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Basic PCR database concepts
\n The PCR database in the FLOW Software contains all relevant data, such as PCR tests, required reagents, required purification protocols, and limit values. The link concept means that the components are linked in a one-to-multiple relationship. For example:\n\n
    \n\t
  • \n\t
    Requested test:
    \n\n\t
      \n\t\t
    • One requested test (or PCR panel) can contain one or more PCR tests.
    • \n\t\t
    • A requested test is linked to one sample material type and one MagNA Pure 96 Purification Protocol. 
    • \n\t
    \n\t
  • \n\t
  • \n\t
    PCR test:
    \n\n\t
      \n\t\t
    • A PCR test can contain one or more PCR targets.
    • \n\t\t
    • In addition, a PCR test is linked to one master mix, one LightCycler® 480 Macro, and one RT mix (Reverse Transcription), if applicable.
    • \n\t\t
    • When the PCR test setup is complete, it can be linked to a requested test. 
    • \n\t
    \n\t
  • \n\t
  • \n\t
    PCR target:
    \n\n\t
      \n\t\t
    • A PCR target can contain one or more controls.
    • \n\t\t
    • All PCR targets linked to one PCR test will be pipetted into the same well on the LightCycler® 480 Multiwell Plate.
    • \n\t\t
    • A PCR target can be linked to an external or internal standard curve for quantification.
    • \n\t
    \n\t
  • \n
\n 
\n
\nCommunication formats FLOW
\n \n
Input file from LIS to FLOW
\nFLOW picks up either the Requested Test or the PCR Target from the LIS. Note that the PCR Test level cannot be used for a LIS order. This is also true when orders are manually entered into the FLOW Order list by the user. The window for entering the worklist manually only allows for selection based on a Requested Test or a PCR Target, and it is not possible to select a PCR Test.
\nIn the context of a LIS order, (2) Target (AssayName) can be either:\n
    \n\t
  • A Requested Test name (in which case all PCR Targets associated with that Requested Test, as well as associated controls, will be ordered for the sample), or
  • \n\t
  • A PCR Target name (in which case only that target, as well as any associated controls for that target, will be ordered for the sample).
  • \n
\n \n\n
Output file for LIS from FLOW
\n\n
    \n\t
  • FLOW picks up the LIS provided data from a .txt file. The LIS information is converted by an ASTM service, so the actual ASTM input will be different. Further details and communication formats can be taken from the FLOW ASTM Manual (Host Interface Manual).
  • \n\t
  • File exchange between FLOW and components is mainly .xml. Only the LC480 communication differs, as the LC480 LIMSServer is used for this purpose.
  • \n\t
  • Export to LIS will be either .txt or .dat, depending on the input (see first point regarding input).
  • \n\t
  • Data output from FLOW can also be a report that is stored as a .pdf, .xls, or .doc.
  • \n
\n \n\n
Control Decision tree
\nThe FLOW Software applies a control concept to assess whether the process steps have been passed or failed. Using the controls is optional, but recommended. Controls include:\n\n
    \n\t
  • Internal control
  • \n\t
  • Positive process control: controls purification and PCR
  • \n\t
  • Negative process control: controls purification and PCR
  • \n\t
  • Positive template control: controls PCR
  • \n\t
  • Negative template control: controls PCR
  • \n
\nThe control decision tree (see Figure 1 below) shows how the test result is evaluated, depending on which controls are linked to the test and the results for the linked controls.\n\n
If no control is added, the FLOW Software analyzes the result based on the Cq threshold defined for the target.
\n 
\n ", "Country": "XG", "Code": "Protocols", "Name": "Protocols" }, { "Language": "en", "Value": "For midsize to high throughput labs that conduct LDT. Use in conjunction with FLOW System.", "Country": "XG", "Code": "Positioning", "Name": "Positioning" }, { "Language": "en", "Value": "The FLOW Software manages the information exchange between all modules of the FLOW Solution (PCR Setup Instrument/MagNA Pure 96 Instrument/LC480 Instrument). Sample test requests are automatically imported from local Laboratory Information Management Systems (LIMS) and distributed by the FLOW Software to all workflow components via .xml file exchange or LIMS interface module (LC 480). Sample test results are reported back to the local LIMS after approval. 

