easy-plex™ 384 system (high-plex) ref 9150 · the easy-plex™ 384 system (high-plex) is also...

9150r05 Instructions For Use August 2016 Page of 1 43

EASY-PLEX™ 384 SYSTEM (HIGH-PLEX) REF 9150

INSTRUCTIONS FOR USE

Before use, these Instructions for Use (IFU) must be read in conjunction with the applicable kit-specific IFU.


TABLE OF CONTENTS

1. WARNINGS AND LIMITATIONS 4 ......................................................................................................................

2. FURTHER INSTRUCTIONS REQUIRED 4 ..........................................................................................................

3. EASY-PLEX™ 384 SYSTEM (HIGH-PLEX) (REF 9150) COMPONENTS 5 ........................................................3.1 OTHER REQUIRED SYSTEM PRODUCTS 6 ..................................................................................................3.2 OPTIONAL ACCESSORY 6 ............................................................................................................................3.3 TECHNICAL AND ENVIRONMENTAL SPECIFICATIONS 6 ...........................................................................

3.3.1 Easy-Plex™ Processor 6 ...............................................................................................................3.3.2 Easy-Plex™ 384 analyser 7 .........................................................................................................

4. INTENDED USE 7 .............................................................................................................................................

5. PRINCIPLE OF THE METHOD 8 .......................................................................................................................

6. TIME REQUIRED TO ANALYSE A SAMPLE 9 ....................................................................................................

7. EASY-PLEX™ PROCESSOR PROCEDURES 10 .................................................................................................7.1 SWITCHING THE PROCESSOR ON/OFF 10 ..................................................................................................7.2 SETUP PROCESSOR DECK 11 ........................................................................................................................7.3 RUN THE PROCESSOR 12 ..............................................................................................................................7.4 PREPARE THE PLATE FOR THE ANALYSER 14 .............................................................................................7.5 EASY-PLEX™ SETUP SOFTWARE - SPECIAL FUNCTIONS 14 .....................................................................

7.5.1 Installing kit templates 14 .............................................................................................................7.5.2 Uninstalling kit templates 16 ........................................................................................................7.5.3 reset tip availability 16 ..................................................................................................................7.5.4 Create support package 16 .........................................................................................................

8. ANALYSERS 17 .................................................................................................................................................8.1 USING A EASY-PLEX™ 384 ANALYSER 17 ....................................................................................................

8.1.1 Switching the analyser on/off 17 ..................................................................................................8.1.2 Startup of the Easy-Plex™ Analyser software (RealTime_PCR Program) 17 ..........................8.1.3 To run the Easy-Plex™ 384 Analyser 18 .......................................................................................8.1.4 Installing Kit templates in 384 Analyser 20 .................................................................................

8.2 USING A ROCHE LC 480 ANALYSER 22 .......................................................................................................8.2.1 Further instructions for Roche LC 480. 22 ...................................................................................8.2.2 To Run a Roche LC 480 Analyser 22 ...........................................................................................

9. END OF RUN PROCEDURE 25 .........................................................................................................................9.1 PROCESSOR CLEAN UP PROCEDURE 25 ....................................................................................................9.2 VERIFICATION OF INSTRUMENT FUNCTION AND SAMPLE INHIBITION 26 .............................................

10. EASY-PLEX™ RESULTS SOFTWARE ANALYSIS 27 .........................................................................................10.1 OVERVIEW OF SOFTWARE OPERATOR INTERFACE CONVENTIONS 27 ..................................................10.2 OPEN 27 .......................................................................................................................................................10.3 REPORT 28 ...................................................................................................................................................

10.3.1 Analysis Report 28 .......................................................................................................................10.3.2 Result Table Report 29 ................................................................................................................10.3.3 Sample Report 29 .......................................................................................................................

10.4 EXPORT 30 ...................................................................................................................................................10.4.1 Results 30 .....................................................................................................................................10.4.2 Summary 31 .................................................................................................................................10.4.3 Analysis Summary 31 ..................................................................................................................10.4.4 Analysis Data 31 ..........................................................................................................................10.4.5 Cycling Data 31 ...........................................................................................................................10.4.6 Melt Data 31 ................................................................................................................................10.4.7 Exchange File 31 ..........................................................................................................................


10.5 VIEW BY SAMPLE 32 ....................................................................................................................................10.6 VIEW BY GENE 32 ........................................................................................................................................10.7 NORMAL SENSITIVITY 33 ............................................................................................................................10.8 HIGH SENSITIVITY 33 ..................................................................................................................................10.9 HISTORY 34 .................................................................................................................................................10.10 OPTIONS 35 ...............................................................................................................................................10.11 ABOUT 35 ....................................................................................................................................................10.12 OTHER SOFTWARE OPERATOR CONVENTIONS 35 ................................................................................

10.12.1 Resizing the window 35 ..............................................................................................................10.12.2 Advanced view 36 ......................................................................................................................10.12.3 Hiding curves 36 ........................................................................................................................

11. INTERPRETING SOFTWARE RESULTS 36 ........................................................................................................11.1 CALLS 36 ........................................................................................................................................................

11.1.1 Present 36 .......................................................................................................................................11.1.2 Present (Low) 36 ...........................................................................................................................11.1.3 Blank 36 .........................................................................................................................................11.1.4 High Fluorescence 36 ...................................................................................................................11.1.5 Check 37 ........................................................................................................................................11.1.6 Rejecting Calls 37 .........................................................................................................................11.1.7 Inhibited 37 ....................................................................................................................................11.1.8 Bad melt 38 ...................................................................................................................................

11.2 CORRECTED MELT 38 ..................................................................................................................................11.3 TAKE-OFF VALUES 38 ...................................................................................................................................11.4 CONCENTRATION 38 ...................................................................................................................................11.5 MULTIPLEXING IN STEP 2 PCR 38 ...............................................................................................................11.6 ALGEBRAIC CALLS 39 .................................................................................................................................

12. MAINTENANCE 39 ..........................................................................................................................................12.1 OPERATOR MAINTENANCE 39 ....................................................................................................................12.2 SERVICE AND CALIBRATION SCHEDULE 39 ..............................................................................................

13. EXPECTED CLINICAL PERFORMANCE 39 ......................................................................................................13.1 INTERFERING SUBSTANCES 39 ...................................................................................................................

14. TROUBLESHOOTING 40 ................................................................................................................................14.1 EASY-PLEX™ PROCESSOR TROUBLESHOOTING 40 ................................................................................14.2 EASY-PLEX™ ANALYSER TROUBLESHOOTING 41 ....................................................................................14.3 RESULTS TROUBLESHOOTING 41 ..............................................................................................................

15. TECHNICAL ENQUIRIES 41 .............................................................................................................................

16. ACKNOWLEDGEMENTS 42 ...........................................................................................................................

17. GLOSSARY 42 .................................................................................................................................................

18. DEFINITIONS 43 .............................................................................................................................................

19. REFERENCES 43.............................................................................................................................................


1. WARNINGS AND LIMITATIONS

WARNING: CAUTION. This device contains moving parts. Do not interact with the deck or open the hood while the device is in operation.

