulate these reagents in this pouch allows us to completely automate the entire process downstream of sample loading.Yeast controlFreeze dried into sample wellPCR1 controlFreeze-dried with Primer Pellet
33The FilmArray – Data FilmArray vs DFA in 387 Pediatric NPAs Pediatric NPA samples were tested by DFA (blue) and by FilmArray (maroon).
3482% of 387 pediatric NPA samples tested positive by FilmArray analysis 2 minutesGetting back to the data from our collaboration with Primary Children’s Medical Center….In this collaboration we tested 387 pediatric NPAs and compared the results to DFA.As you can see the FilmArray has a much higher rate of positive calls vs. DFA. 82% of the samples tested on the FilmArray were positive versus only 47% positive tested via DFA.Of course on the FilmArray we are able to test for more organisms than DFA. Most of the positives we saw were due to a few major contributors : Rhino Virus, Adeno Virus, RSV, and Influenza A Virus.
3532% tested positive for 2 or more pathogens by FilmArray analysis 1 minuteOne of the more interesting observations from this data set is the rate of multiple positives detected on the FIlmArray. 32% of the 387 pediatric NPAs we tested in collaboration with PCMC tested positive for 2 or more pathogens – compared to only 2.1% by DFA.What are the clinical implications of this? Great question. We don’t know. But isn’t exciting to finally have the tools to address that question.