Fig. 2: FLOW Flex configuration

The FLOW Solution is available in different configurations. The FLOW Classic configuration (see Figure 1) consists of five individual components:

  • 1 MagNA Pure 96 System,
  • 2 FLOW PCR Setup System (for Primary Sample Handling and PCR Setup)
  • 1 LC 480 Instrument
  • 1 FLOW Software
 
FLOW classic can be extended with up to 3 MagNA Pure 96 Systems and up to 5 LC 480 Instruments for increased throughput.

Fig. 1: FLOW Classic configuration

In a FLOW Flex configuration (see Figure 2 ), one FLOW PCR Setup System covers both the primary sample handling and PCR setup steps in the workflow. A FLOW Flex configuration can be upgraded to a FLOW Classic configuration when needed. 
 
For maximum performance with variable workflow options and including backup solutions, both FLOW Classic and FLOW Flex can be used in parallel with the FLOW Fusion application (see Figure 3 below) for optimized data exchange between two FLOW installations (FLOW Classic and/or FLOW Flex).

Figure 2: FLOW FLEX configuration

The FLOW Fusion application requires a FLOW Software Additional License. For further details, contact your local Roche representative.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "
Overview of the FLOW Solution workflow
The FLOW Solution offers a standardized workflow and automated data solution starting from primary samples to qPCR results. Along with the flexibility and scalability to meet versatile test demands, the IT-supported workflow reduces error, shortens turnaround time, increases safety, and enhances laboratory efficiency. The workflow enabled by the FLOW Solution is perfectly suited for laboratories particularly with a high number of samples and a high variety of targets to be tested.Fig. 1: Workflow overviewFig. 2: Flow Fusion workflowFig. 3: Input and output data FLOWFig. 4: O: drive structure and important locations
 ", "Country": "XG", "Code": "General Considerations", "Name": "General Considerations" }, { "Language": "en", "Value": "The FLOW Software manages data transfer with the connected components for primary sample handling, purification, PCR setup, and amplification and detection via a file sharing approach, in which the O: drive of the FLOW Software Control Unit is used as a shared network drive. It also facilitates the result approval workflow and the transfer of orders and results from and to the customer LIMS system.



In addition, the FLOW software offers extensive options for statistical analysis of FLOW result data.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "The FLOW Software manages the entire laboratory workflow. It communicates with your LIMS system to receive sample/analysis requests, provides and gathers information from the four connected systems (FLOW PCR Setup Instrument in primary sample handling mode, MagNA Pure 96 Instrument, FLOW PCR Setup Instrument in PCR setup mode, LightCycler® 480 Instrument), and reports test results. The FLOW Software ensures that all components can communicate with each other via XML files or LIMS interface modules.
The FLOW Software is intended for general laboratory use.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "FLOW Classic configuration
You can stagger up to 4 FLOW runs over one 8 hour working day. This is a simple setup with one instrument each. If you add more MP 96 and Roche qPCR Instruments, you can get a higher throughput
(see Figure 1, below).Fig. 1: FLOW Classic configuration

FLOW FLEX configuration
You can stagger up to 3 FLOW runs over one 8.5 hour working day. This is a simple setup with one instrument each. If you add more MP 96 Systems and Roche qPCR Instruments, you can get a higher throughput.
See Figure 2, below.Figure 2: FLOW FLEX configuration

Example calculations based on a standard batch size of 96 samples and duplex PCR

  • FLOW Classic:
    • Up to 4 full FLOW runs per day
    • 4x96 samples x 2 targets = 768 individual results per day

  • FLOW Flex:
    • Up to 3 full FLOW runs per day (9 hour shift).
    • 3x96 samples x 2 targets = 576 individual results per day.
 
Additional components will increase the throughput of the individual batch but will also increase the runtime of components (especially PCR setup). Therefore, the increase in throughput is not linear with regard to addition of components.

Alternative setups, such as two FLOW Flex installations, might reach a higher throughput than 1 FLOW Classic installation with 2 MP96 Systems and 2 Roche qPCR Instruments.
", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "Control concept

The FLOW Software applies a control concept to assess whether the process steps have been passed or failed. Using the controls is optional. If no control is added, the FLOW Software analyzes the result based on the Cp threshold defined for the target.