WARNING: PINCH POINT. Please take care when opening and closing the hood.

WARNING: BIOHAZARD. Some samples used with this instrument may contain infectious agents. Handle such samples with care in accordance with relevant safety regulations.

• WARNING: The Easy-Plex™ 384 System (High-Plex) must only be moved from its installed location by an authorised technician or representative, otherwise it may compromise safety and correct function.

• WARNING: During the Analyser operation, the thermal block and hot cover module may get to temperatures of 100-105°C. Do not attempt to retrieve the STEP 2 PLATE until Program has been completed is displayed.

• WARNING: The IVD performance claims of the Easy-Plex™ 384 System (High-Plex), or any of its components, or reagents, will not be extended to uses in a manner contrary to the IFU.

• WARNING: Always use appropriate personal protective equipment (PPE) where there can be contact with skin when handling potentially infectious substances or surfaces (such as human samples or reagents).

• WARNING: IVD claims do not extend to any changes made by the operator to a result (i.e. “Reject” or “Confirm” or "Present (Low)" as a result). Any operator changes will be clearly indicated in the Analysis Report (see Section 10.3.1 Analysis Report).

• WARNING: Do not remove grease from the linear bearings and bearing rails that move the robot arm (X, Y and Z axis). Wiping the grease may affect the performance of the robot and encourage corrosion.

• Note: The Processor deck may be cleaned with 70% ethanol and DNAoff.

• Note: Do not use abrasive materials when cleaning, as the polycarbonate material of the Processor lid is easily scratched. A clean, soft cloth is recommended.

• The products are intended for use by qualified laboratories only. Staff in the laboratories using AusDiagnostics products are expected to have received training in the risks of handling specimens of human origin and the protective measures to be taken.

• Good Laboratory Practises (GLP) should be followed when using these devices.

2. FURTHER INSTRUCTIONS REQUIRED The Instructions for Use (IFU) for the Easy-Plex™ 384 System (High-Plex) includes information that applies to all Easy-Plex™ 384 System (High-Plex) product operators. Kit-specific IFUs are also required to be read before use, and are not included in this document. Please refer to the AusDiagnostics website: www.ausdiagnostics.com for kit-specific IFUs.

The Easy-Plex™ 384 System (High-Plex) is also compatible with the Roche LC480 instrument as a 384-well analyser. Please read the appropriate Roche LC480 IFU and safety documents before use. The Easy-Plex™ 384 System (High-Plex) IFU only provides procedures for running a Step 2 Plate in a Roche LC480.

Warranty limitation information may be obtained by contacting AusDiagnostics (see section 15. TECHNICAL ENQUIRIES).

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http://www.ausdiagnostics.com


3. EASY-PLEX™ 384 SYSTEM (HIGH-PLEX) (REF 9150) COMPONENTS Components of the Easy-Plex™ 384 System (High-Plex) are not sold separately by AusDiagnostics. Where applicable, the AusDiagnostics 384-well Plate Analyser may be substituted for a Roche LC480. All options are provided below:

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*If a Roche LC480 Analyser is used, the operator must have the appropriate Roche software.

3a

3b

COMPONENTS FUNCTION

Computer with software

Easy-Plex™ Assay Setup software (Version 1.9.2) To set-up the Processor function

Easy-Plex™ Analyser software* (Version 7.7) To set-up the Analyser function

Easy-Plex™ Results software (Version 1.6.1) For reporting and analysing results

Easy-Plex™ Processor Automates the assay set-up and Step 1 PCR

384-well Plate Analyser Runs and analyses the Step 2 PCR

Easy-Plex™ 384 Analyser Exports files as .384

Roche Analyser LC480 Exports files as .ixo

1

2

3a

3b

1

2

3a

3b


3.1 OTHER REQUIRED SYSTEM PRODUCTS The following products must be used with the Easy-Plex™ 384 System (High-Plex), and may be purchased separately from AusDiagnostics or other suppliers. This list does not include kit-specific products.

3.2 OPTIONAL ACCESSORY A barcode reader (REF 9340) may be used with the Easy-Plex™ 384 System (High-Plex) for inputting sample names and LOT numbers into the Easy-Plex™ Setup Software (see Section 7.2 Setup Processor Deck). A symbol will denote when a barcode reader can be used. A barcode reader can be connected via the USB 2.0 port in the computer. Please read the information provided upon delivery for instructions on safe installation and use.

3.3 TECHNICAL AND ENVIRONMENTAL SPECIFICATIONS The Easy-Plex™ 384 System (High-Plex) complies with the requirements described in IEC 61326-2-6:2013. For further information, please contact AusDiagnostics.

All technical information for the Plate Spinner and barcode reader is included in the sales packaging.

3.3.1 Easy-Plex™ Processor The Easy-Plex™ Processor is supplied with a computer and the relevant software to run the entire system. The specifications for the Easy-Plex™ Processor are given below.

REF NAME FUNCTION QTY

9042Plate Spinner Centrifuge (Axygen) For spinning down Step 2 Plate contents 1

9020 Sealing films for 384-well plate For protecting Step 2 Plate contents 60 films

9302 Robot Tips (Axygen)Non-conductive 100 µl tips used by the Easy-Plex™ Processor

10 racks of 96

9303 Robot Tips (Axygen)Non-conductive 100 µl tips used by the Easy-Plex™ Processor

50 racks of 96

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SYSTEM FEATURE SPECIFICATIONS

Thermocycler block format Embedded 24-well blockAmbient temperatures 18 - 35°CThermal temperatures Block: 35 - 100°CTemperature accuracy 0.5°CMax heating speed 2.5°C/secMax cooling speed 1.5°C/secPipetting head Single channeled, 20-150 µl/secPrecision CV<2% 10 µl (wet-to-wet transfer)Tips 96-racked 80 µl low retention tipsTip waste handling All tips discarded externally; no waste accumulation within deck areaUV sterilization UV light system for deck sterilisation within closed hoodHood Integrated strut-supported tinted acrylic hood for access on 3 sides

Dimensions H (hood closed) 390 mm (15.4’’) H (hood opened) 770 mm (30.3’’) W 490 mm (19.3’’) D 580 mm (23’’); 640 mm (25.6’’) including cables


3.3.2 Easy-Plex™ 384 Analyser The specifications for the Easy-Plex™ 384 Analyser is given below.

4. INTENDED USE The Easy-Plex™ 384 System (High-Plex) consists of multiple components (see Section 3. Easy-Plex™ 384 System (High-Plex) (REF 9150) components), and is intended for in vitro diagnostic (IVD) use by suitably trained personnel in qualified laboratories using IVD High-Plex products in Australia (ARTG Identifier 177847) and the European Union (CE). The Easy-Plex™ 384 System (High-Plex) is sold under a single catalogue number; its components are not sold separately. Easy-Plex™ 384 System (High-Plex) products are semi-automated qualitative tests for the identification of pathogens in nucleic acid extracts from appropriate specimen types.

Weight 30 kg (66 lbs)Voltage / Frequency 100-240V AC: 50/60 HzPower 300 VAFuse F5A 240VComputer interface Serial DB9 RS-232



Block format Standard 384-well blockTubes type 384-well 0.045 ml PCR plateAmbient temperatures 18-26°CThermal temperatures Block: 0-100°C, heated lid: 105°CTemperature accuracy 0.2°CMax heating speed 2.1°C/secMax cooling speed 1.0°C/secLight source Light emitting diodeDetector Charged coupled device (CCD) matrixExcitation range 480 - 680 nmDetection range 510 - 710 nmDimensions H 540 mm (21.3’’)

W 210 mm (8.3’’) D 540 mm (21.3’’); 600 mm (23.6’’) including cables

Weight 27 kg (59.5 lbs)Electrical 220-240V AC: 50 HzPower Not over 550WComputer interface USB 2.0


5. PRINCIPLE OF THE METHOD

Easy-Plex™ Systems are based on the principle of Multiplexed Tandem Polymerase Chain Reaction (MT-PCR). MT-PCR1 employs two sequential PCR steps, which are referred to as “Step 1” and “Step 2”.

Samples (typically nucleic acid extracts) are loaded into kit-specific Step 1 Tubes, which contains all primers required for Step 1 PCR. Step 1 is a short multiplexed pre-amplification reaction using primers homologous to all targets offered by the product. Each pair of nested primers are specific to one target.

The Easy-Plex™ Processor automates the process of adding Step 1 Mastermix, oil and water, and performing the PCR reaction. Step 1 Mastermix contains enzymes in buffer specific for Step 1 PCR. Oil is added to prevent evaporation of the mixture during PCR.

PCR products from Step 1 are diluted with water and Step 2 Mastermix into separate dilution plate wells by the Processor. Step 2 Mastermix contains enzymes in buffer specific for Step 2 PCR. Diluted PCR products are then aliquoted into separate wells of a kit-specific Step 2 Plate.

The Step 2 Plate contains separate primer pairs for every target offered by the kit. Step 2 primers are designed to be ‘nested inside’ Step 1 primers, which increases the sensitivity and specificity of the assay, whilst diluting out potential inhibitory and cross-reacting substances. After all diluted samples have been transferred the operator must seal and spin down the contents of the Step 2 Plate. The Step 2 Plate is then transferred to a 384-well real-time PCR Analyser, in which it performs Step 2 amplification and monitors the level of fluorescence.

The dye present in the Step 2 reaction will emit fluorescence once it is intercalated in double-stranded DNA. The fluorescence increase during amplification is monitored by the Analyser. After the run is complete the fluorescence is analysed by the Easy-Plex™ Results Software which automatically reports if a pathogen is present or not.

STEP 1 TUBE

STEP 2 PLATE WELLS

STEP 1 PCR

STEP 2 PCR

TARGET

PRIMERS

ALIQUOTTED BY EASY-PLEX™ PROCESSOR


6. TIME REQUIRED TO ANALYSE A SAMPLE Approximate hands-on time required when running a full plate is < 1 min per sample.

The Easy-Plex™ Processor can be reloaded with a second batch of samples while the Easy-Plex™ 384 Analyser is performing Step 2 analysis.

*The time is less for partial batches

PROCEDUREMAX. TASK

TIME *HANDS ON

TIME *

Set up Easy-Plex™ Processor with reagents and samples (performed by operator) including starting the run in the software 5 min 5 min

Step 1: Multiplex PCR in Step 1 Tubes and aliquot into Step 2 Plate (performed by Processor) 114 min 0 min

Sealing of Step 2 Plate and plate transfer (performed by operator) 1 min 1 min

Clean up of Easy-Plex™ Processor deck (performed by Processor and operator) 6 min 2 min

Step 2: Real-Time PCR (performed by 384-well Analyser) 75 min 0 min

Data analysis and reporting 3 min 3 min

U.V. treatment of Easy-Plex™ Processor deck 15 min 0 min

TOTAL TIME 219 min 11 min


7. EASY-PLEX™ PROCESSOR PROCEDURES

7.1 SWITCHING THE PROCESSOR ON/OFF The on/off switch for the Processor is located at the rear of the device (shown in the image below). The Processor may be switched off after the Processor clean-up procedure has been completed (see Section 9.1 Processor Clean Up Procedure).

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SIDE

REAR


7.2 SETUP PROCESSOR DECK

Figure 1. The Easy-Plex™ Processor deck set up for a High-Plex run. Sample placement in the autosampling block and the Step 1 Tubes in the thermal cycler are shown on the right.

1. Decide how many samples will be analysed during the run. The number of Step 1 Mastermix , Step 2 Mastermix , water , and oil tubes required is dependent on the number of samples being analysed (see table below).

2. Remove the appropriate number of Step 1 and Step 2 Mastermix tubes from the freezer. While the Mastermixes are thawing, proceed with the sample preparation and Easy-Plex™ Processor setup.

IMPORTANT: Once the Mastermix is thawed, it cannot be re-frozen. If Mastermix received has been thawed, please discard and contact AusDiagnostics for a replacement (see Section 15). 3. Set up the Easy-Plex™ Processor as shown in Figure 1.

4. Add 4 mL of bleach containing 0.4% available chlorine to the Bleach Container and place in the reagent block (Figure 1).

5. Attach a Tip Disposal Bag to the waste chute (see Section 7.1 Figure for location on system).

6. Fit a Dilution Plate as per Figure 1. The Dilution Plate can be orientated in either direction. 7. Open the package containing the STEP 2 PLATE and fit it onto the Processor deck (labelled High-Plex

plate in Figure 1), being careful that the A1 well is in top left corner. The barcode label on the STEP 2 PLATE packaging will be used to identify the LOT number when starting the Easy-Plex™ Assay Setup software.

8. Choose the required number of STEP 1 TUBES needed. • The STEP 1 TUBES pack must have matching colour, catalogue number and version number to the

STEP 2 PLATE label. IMPORTANT: Do not use Step 1 or Step 2 kit components with different catalogue and/or version numbers.

• If the strip of STEP 1 TUBES has more wells than needed for the run, cut the strip to the appropriate length. Return any remaining, capped and unused wells to the STEP 1 TUBES pack to store for future use.

No. of samples for analysis

No. of Step 1 Mastermix tubes

No. of Step 2 Mastermix tubes No. of Water tubes No. of Oil tubes

1 - 8 1 1 1 1

9 - 16 2 2 2 2

17 - 24 3 3 3 3


• If the strip of STEP 1 TUBES has fewer wells than needed for the run, add the appropriate number of wells from other identical STEP 1 TUBES. If multiple batches of STEP 1 TUBES in the same run are used, keep a record of all LOT numbers as they need to be entered in the Easy-Plex™ Assay Setup software.

IMPORTANT: Capped 8-well strips of STEP 1 TUBES are not individually labelled. Immediately return any unused STEP 1 TUBES to their pack. 9. Add the samples to the Easy-Plex™ Processor deck as per the kit-specific IFU. Note: Manually pipetting nucleic acid extracts into STEP 1 TUBES is best performed in a biological safety cabinet or PCR setup area. 10. Regardless of sampling method (robotic or manual), place the STEP 1 TUBES in the thermal cycler.