In the PCR database configuration, the operator can specify the following controls:
 
Internal Control (IC):
  • Monitors the nucleic acid isolation efficiency; the control material is contained in each sample.
  • Controls the purification on the MagNA Pure 96 Instrument and the PCR on the LC480 instrument.

Positive Process Control
  • Comprises sample material that is positive for the target.
  • Controls the purification on the MagNA Pure 96 Instrument and the PCR on the LC480 instrument.

Negative Process Control:
  • Comprises sample material which is negative for the target.
  • Controls the purification on the MagNA Pure 96 Instrument and the PCR on the LC480 instrument.

Positive Template Control (PTC):
  • Comprises nucleic acid which is positive for the target.
  • Controls the PCR on the LC480 instrument.

Negative Template Control (NTC):
  • Reaction mix without template nucleic acid.
  • Controls the PCR on the LC480 instrument.
", "Country": "XG", "Code": "Control Reactions", "Name": "Control Reactions" }, { "Language": "en", "Value": "
Workflow
 
LIS input
Duplicate requests from LIS may cause issues with the result approval and final FLOW orderlist cleanup step. Multiple, identical requests from LIS must be avoided (identical requests may have also been added via the Enter Worklist option).
 
If the same sample ID is tagged to two individual sample types from the same source, they must be labeled uniquely to allow proper identification by the FLOW Software. 
Replicate handling for RT-PCR tests
If a 2-step RT mix is linked to a PCR Test containing replicates, the replicate requests will only be created for the actual PCR and not for the RT step.
Eluate archiving
Every purification plate created is automatically added to the archive. Eluates can be assigned to a new worklist by using the Enter Worklist - Archived Samples function. The user can search for Sample ID or Plate ID or Query all. Selected samples can be assigned to all targets available in the database.
Note that the calculations regarding remaining eluate available in a plate are not correct. The dead volume of the eluate is not taken into consideration in the calculation, which may lead to pipetting failure on the PSU. If multiple wells have been extracted, the FLOW Software will automatically select the last well to pipette, as this will contain more volume than the first well.
When targets are requested from the archive, the internal control also needs to be requested manually.  As long as the internal control is manually ordered for the sample at the same time as the target, the software will consider the IC result as part of the control evaluation for the target result.
Sample pipetting layout in the MP96 processing cartridge
The user can decide whether the worklist created for the PSH should be used for the pipetting pattern (alphanumeric), or whether the instrument should pipette the samples as loaded on to the PSH.  This setting can be adjusted in Application Configuration Editor. Note that choosing to pipette based on the sample loading order may slow down PCR Setup.
Process controls are pipetted in the same order they were configured and saved in DB-tblPCR_Control table, regardless of the type or alphabetical letter. Samples are pipetted after controls.
Sorting of controls, samples, and standards on the PSH
On the PSH, the controls are pipetted first; then the samples are sorted and pipetted alphanumerically as shown in the worklist. Standards pipetted on the PSH will be treated as samples.
PCR Plate Layout  in PSU
The user cannot influence the PCR Plate Layout on the FLOW PCR Setup System. Sorting is per target, for example, Sample 1 Target 1; Sample 2 Target 1; Sample 3 Target 1, etc. This allows for easier recalculation of target results in the LightCycler® 480 Software if a reimport is necessary.
  • Samples and controls are pipetted together for each PCR Test.  That is, all samples and controls are first pipetted for PCRTest1, then pipetted for PCRTest2, and so on. The first PCR Test pipetted is the first one configured and linked to the first alphabetically ordered Macro - as appears in tblPCR_Test table.
  • For a specific PCR Test, PTCs are first pipetted on the plate in the order they were configured, followed by NTCs in the order they were configured, and then by Process Controls in alphabetical order (regardless of whether they are PCs or NCs).  After all controls are pipetted, then the samples are pipetted.
  • If there are PCR Tests configured for multiple Macros, for example, MacroA and MacroB, resulting in 2 PCR Plates, the controls/samples linked to a first configured PCR Test that is linked to the first alphabetically ordered MacroA will be pipetted first, followed by all samples/controls linked to all other PCR Tests linked to MacroA. The 2nd MacroB, with all PCR Tests linked to it, is pipetted in the configured order.
Creating a PSU worklist
Creating a PSU Worklist involves the following rules:
  • Samples/ controls are pipetted by PCR Test.
  • Samples/controls are pipetted on separate plates per Macro.
  • Controls are pipetted before the samples for each PCR Test.
  • All replicates are pipetted on the same plate.
  • Template controls are pipetted on each plate for each PCR Test.
  • Process controls are only pipetted on the 1st plate for a PCR test as long as samples are linked to that PCR Test on multiple plates.
  • Up to 5 PCR plates can be created by the PSU in one run.
  • Only up to 3 purification plates can be considered for the worklist.
PCR setup runtimes
The PCR setup and sample transfer runtimes strongly depend on the programmed setup. The number of samples, as well as number of components and plates to be pipetted, significantly affects the runtime. In general, a setup with 1 plate is considered time neutral with 1 MagNA Pure 96 purification run (1 hour).
 