Ensure the STEP 1 TUBES are uncapped. The first sample should always be in the top left of the cycler (see Figure 1) and subsequent samples placed in the column.

11. Push down the black thermal cycler cover over the thermal cycler block.

12. Once the Step 1 and Step 2 Mastermix are thawed, mix the tubes by inversion five times.

13. Spin the Mastermixes, water and oil at low g force for 5 seconds to remove any liquid from the lid. Place all of the tubes in the corresponding colour coded positions in the reagent block (Figure 1) and remove the lids.

7.3 RUN THE PROCESSOR

! 1. Open the Easy-Plex™ Assay Setup software icon on the desktop.

2. Set the correct 384-well Analyser as default; this will only have to be done once. To set the 384-well Analyser, go to Tools > Options.

3. Options: a) Choose the correct Analyser (AusDiagnostics Easy-Plex™ 384 Analyser or Roche LC480). If using

the Roche Analyser, confirm that the correct system (I or II) is selected. b) Choose the preferred output file save location.


c) Select OK to save and exit Options.

4. Add the following information to the Easy-Plex™ Assay Setup:

a) Operator Name: Operator’s name b) Select Test Kit: Select the correct test kit from the drop down menu.

It is very important that the selected file number is identical to the catalogue and version number of the kit (found on the immediate labels for the STEP 1 TUBES and STEP 2 PLATE). If the pull down menu does not contain a file corresponding to the STEP 2 PLATE, see Section 7.5.1 Installing kit templates.

c) Enter Sample IDs: Enter the sample ID numbers by either uploading a .CSV or .TXT file, scanning the sample barcodes or typing the sample names directly into the Easy-Plex™ Assay Setup software.

Remember that the samples are loaded in columns, with sample 1 in the top left corner of the sampling block or thermal cycler. Note: Sample names cannot be identical or the Easy-Plex™ Assay Setup software will not recognise the repeated sample.

d) Sample Size: Please refer to kit-specific IFU for appropriate sample size options.

e) Sampling: Please refer to kit-specific IFU for appropriate robotic sampling options.

f) Sensitivity: Select the Step 1 cycling performance. IVD performance claims are only applicable for “Normal” or “IVD” sensitivity selection.

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g) Output File: Output file name may be edited here. To change the save location, see Section 7.3, point 3b.

5. Select Start to proceed.

6. The software will prompt the operator to check that all materials are on the deck and to enter the LOT number for the STEP 2 PLATE, the Mastermixes and the STEP 1 TUBES, and to confirm the sample names. Select OK after each input.

7. Once the checklist is completed, select OK to start the Processor. The Processor will start.

IMPORTANT: The run must be started within 30 minutes of thawing the Mastermix.

7.4 PREPARE THE PLATE FOR THE ANALYSER 1. When the run has finished, the Easy-Plex™ Assay Setup software will offer to add bleach to the STEP 1

TUBES. First, remove STEP 2 PLATE from the Processor.

Note: To optimise run times, start the procedure to bleach the STEP 1 TUBES after removing the STEP 2 PLATE. Please see Section 9.1 Processor Clean Up Procedure.

2. To seal the STEP 2 PLATE, remove the protective sheet from the sealing film (REF 9213) and place the adhesive side of the sealing film onto the STEP 2 PLATE. By using a plate roller or scraper, spread from the centre of the plate outwards to prevent the creation of air pockets. Pay special attention to the corners.

IMPORTANT: CONFIRM EACH WELL IS SEALED.

3. Place the sealed STEP 2 PLATE in the plate spinner, with the seal facing inwards to spin all liquid to the bottom of the wells. Placing the plate in the wrong orientation in the spinner leads to errors and invalid runs. Spin plate for 20 seconds. Check that there are no bubbles remaining in the wells. Spin for an additional 20 seconds until no bubbles remain.

4. Proceed to Section 8. Analysers for instructions on using the relevant 384-well Analyser. IMPORTANT: The Analyser must be started within 30 minutes of the Processor finishing.

7.5 EASY-PLEX™ SETUP SOFTWARE - SPECIAL FUNCTIONS

7.5.1 Installing Kit Templates If the pull down menu of the Easy-Plex™ Setup Assay software does not contain a file corresponding to the catalogue and version number on the mylar bag containing the STEP 2 PLATE, the correct file must be installed. If the correct template file has not been installed, please contact AusDiagnostics directly for assistance (see Section 15 for contact details).

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If an AusDiagnostics customer liaison or technician is not available to install your template, it may be installed as follows:

To add a new template select on File > Install Kit Template.

Once the correct product template has been located (i.e. a *.mt file) select Open.

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The Easy-Plex™ Assay Setup Software will confirm that the product template has been installed.

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7.5.2 Uninstalling Kit Templates If a kit template needs to be uninstalled, the operator can do this by selecting the template from the Select Test Kit dropdown menu on the homepage in the Easy-Plex™ Setup Assay software.

In this example (below) the Faecal Pathogens C (16-well) template needs to be uninstalled.

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After selecting the template, select File > Uninstall Selected Template. A final warning message will confirm the uninstalling of the template.

After selecting Yes, the template will have uninstalled successfully.

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7.5.3 Reset Tip Availability The Easy-Plex™ Assay Setup software records how many tips are left at the end of each run and this is recalled at the start of the next run. The Easy-Plex™ Processor will automatically move to the recorded first available tip location.

If needed, the tip availability can be reset to three full racks by selecting Tools > Reset Tip Availability.

7.5.4 Create Support Package In the event of a software crash or a problem with the Easy-Plex™ Processor, a support file should be created to email to AusDiagnostics – the support file is a detailed log file of the Processor operations at the time of the crash. Select Help > Create Support Package. It is helpful if the operator can provide a description of what the Easy-Plex™ Processor was doing at the time of the problem.


8. ANALYSERS 8.1 USING A EASY-PLEX™ 384 ANALYSER

8.1.1 Switching The Analyser On/Off The on/off switch for the Analyser is located at the rear of the device (shown in the figure below).

8.1.2 Startup Of The Easy-Plex™ Analyser Software (Realtime_Pcr Program) 1. Ensure that the Analyser is switched on.

2. On the computer desktop, select the RealTime_PCR icon.

3. Select Operator and select Device handling.

4. A List devices window will appear. Choose which Analyser to use (the serial number can be found in the top left corner of the Analyser screen). Select Connect.

5. The device will be connected to the computer and the indication in the status line of the operation

procedure will be changed to


6. After automatic instrument testing, its status indication will be either:

: A green background, to indicate that the instrument is on and is ready for operation.

: A yellow background, to indicate that the instrument has recently been switched on, and will go through a warm-up (as displayed with a “WARMING” notice on the device screen). After warm-up (max 10 min), the background colour will change from yellow to green.

IMPORTANT: If the device is switched on with a yellow status indication, but not “WARMING” as indicated, please contact AusDiagnostics. Also see Section 14. Troubleshooting.