Several different setups for 96 and 384 wells have been tested. Conditions were:
  • 1 PCR plate (96 or 384)
  • PCR Tests from 1-4
  • Each test made out of 3 components
 
PCR Setup results (examples):
  • One 96-well plate with 24 samples assigned to 4 tests: 11 minutes.
  • One 384-well plate with 95 samples assigned to 4 tests: 35 minutes.
 
Sample transfer results (examples):
  • One 96-well plate: 6-16 minutes.
  • One 384-well plate: 45 minutes.
 
Note that the run times are influenced by multiple parameters, such as last used tip layout, position of tubes on the carrier, layout of the purification plate, etc. The above listed times are examples for specific setups. Other setups may lead to different results. The benchmark should be: PCR Setup from 1 PCR Plate can be pipetted time neutral with MagNA Pure 96 purification.


Example measurement PSH/PSU runtime:

Setup:
  • 96 samples (001 to 096) loaded on the PSH, 200 μl Generic sample type transferred into the one MP96 Processing Cartridge. Samples were loaded in the rack in alphanumerical barcode order (faster as no sorting is required).
  • 8 different PCR test/master mixes ordered for each of the 96 samples, 15 μl reaction mix (loaded as ready-to-use on the PSU) + 5 μl eluate in each well of a 384-well plate.
  • No controls were ordered.
 
PSH pipetting time: 
  • 15 minutes to pipette the 96 samples into the MP96 Processing Cartridge.
 
PSU pipetting time: 
  • Dispensing of the 8 reaction mixes (4 mixes in each 384-well plate):
    • When multi-dispensing the reaction mixes with one tip: 45 minutes per plate, resulting in a total of 90 minutes for the pipetting time.
    • When multi-dispensing the reaction mixes with two tips: 43 minutes per plate, resulting in a total of 86 minutes for the pipetting time.
  • Dispensing of the samples: each of the 96 samples from the MP96 Output Plate is dispensed eight times, four times in each LC480 384-well plate. 45 minutes are required per LC480 384-well plate, resulting in a total of 90 minutes for the pipetting.
Standard curve addition
When samples are sent for retest, the internal standard curve is added again automatically (PSU standard curve).  For tests with an external standard curve and calibrator, the calibrator is added automatically to the worklist when samples are sent for retest.
According to our tests with an external standard curve and a PSU retest, the calibrator is included in the PSU Worklist Setup automatically.
If multiple LightCycler® plates are pipetted for the same target, only one plate contains the standard curve.
Standards from internal curves added on the PSH are not added automatically at PSU retest, but must be assigned from the archive plate manually.


Automated ordering of retest/reflex tests
There is no option in the software for automatically and selectively ordering a new specific test for a sample that is \"reactive\" for another test.


PSU retest on already approved samples
Retesting, that is, testing a sample for the same target, of samples that have already been approved is not possible.  If a sample requires retesting, the order for the retest should only be done via the Result screen before approval of the sample results, that is, either as a PSH retest or a PSU retest. Reflex testing, that is, assigning a new target to a sample, must be ordered via the Archive option. Go to FLOW Workplace - Orderlist and Enter worklist. In the archived samples section, the samples can be selected again and linked to a target. The selected samples will appear as ON_HOLD to the FLOW Orderlist.


IC addition for retests/reflex tests
For PSH retests, the IC will be linked automatically once the Start button is clicked. In contrast, for PSU retest or reflex test, the IC will need to be additionally requested by the operator as it will not be added automatically.


Insufficient Internal Control (IC) loaded during the primary sample handling (PSH) step
If insufficient IC is loaded during the PSH step, the software will request more IC.  Load additional IC on to the instrument and confirm by clicking <OK>.
If <CANCEL> is clicked, the following will occur:
  • The MP96 order file will be created and will contain the comment <Comment>1</Comment> for all samples indicating they all failed.
  • Samples will be returned to the status NOT_ALLOCATED.
 
The MP96 order should be automatically removed from the MP96 Order folder on the: O: drive when the next run starts.  However, check the MP96 order folder to ensure that it has been cleared before starting the next run. In addition, the MP96 order should be deleted from the MP96 Database if it has been automatically imported, for example, via the Host Module.
Generally, it is best to refill the IC when requested during the PSH, and to not click <CANCEL>.
ON_HOLD at PSU level
ON_HOLD Samples with reported errors during PCR setup, samples sent for a retest/reflex test on PCR setup level, and samples exceeding the capacity of PCR plates to prepare in one run on the FLOW PCR Setup Instrument, are added to the ON_HOLD status. These samples are added to the next worklist for the FLOW PCR Setup Instrument as soon as a new batch of samples in the FLOW process reaches this step (PURIFICATION_STARTED). Alternatively, ON_HOLD samples can also be processed independently of other batches by manual override when using the Start button (choose Start > No).


Clot detection
The primary sample handling method will check twice if pipetting is possible when a clot is detected. Afterward, the sample will be automatically excluded. However, the sample can be pipetted manually after the primary sample transfer is complete.


Sample mixing
Sample mixing is used for Whole Blood and Cell Suspension sample types. If these sample types are assigned by a Requested Test, the samples will be mixed accordingly during primary sample handling.
If different sample types are to be tested for the same target, programming for this may be a challenge. The same targets would need to be added multiple times, using different names in the PCR Database for each sample type, and then linked to separate Requested Tests to allow for selecting of different sample types.
 
 
Result

 
 Control behavior
  • Process Controls:
    • Negative Process Controls will be shown but not evaluated for the target IC. The operator needs to take appropriate actions in case of a failure.
    • If process controls are present, they are added automatically to every MagNA Pure 96 Purification Cartridge in a single sample transfer run. On the PCR level, if multiple PCR plates are prepared, the process controls are only added to the first PCR Plate. The evaluation is done via cross-plate check.
    • If multiple process controls for the same target but from different purification plates are located on one LC480 plate, for example, due to ON_HOLD integration, the process control from the corresponding purification plate is used for result interpretation of the samples.
  • Internal Controls:
    • If a sample is assigned to multiple tests that require different internal controls, two individual purifications will be prepared during the sample transfer on the PSH.
    • If the same internal control is used for different PCR Tests or Requested Tests, multiple internal control targets with different names must be defined to allow proper linking. On the Requested Test level, the same barcode can be defined for Internal Control spiking on the PSH, but on target level, the names must be different.
Result calling
Uncertain calls are transferred from the LightCycler® 480 Instrument to the FLOW Software. As uncertain calls do contain Cq values, these are treated as valid results by the FLOW Software. If uncertain results are transferred for controls, the linked samples will not be flagged as control failed.


Calculation of the “Delta cq” value for external standard curve
The “Delta cq” value is calculated based on the difference between the internal standard/calibrator cq and the extrapolated cq value of the external curve for this concentration.


Standard curve calculation
The FLOW standard curve calculation uses the same method as the LightCycler® 480 Application Software. The calculation is done using the AbsQuant 2nd Derivative Max Algorithm, containing a linear and non-linear regression. In FLOW, only a linear curve is displayed, although a non-linear regression may have been used for certain data points.
The LightCycler® 480 Software uses more decimal points than the FLOW Software for the calculation. This can lead to minor differences between the calculated values. 
 