: A red background, to indicate that the instrument is either switched off at the moment of program startup, or is not connected to the computer, or has not been selected in the List devices window, or another instance of the software is open.

7. If the status indication is green or yellow, the STEP 2 PLATE may be loaded into the device.

8.1.3 To Run The Easy-Plex™ 384 Analyser 1. If using the kit for the first time, see Section 8.1.4 Installing kit templates in 384 Analyser before

proceeding any further. Once the Analyser has been connected and is ready to use, select Import.

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2. Select the correct Step 1 output file from the Easy-Plex™ Processor (.xml). Select Open.

3. The Analyser software will load the run setup from the imported file. In this example, 24 samples will be analysed, so 24 columns of the STEP 2 PLATE are highlighted in blue demonstrating which wells are to be analysed.

4. Select the Apply button.

5. The Easy-Plex™ 384 Analyser can be opened in two ways:

I. Using the Analyser software in the Start program tab, select the Open block icon on the bottom right of the window. Select the Close block icon to close the Analyser.

or II. Using the buttons underneath the Analyser screen, select the button on the left with the yellow light.

This provides two options Exit (left button), or Open (middle button). Press the middle button once.

a) Place the sealed STEP 2 PLATE in the Analyser. b) To close the Analyser, press the left button to provide two options Exit (left button) and Close

(middle button). Press the middle button once.

IMPORTANT: Be careful that the A1 well is located in the top left corner.


6. In the Start Program tab, select Start Program to initialise the Step 2 PCR.

7. Save the results files in the desired directory.

8. The software tracks the cycling in real time.

8.1.4 Installing Kit Templates In 384 Analyser The first time a new kit is run, the template needs to be installed in the Easy-Plex™ 384 Analyser.

1. On the computer desktop, select the RealTime_PCR icon.

2. Connect to the Easy-Plex™ 384 Analyser


3. Select Tests > Copy group of tests

4. Select the “from *.ini file” from the drop down menu

5. Go to the Easy-Plex™ Assay Setup output directory. The location of the output directory can be retrieved by opening the Easy-Plex™ Assay Setup software, and opening Tools > Options (see Section 7.3, point 3b).

6. Select the *.ini file for the new Kit template


7. Check the desired *.ini file, choose for ALL users (1) and then select the copy button (2).

8. The new template is now installed and the *.xml files can be imported for that template.

8.2 USING A ROCHE LC 480 ANALYSER

8.2.1 Further Instructions For Roche LC 480. Please read the appropriate Roche LC480 IFU for appropriate safety, maintenance and set-up instructions.

8.2.2 To Run A Roche LC 480 Analyser 1. Once the Step 1 reaction has completed, seal the STEP 2 PLATE correctly (see Section 7.4 Prepare the Plate for the Analyser), spin it in a plate-spinner (seal facing inside) to get rid of any residual bubbles, and insert it into the LC480 instrument. The LED lights on the front of the cycler should be unblinking green.

2. Select the LightCycler 480 icon on the desktop.


3. After filling in the User Name (“admin”) and Password (“LightCycler480”), go to the Navigator window. The operator can access this window by selecting it from the drop-down menu located underneath Overview.

4. The operator now needs to import the *.ixo file created by the Easy-Plex™ Processor.


After selecting the Import option in the Navigator window, the operator can now select the desired *.ixo file.

5. After the *.ixo file has been imported, the operator can start the run by selecting Start Run in the Experiment tab. Save the experiment in the Experiments output directory. The run will now initialise.

6. After the run has finished on the LC480 software, the results need to be exported as an *.xml file. The operator can do this by simply selecting the Export button in the Experiment tab.


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The exported *.xml file needs to have the exact same name (save for the extension) as the *.ixo file created by the Easy-Plex™ Processor after the Step 1 reaction. It also needs to be saved in the same output directory as its corresponding *.ixo file. Once this has been verified and the *.xml file extension has been selected (1), select the Save button (2).

7. The run is now ready to be viewed and analysed in the Easy-Plex™ Results software.

9. END OF RUN PROCEDURE 9.1 PROCESSOR CLEAN UP PROCEDURE 1. When the run has finished, the Easy-Plex™ Assay Setup software will offer to add bleach to the STEP 1

TUBES. After the STEP 2 PLATE has been removed (See Section 7.4 Prepare the Plate for the Analyser), the STEP 1 TUBES can be bleached.

2. Select Continue for the Processor to add bleach to the STEP 1 TUBES.

3. After bleach has been added, dispose of the tubes and dilution plate in the waste bag.

4. Wipe any spillages on the surface of the Easy-Plex™ Analyser deck (including the thermal cycler cover) with a non-corrosive nucleic acid denaturing reagent (e.g. DNA-OFFTM).

5. Remove the tip ejector chute and rinse any deposit away in running water.

6. Seal and dispose of the waste bag according to safety regulations relevant for biohazard waste. One bag is provided for every two runs.


7. The Processor deck should be irradiated with the UV light between runs to inactivate any DNA material on the deck surface. Close the Processor lid before turning on the UV lamp. The UV lamp is turned on by selecting Tools > UV Lamp Control. Select Start to turn on the UV lamp.

9.2 VERIFICATION OF INSTRUMENT FUNCTION AND SAMPLE INHIBITION To verify the validity of the results, the cycling curves for the internal control, SPIKE, must be checked. This is done by using the “View By Gene” option and toggling to the last ‘Gene’.

The SPIKE cycling curves must take-off between 10 - 20 cycles with an S-shaped curve (see figure below).

The results for a sample are invalid if:

• SPIKE is shown as “Inhibited” in the results box and no pathogen was detected. In this case, it is recommended that the sample should be re-extracted if appropriate and the analysis repeated.

• The Sample Adequacy Control or Human DNA Control (only applicable to certain kits) is negative and no pathogen detected. In this case, it is recommended that the sample should be re-collected and re-extracted if appropriate and the analysis repeated.

• Target has a take-off value of <10 and there is a melt curve with no corresponding cycling curve. In this case, it is recommended that the sample should be diluted 1:500 and the analysis repeated in a later run for confirmation.


If needed, please contact AusDiagnostics for technical advice (see Section 15. Technical Enquiries).

10. EASY-PLEX™ RESULTS SOFTWARE ANALYSIS

10.1 OVERVIEW OF SOFTWARE OPERATOR INTERFACE CONVENTIONS The software’s operator interface displays recognisable and straightforward elements (i.e. buttons) with a defined functionality:

10.2 OPEN When selecting Open, the operator can open a desired results file by selecting it from the directory where the run file was saved in.

BUTTON FUNCTION

Opens a run file.

Generates “Analysis”, “Result Table” and “Sample” reports.

Exports the run information and data as “Results”, “Summary”, “Analysis Summary”, “Analysis Data”, “Cycling Data” and “Exchange File”.

Views the results by sample.

Views the results by gene.

Displays a normal analysis sensitivity.

Displays a high analysis sensitivity.

Skips to the first sample or gene.

Toggles left between samples or genes.

Toggles right between samples or genes.