 
Configuration

 
 Using 96- and 384-well plates in parallel on FLOW
It is not possible to use 96- and 384-well plates in parallel on FLOW. However, it is possible to run both plate types on one FLOW Solution, as long as a workflow using one type of plate is first run to completion before setting up a new run for the other plate type.
This could be done as follows: Create two independent databases, one for 96- and one for the 384-well format. Finish one complete FLOW process with one plate format type. Then change the database to switch to the 384-well format.
The procedure described above is generally not recommended. If both plate types should be used, it is best to set up two FLOW Solutions, one for each plate type.
MagNA Pure 96 Processing Cartridge sorting
If multiple MagNA Pure 96 Processing Cartridges are pipetted on the FLOW PCR Setup Instrument in primary sample handling mode (different purification protocols, more than 96 samples), and the number of MagNA Pure 96 Instruments setting in FLOW is set to 1, only one plate will be set to PURIFICATION_STARTED. The second plate will remain PSH_COMPLETE until the first plate has reached status PSU_STARTED. This means that orders are not merged at the PSU level.
 
The FLOW Software will create the first MagNA Pure 96 Order from the MagNA Pure 96 Processing Cartridge loaded on position 1 on the carrier. The barcode (number) is not taken into consideration for sorting.
Multiple MP96 purifications and merging at PSU level
A standard FLOW workflow with 1 MagNA Pure 96 Instrument will always create a single MP96 Order file, even if more than 96 samples have been processed during primary sample handling. If more than 96 samples are processed by the PSH software, samples pipetted into the first MP96 processing cartridge will be set to status PURFICATION_STARTED, whereas samples pipetted into the other processing cartridges will remain in the status PSH_COMPLETE until the sample status from the first processing cartridge switches to PSU_STARTED (which occurs when the MP96 has completed the purification, and the PSU received the worklist_samples file).
If multiple MP96 instruments are defined in the FLOW PCR database (up to 3 is possible), orders will be merged for the PSU worklist. In this case, FLOW will wait until all MP96 Orders (up to 2 or 3, depending on the PCR database configuration) have been processed, and then will create a merged worklist_samples for the PSU. This process can also be \"simulated\" by defining multiple MP96 instruments in the PCR database, but running the purifications one after the other on a single MP96. A merged PSU worklist_setup will be created after all purifications are complete.  However, this will have an impact on the overall turnaround time.
FLOW Fusion
FLOW Fusion cannot compare and align existing purification archives, sample retests, or statistical analysis calculations from different FLOW Solutions. FLOW Fusion only checks the sample status PSH_COMPLETE and the actual result approval. Any other status in between the two FLOW lines is not cross-checked, as the status is set to ALLOCATED. There is also no trigger to transfer this information. Therefore, FLOW databases only have the PURIFICATION_ARCHIVE from the MP96 result file that was imported as a reference for purification archives. In a FLOW Fusion environment, the MP96 result files are only imported to one FLOW database. As a result, retests/reflex tests based on an eluate cannot be assigned to the other line. 
Options for FLOW lines in parallel
 Two FLOW lines with identical programming of the PCR database (clones):
  • FLOW Fusion
 
Two FLOW lines dedicated to different targets/sample types:
  • FLOW Fusion not required. The FLOW Software will only import requests that are known in the database. All others will be ignored. If the same LIS request is sent to both lines, the sorting will happen automatically.
Using multiple databases on the LightCycler® 480 Instrument
If multiple databases on the LC480 are in use, the LIMS Interface Module automatically logs into the last used database. Ensure that this database contains the requested LightCycler® 480 Macros, as defined in in the FLOW PCR database. Furthermore, password and username entered in the FLOW PCR database must match the last logged in database on the LC480.
If the LC480 is used for FLOW only, use only one database. If LC480 is used in both standalone mode and in FLOW, use at least two databases.
Do not set the LC480 Instruments to virtual in the LightCycler® 480 Software. Otherwise, the FLOW Software cannot access the instrument and no error is reported.
 
 
Settings (PCR Database)

 
Internal Control definition
Per definition, an Internal Control should control the whole process including purification and amplification, ideally in the same PCR reaction as a multiplexed target.
For unstable Internal Control targets, several workarounds may be useful:
  • Spiking the IC on the MagNA Pure 96 Instrument: Define target IC in the FLOW DB as usual. Do not define a barcode for the IC on Requested Test level. Once the order for the MagNA Pure 96 is imported, modify it by adding the Internal Control in the MagNA Pure 96 Software. After modification of the order, save it to proceed. On the MagNA Pure 96, the Internal Control is added to lysis buffer before being added to the sample, therefore the risk of degradation is minimized.
  • Spiking on the PSU: Define target IC in the FLOW DB as usual. Do not define a barcode for the IC on Requested Test level. Define the IC as additional component on reagent level, or as primer or probe on PCR Target level, or, for ready-to-use mixes, add it manually to the reaction mix.
 
If an Internal Control requires reporting back to LIS, it needs to be defined as a standard target. In this case, the Internal Control evaluation needs to be done manually by the operator.
Per default, no control data is sent back to LIS. The control data is only used for the result interpretation in FLOW.
Dead volume of elution plate wells (μl)
The dead volume of elution plate wells setting in the PCR Database configuration is based on a 6 x 5 μl pipetting scheme, enabling 6 reactions out of 50 μl eluate. The setting may require adjustment if higher eluate volumes are used for the PCR setup. This recommendation is based on field feedback for failing eluate transfer when using 8 or 10 μl eluate in the PCR setup. Contact your local Roche representative for more information about how to adjust this setting.


Master mix terminology
Linking the same ready-to-use reaction mix to different PCR Tests or Requested Tests is not possible, due to the requirement of having the same name for the PCR Test and Master Mix Barcode.

Maintenance

 
Database size
Regular archiving is highly recommended when using the FLOW Software. The working database size should not exceed a value of 2 GB to prevent performance related issues. For archive databases, the maximum value should be set to 4 GB.
Statistical Analysis Module (SAM)
Once archived, control data can no longer be imported into the QC database of the working database. This means the QC analysis needs to be set up in a way that ensures the data is successfully imported before being moved to the archive. This could be set using the automated import job options.
 
FLOW PSH configuration vs. PSH standalone 
  • FLOW PSH configuration:
    • Primary sample handling orders are sent by the FLOW Software. The data flow is fully automated between LIS - FLOW Software and primary sample handling. The workflow contains primary sample handling, purification, PCR setup, amplification and detection, result review, and approval and reporting to LIS (see Figure 1, below).
  
  • PSH Standalone:
    • The orders are individually created by the operator, either worklist-based or direct input by barcode scanning on the instrument. The automated data transfer only covers the communication between primary sample handling and MagNA Pure 96 purification. The workflow stops after the sample purification (see Figure 2, below).
 
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  1. FLOW Software DVD
  2. FLOW Dongle (1)
  3. FLOW Terminal Dongles (2)
", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "
The FLOW Solution Troubleshooting Guide provides an overview of tips and tricks to overcome workflow interruptions in the FLOW Solution. Before running experiments on the FLOW Solution, operators should become familiar with the specifications for sample materials, pretreatments, detection and analysis formats, and the control concept of the FLOW Solution.
 
The FLOW Solution Troubleshooting Guide covers:
  • General recommendations
  • Troubleshooting before a run
  • Troubleshooting during a run
  • Post run troubleshooting
  • How to create support data
 
This section describes additional tips and tricks that have not yet been added to the FLOW Solution Troubleshooting Guide. 

Addition to “The MP96 orderfile is not created”
 
Scenario: An old PSU worklist blocked the creation of the new MP96 orderfile and PSU worklist_setup. The MP96 plate was processed independently on the MagNA Pure 96 System, but the user wanted to get the MP96 plate information recognized by the FLOW software again so that the workflow could continue.
 
The workflow can be resumed as follows:

  1. Remove the old, blocking worklists from the PSU worklist folder.-> The FLOW Software will create an MP96 and PSU orderfile
  2. Export the MP96 result file from the independent run to a local folder. Rename the file to #8Barcode of the MP96 processing cartridge so that it matches the MP96 orderfile name. In addition, add the sample IDs to the .xml file to match the original FLOW order. After renaming and editing, copy the resultfile back to the MP96 results folder on the O: drive.

This manual intervention is not traceable by the FLOW Software. Any modification of MP96 order or result files are the sole responsibility of the operator.
 

 Addition to Post run troubleshooting
 
Scenario: The monitor.lok file is blocking the LIS result reporting. The error message in the FLOW Annunciator is either:

  • Unable to create SRIMPORT.DAT. In this case, rename file SRIMPORT.tmp to SRIMPORT.DAT and try loading it through Monitor again later.
        or
  • Unable to delete the Monitor.Lok file in “O:\\Morpheus\\AuroraSRImport”. In this case, delete the file manually.
 