Skips to the last sample or gene.

Sets the minimum concentration value for a positive call.

Monitors changes in melt temperature by displaying a dot plot of melt temperature vs. date for each gene.

Displays current Easy-Plex™ Results software version.


10.3 REPORT The Report function allows the results from the run to be summarised in 3 pdf-based report types: “Analysis”, “Result Table” and “Sample”. All these report types can be printed through the pdf-toolbar.

10.3.1 Analysis Report The Analysis Report summarises the results per Sample ID. The kit name, catalogue and version number are featured in the document’s title. Operator name and date of experiment are displayed on the first page, whereas assay sensitivity, used sample volume, reaction plate batch numbers and output directory locations are displayed at the bottom of the document. Step 1 and 2 mastermix batch numbers, and Step 1 reaction tube batches are listed in every Sample ID table (note: the run’s *.esetup file needs to be present in the same file directory as the instrument data file in order for this information to be displayed).

A call can either be “Present”, “Check” or blank (i.e. not detected).

Intensity gives a 5 star-based representation of how much of the target gene is present in the sample.


10.3.2 Result Table Report This report type summarises all obtained results in one table, by listing all the different Step 2 reaction wells and their respective colours, samples, genes, calls and relative concentrations (in arbitrary units).

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10.3.3 Sample Report Similar to the Analysis Report, Sample Report summarises the results per Sample ID. However additional data such as concentration, corrected melt temperature, and take-off values are also listed. Each sample is shown on a separate page. Run information (operator, sample volume used, Step 1 sensitivity, analysis sensitivity and data file directory) is listed underneath each Sample ID table. Note: the run’s *.esetup file needs to present in the same file directory as the instrument data file in order for this information to be displayed. Amplification (fluorescence vs. cycle) and melt temperature (-dF/dT vs. temperature) curves are also displayed at the bottom of every page.

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10.4 EXPORT The export function allows the operator to either export the results as raw data in a *.csv file (which can be opened by any spreadsheet application), an XML file (for LIMS systems), or save the results as an Easy-Plex™ Exchange (*.epx) file.

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10.4.1 Results By exporting the run data as a Results *.csv file, the operator can access this information through any spreadsheet application (e.g. Excel, Numbers, etc.). The data is displayed over five columns, with sample ID information listed in the second column adjacent to well number, gene names, calls and concentrations. This format is useful for import into a LIMS reporting system.


10.4.2 Summary Summary exports concentration results, sorted by gene names and by sample IDs. The first column of the spreadsheet contains the file ID, whereas the last column includes any diagnosis output.

10.4.3 Analysis Summary Analysis Summary gives a summary of take-off and corrected melt values, sorted by gene names and by sample IDs. The first column of the spreadsheet contains the *.384 file ID.

10.4.4 Analysis Data Exporting Analysis Data generates an *.eresults file in XML format, which can be opened by any text editing program. Analysis Data contains additional information output and all the parameters needed for results analysis. This format may be useful for interfacing to a LIMS.

10.4.5 Cycling Data Cycling Data exports unprocessed cycling data as a *.csv file.

10.4.6 Melt Data Melt Data exports unprocessed cycling data as a *.csv file.

10.4.7 Exchange File Exporting the run as an Exchange File generates an *.epx results file. This file type can be opened by the Easy-Plex™ Results software. Its file size (<100KB) is significantly smaller than the original data file from the Analyser instrument, which makes this *.epx file the preferred file type when attaching result files to emails or when saving hard disc space. Note: in order to retain the Report run information in the *.epx file (such as operator name, batch numbers, sensitivity type, etc.), the *.esetup run file (generated by the Easy-Plex™ Assay Setup software) needs to be present in the same file directory as the actual data containing results file when exporting the Exchange File.

After saving the *.epx file in the desired directory, the operator is prompted with a message asking whether the original input files (i.e. the data containing file and the *.esetup file) need to be removed. This may be selected to save disk space and avoid the duplication of results files, however .384 files may be required if technical assistance is requested.


10.5 VIEW BY SAMPLE The cycling curves (top) and the melt curves (bottom) for all of the targets that have amplified are displayed for one sample. The expected melt temperature range for each target is displayed by a highlighted area.

Hovering over a particular curve shows the gene to which the curve represents, highlighted in the adjacent table on the left.

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10.6 VIEW BY GENE With this setting, all of the samples that amplified per target are shown. The expected melt range is also highlighted for each target.

Hovering over a particular curve shows the sample to which the curve represents, highlighted in the adjacent table on the left.


The navigator buttons “<” and “>” toggle the display between samples or genes. Furthermore, in both View by Sample and View by Gene specific sample IDs or genes can be selected by selecting the main title:

10.7 NORMAL SENSITIVITY In Normal Sensitivity the fluorescence melt temperature threshold for detection is set at 5% of the total fluorescence. Any targets with a fluorescence higher than this value will be displayed as normalised peaks.

10.8 HIGH SENSITIVITY In High Sensitivity the fluorescence melt temperature threshold for detection is lowered to 2% of the total fluorescence and lower acceleration rates are valid. Any targets with a fluorescence higher than this value that comply with expected melt and acceleration parameters will call present and be displayed as normalised peaks.

WARNING: IVD claims are for the software set at normal sensitivity.


10.9 HISTORY The History function saves all positive calls for each gene and assembles the results in a dot plot (corrected melt temperatures vs. time). Furthermore, melt peaks from valid PCR reactions are recorded in the history even if they were not in range. These out of range peaks are shown on the history chart as grey triangles.

Changes in melt temperature over time would be an indication of changed target sequence or possibly a change in instrument function. If grey triangles are observed or significant jumps in the melt temperature of positive controls are observed, please contact your AusDiagnostics representative. Hovering over a dot displays the file name from which the positive result was taken. Results for different genes and kits can be selected through the appropriate dropdown menus. Additionally, a timeframe (All, Last 90 Days or Last 12 Months) can be selected for the preferred data display.

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When SPIKE is selected as a target under the Gene dropdown menu, a dot plot of its take-off values vs. time is displayed. This will allow the operator to monitor any possible sample inhibition (marked by delayed take-off values).


10.10 OPTIONS Options allows the operator to make a distinction between low level and high level targets. By setting a concentration threshold, all targets below this value will be called “Present (Low)” instead of “Present”. IMPORTANT: the default minimum concentration is set at 0, unless changed manually. Once changed, this threshold concentration value will apply to all templates opened in the Easy-Plex™ Results software.

10.11 ABOUT About informs the operator which version of the software is currently in use.

10.12 OTHER SOFTWARE OPERATOR CONVENTIONS

10.12.1 Resizing The Window In order to expand the listed information, the window can be resized by hovering the controller mouse at the right hand side of the results table and dragging the window to the left or right.


10.12.2 Advanced View Operators can toggle in between Standard View and Advanced View by right-clicking the toolbar in the left hand side of the results window pane, and selecting the preferred view. Advanced View shows Corrected Melt, Take-Off values, Rejection User and Rejection Comment (see Section 11.1.6 Rejecting Calls).

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10.12.3 Hiding Curves It is possible to turn off the display of the cycling and melting curves for particular targets in the results window. This is done by right-selecting a target and deselecting “Show Curves”.

11. INTERPRETING SOFTWARE RESULTS 11.1 CALLS

11.1.1 Present A target is considered present when the cycling curve displays amplification that falls within predetermined parameters.

For some kits where dual infection is anticipated, the relative concentration of each positive gene is expressed as a percentage. The result is displayed in the Call column e.g. "Present (95%)". Percentages are rounded to the nearest 1% with <1% shown for smaller values.

11.1.2 Present (Low) A concentration threshold can be implemented through the options function to distinguish low level from high level targets (see Section 10.10 Options). Any targets with a concentration lower than the set threshold, will be called “Present (Low)”. All report types will contain the text “Present (Low)” rather than “Present” to indicate operator involvement.

11.1.3 Blank A target is considered undetected when there is no amplification within the predetermined parameters.

11.1.4 High Fluorescence The high fluorescence condition is displayed when a well has a significantly higher fluorescence than the average fluorescence of all of the SPIKE reaction controls. This may indicate a high fluorescence particle (e.g. dust contamination) in the plate wells.


11.1.5 Check In some cases when the cycling curve acceleration is less than preset parameters, targets will be marked as “Check” rather than “Present”, and no concentration estimate is provided. The operator may right select on the “Check” call and change it to “Confirmed”. The normalised melt peak will remain visible in both the melt graph and in the sample report. All report types will contain the text “Confirmed” rather than “Present” to indicate operator involvement.

Note: During the determination of the clinical performance of IVDs, “Check” results were excluded from the analysis.

11.1.6 Rejecting Calls A call may be rejected by right clicking the “Present” call and changing it to “Rejected”.

Upon rejecting a call, a message will prompt the operator to enter his or her name, and reason for rejecting the call. These details will subsequently be added to Analysis Report, and the call will change from “Present” to “Rejected” for that particular target.

11.1.7 Inhibited SPIKE, the internal control, is included in every assay. The detection of the SPIKE target controls for the integrity of the reagents (polymerase, primers, etc.), equipment function (thermal cyclers) and the presence of inhibitors in the sample.

The SPIKE cycling curves must take-off between 10-20 cycles with an S-shaped curve. If the SPIKE cycling curve has a take-off value greater than cycle 20, then this indicates significant inhibition. A SPIKE take-off value greater than 5 cycles above the median is marked as Inhibited.


Please consult the kit-specific IFU (Section 9. Controls) for additional information about controls.

11.1.8 Bad Melt If SPIKE’s melt temperature is more than 0.5°C different from the mean, a Bad Melt is reported and the following warning message is displayed “Normaliser reaction not within specifications”. This indicates an incorrect reaction mix and the sample should be retested.

If this recurs, please contact AusDiagnostics.

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11.2 CORRECTED MELT Melt temperatures of all the different targets are adjusted according to the difference between the raw melting temperature and the known melting temperature of SPIKE (81.8°C). Corrected melts are used in the calling algorithm to allow for small changes in running conditions on individual systems.

11.3 TAKE-OFF VALUES The take-off value is derived by stepping back from the point of maximum second differential, d2F/d2T, (i.e. maximum acceleration) to the point where the fluorescence emerges from background.

11.4 CONCENTRATION Molecular target concentrations expressed as arbitrary units, and are calculated relative towards the internal control SPIKE, which amplifies a known amount of target molecules. As the concentration of SPIKE is not measured these values are in arbitrary units.

11.5 MULTIPLEXING IN STEP 2 PCR The Results software allows for multiplexing in Step 2 PCR. If two reactions are combined so that different melt peaks are expected for different genes or genotypes, the results software can automatically call the correct ID. If the corrected melt is in a region of overlapping melt windows, then a list of genes or genotypes is displayed with the gene having the closest melt shown first.


11.6 ALGEBRAIC CALLS Occasionally it is useful to be able to make a call based on an algebraic equation. For instance if clinical symptoms are only present if a pathogen is above a critical level, or if a combination of different genes is present. Such expressions can be built into the Results software and give rise to an algebraic call in the information box in the bottom left hand corner of the window. Where applicable, please see the kit-specific IFU.

12. MAINTENANCE 12.1 OPERATOR MAINTENANCE Ensure that there is nothing underneath the Processor, for example paper, lint/fluff or pipette tips.

The Easy-Plex™ Processor, computer and Analyser should be completely switched off at least once a week.

The Processor, computer and Analyser should be kept clean as per laboratory procedures.

Please see Section 9.1 Processor Clean-up Procedure.

12.2 SERVICE AND CALIBRATION SCHEDULE A preventative maintenance contract is required to ensure the system is performing to specifications. Preventative maintenance for the Easy-Plex™ 384 System (High-Plex) occurs twice a year, consisting of one minor and one major service.

It is not recommended that operators calibrate the Processor. Damage caused by operators incorrectly calibrating the Processor will not be covered by the maintenance contract.

13. EXPECTED CLINICAL PERFORMANCE For full details of clinical performance, please refer to the kit-specific IFU (Section 9. Expected Clinical Performance).

13.1 INTERFERING SUBSTANCES The accepted primary method of PCR interference is via interaction with, or inhibition of, the polymerases responsible for the PCR reaction2,3,4. As any effects will not be primer-specific, the potential interference of substances in the RNA and DNA mastermixes can be determined using either the NONO or SPIKE assays, respectively.

A list was compiled of substances which may be present in clinical sample types used for all AusDiagnostics kits. Variables such as sample collection method, types of samples (e.g. sputum, skin lesions), and exogenous (e.g. OTC medicines, skin lotions) and endogenous (e.g. blood, mucin) substances were considered.

Solutions for each exogenous (1% solution) and endogenous (1%, 5% or 10% solution) substance were prepared and supplemented with human RNA and synthetic DNA. The concentrations of substances in these solutions were all significantly higher than concentrations commonly found in clinical samples. The solutions were then extracted, run and analysed for PCR inhibition.

There was no interference of the RNA or DNA mastermixes by the exogenous substances tested. The two endogenous substances, mucin and blood, were tested at two concentrations. There was no inhibition detected except for 10% mucin, ethanol and an ethanol-based product (Listerine) with the RNA Mastermix. These results only provide an indication of potential inhibition, as interference by rarely used substances may not be identifiable. The nucleic acid controls included in most High-Plex kits will detect interference caused by the sample.

SUBSTANCE KEY INGREDIENTS

Ethanol EthanolSwab media Sodium thioglycollate, sodium chloride, disoldium phosphateCharcoal swab media Charcoal, agar, sodium chloride

SUBSTANCE


14. TROUBLESHOOTING For more assistance or if any issues recur, please contact AusDiagnostics.

Phone: +61 2 9698 8030

Email: [email protected]

14.1 EASY-PLEX™ PROCESSOR TROUBLESHOOTING

Rhinocort (nasal spray) BudesonideAtrovert (nasal spray) Ipratropium BromideSoothers (lozenges) Menthol, eucalyptus oilBenadryl Chest Forte (cough medicine) Bromohexine hydrochlorideDuro-tuss Dry Cough (cough medicine) Pholcodine, sorbitolListerine (mouth wash) Ethanol, benzoic acid, thymol, cineoleThursday Plantation Aloe (cream) Aloe barbadensisPalmer’s Shea butter (cream) Palmitic acid, stearic acidBanana Boat Sport (cream) Octyl methoxycinnamate, 4-methyl benylidine, zinc oxideVirasolve (cold sore cream) Idoxuridine, lignocaine hydrochloride, benzalkonium chloridePharmacy action anti-fungal cream Clotrimazole, benzyl alcoholProchlorperzine maleate (oral medication)

Apo-prochlorperazine

Chemist's own stomach ache and pain relief (oral medication)

Hyoscine butylbromide

Imodium (oral medication) Loperamide hydrochlorideAmpicillin (antibiotic) AmpicillinGentamycin (antibiotic) GentamycinBlood Human whole bloodDissolved mucus Bovine mucin

KEY INGREDIENTSSUBSTANCE

ISSUE / ERROR MESSAGE CAUSE / RESOLUTION

“ No devices found. No Easy-Plex™ Assay Setup devices have been found. Do you wish to perform the run on a software simulation? “

Select No. Confirm the Processor is turned on and that the serial cable is firmly plugged into the computer and Processor. Select Start in the Easy-Plex™ Assay Setup. If message persists contact AusDiagnostics.

Inconsistent volumes in wells Disregard results. Check the deck equipment is loaded correctly (flat on deck). Ensure all reagents have been spun before repeating run.

Software crashed due to unexpected error

Create support package by selecting Help > Create support package, and email it to AusDiagnostics. Close and restart the software.

Bent pipette tips Disregard any results. Reset the pipettor head by turning the Processor off and back on. Confirm Step 1 strip tube lids and reagent lids are removed before repeating the run.

Processor runs are taking longer than usual

Possible issue with the cycler. Contact AusDiagnostics.


14.2 EASY-PLEX™ ANALYSER TROUBLESHOOTING

14.3 RESULTS TROUBLESHOOTING

15. TECHNICAL ENQUIRIES For assistance or if any issues recur, please contact AusDiagnostics. Email: [email protected]


“Attention! Discovered mistake when reading the block parameter from instrument…”

Confirm the Analyser is turned on and that the USB cable is firmly plugged into the computer and Analyser. Restart the software.

“Attention! Block not found in working order”

Open the plate tray and close again. Restart software. Select start.

“Attention!…” Restart the Analyser. Error messages may appear if the Analyser has not been switched off at least once a week.

Dark residue present on the plate and on the Analyser block, with no valid results.

Plate was spun with the incorrect orientation in the plate spinner. Run must be repeated with attention to plate orientation during the spinning step.

Contact AusDiagnostics. This is most likely an Analyser malfunction.

The status indication remains yellow after 10 min.


No results for SPIKE, but another target has peaks in every sample

Disregard any results. Step 2 Plate was inserted in wrong orientation in the Analyser block. Before repeating run make sure the plate is orientated so that A1 is at the top left corner.

SPIKE did not give a positive result Make sure that water and Step 2 master mix tubes are spun before removing lids. After confirming the system is functioning correctly (e.g. consistent volumes in tubes, reagent tubes in correct position on Processor deck), a negative SPIKE is most likely due to PCR inhibition. Please see Section 9.2 Verification of Instrument Function and Sample Inhibition.

No fluorescence measured for any target Repeat run after restarting the Analyser and checking that oil and water tubes have been correctly set up. Incorrectly set up oil and/or water tubes, or Analyser malfunction, are the most likely causes of this issue.

AusDiagnostics UK Ltd Unit 3 (Ground Floor) Anglo Business Park, Asheridge Rd, Chesham Bucks, HP5 2QA UK Tel: +44 (0) 1494 300121 Mob: +44 (0) 7896 534843

AusDiagnostics Pty Ltd 205 Victoria Street BEACONSFIELD NSW 2015 Australia Phone: +612 9698 8030 Fax: +612 9319 1661


16. ACKNOWLEDGEMENTS Edited by: M. Craggs, P. Woodbridge and J. Zhang. Approved by: A. Johannsson

17. GLOSSARY

Manufacturer

Authorised representative in the European Union

Catalogue Number

Version number

LOT Lot/Batch code

GTIN This product has been assigned a unique Global Trade Item Number.

Indicates that the relevant product is intended for in vitro diagnostic use in Australia and is included on the Australian Register of Therapeutic Goods (ARTG).

Indicates that the relevant product is intended for in vitro diagnostic use in the European Economic Area and is compliant with the European IVD Directive 98/79/EC.

WARNING: BIOHAZARD.

WARNING: PINCH POINT.

C-Tick mark for Australia

WARNING. Please read the indicated section carefully.

Please consult the identified instructions for use before use

Do not re-use

Storage temperature range (upper and lower limit)

Storage temperature range (upper limit only)

Positive Control

Expiry date (yyyy-mm-dd)

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18. DEFINITIONS A run: All steps from starting the Easy-Plex™ Assay Setup Software (see Section 7.3 Run the processor) to generation of the Easy-Plex™ Results file is considered “a run”.

IFU: Instructions for Use.

WARNING: Indicates a statement that alerts operators about a situations that, if not avoided, could result in hazards or other serious adverse consequences from the use of the device.

IMPORTANT: Indicates a statement that alerts the operator to special care or special activities necessary for the safe and effective use of the device.

Note: Indicates additional information.

Operator: The individual who is interacting with the Easy-Plex™ 384 System (High-Plex).

Primers: Short synthetic oligonucleotides specifically designed to bind to and amplify specific gene sequences under conditions provided during PCR.

Target: A gene or sequence that primers are designed to hybridise to.

Kit: A set of kit components that are intended to be used together to detect a specific group (“panel”) of targets.

Kit-specific IFU: Instructions for Use which contains information specific to a kit, or multiple similar kits.

19. REFERENCES 1. Stanley, K.K. & Szewczuk. E. (2005) Multiplexed tandem PCR: gene profiling from small amounts of

RNA using SYBR Green detection. Nucleic Acids Research. 33: e180. 2. Kermekchiev, M.B. et al. (2009). Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA

amplification form whole blood and crude soil samples. Nucleic Acids Research. 37 (5): e40.

3. Opel, K.L. et al. (2010). A study of PCR inhibition mechanisms using real time PCR. Journal of Forensic Sciences. 55(1):25-33.

4. Schrader, C. et al. (2012). PCR inhibitors - occurrence, properties and removal. Journal of Applied Microbiology. 113: 1014-1026.

A barcode reader may be used to input sample names or LOT numbers in this step.

IFU Instructions for use

WARNING Indicates a statement that alerts users about a situations that, if not avoided, could result in hazards or other serious adverse consequences from the use of the device

IMPORTANT Indicates a statement that alerts the user to special care or special activities necessary for the safe and effectives use of the device

Note Indicates additional information

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