The result reporting workflow works as follows: Result approval in the FLOW Software will trigger the creation of an SRIMPORT.dat file in O:\\Morpheus\\AuroraSRImport. This monitor application (magnifying glass in windows task bar) screens this location every minute for unprocessed SRIMPORT.dat files. Once a file is present, the monitor will convert it to a LIS result file. During conversion, the SRIMPORT.dat is not accessible for any additional approvals, and a monitor.lok file is created. When processing is finished, the monitor.lok file is automatically removed, and the SRIMPORT.dat content is moved to SRIMPORT.old.
 
When results are approved while the monitor is processing a recently created SRIMPORT.dat file in the O:\\Morpheus\\AuroraSRImport folder, the data cannot be added to the SRIMPORT.dat file. This will trigger the creation of a SRIMPORT.tmp file.
 
To get these results reported back to LIS, rename the SRIMPORT.tmp to SRIMPORT.dat, so that it can be processed by the monitor application.
 
If the monitor.lok file cannot be deleted, the monitor application might need to be stopped or closed. Click on the magnifying glass icon in the windows taskbar, and close the monitor application. Delete the monitor.lok file, rename the SRIMPORT.tmp to SRIMPORT.dat, and restart the monitor application either out of the FLOW Software, by clicking on the monitor icon, or by restarting the FLOW Software.

Addition to “Purification”
If the MP96 has a problem during processing, or the safeguard warnings have been overruled by the operator, the samples are reported back to the FLOW Software with a “Failed” status. The sample status will be set back to “NOT_ALLOCATED”, but the FLOW Software will still create a worklist_samples for the PSU. This is due to the fact that not all samples may have failed during MP96 processing, or additional samples from other plates may be part of the PCR setup. However, even if no sample is to be processed, the worklist_setup file is created to finish the PCR setup workflow (reagents have already been added to the PCR plate). The operator can decide to either finish the run on the PSU, or to manually delete the created PSU worklists and start with a new FLOW process.

Addition to “PCR Setup”
If the PSU has a problem during processing, and no sample has been pipetted, the plate may still change to status PSU_COMPLETE if, for example, controls have been pipetted successfully. In this case, if the plate is not run on the LC480, it may block the running of the next plates on the LC480, as the FLOW Software still expects this plate to be loaded to the LC480. In this case, contact your local Roche representative for support to resume the workflow and remove the blocking plate from the database.

Addition to “LC480”
 
Recovery procedure in case of LC480 Instrument run abort
An LC480 run abort at the early stage can be recovered in FLOW by the following procedure: Prerequisite: Sample status in FLOW is LC480_STARTED.

1. Stop LC480 LIMS Interface Module and start the plate in the LightCycler® 480 Software manually using the same conditions as defined in the FLOW Software, that is, use assigned macro. The experiment must be performed by the same user as defined in FLOW, and the file needs to be stored in the correct location (default User/Experiment).

2. After the run, adjust the file as follows:
  • The filename has to correspond to the FLOW terminology (expBARCODE).
  • In the sample editor of the experiment, the correct sample information and location needs to be imported. To do this, take the sample names and location from the relevant .txt file located on O:/LightCycler/Data/Sent folder. Open the text file in Excel, define the column separator as “|”. Store the file in .txt. format, use the import function in the LightCycler® 480 Sample Editor, and assign the correct headers (Sample Name and Pos). Save the file again once the sample information has been imported and ensure that it is stored in the correct location (default User/Experiment).

3. Close the LightCycler® 480 Software and open the LC480 LIMS Interface Module. The FLOW Software will automatically retrieve the result file.

Addition to “Data flow”
If result files from individual components should be transferred to multiple FLOW Servers, such as when an instrument is used as a backup in another FLOW line, the file paths can be adjusted to locally export the files. From there, the files can be copied to the desired O: drive location of the respective FLOW Server.
This manual intervention is not traceable by the FLOW Software. Any modification of individual results files is the sole responsibility of the operator.

Addition to \"LIS Import Issues\"
If pending orders were deleted due to a reset unread tables, the provided LIS files can be imported again by copying them from the O:\\LIS\\Worklist\\Processed folder back to O:\\LIS\\Worklist.

Miscellaneous
 
PCR Database/Preparation of master mix/Decimals
The operator programmed 3.7 µl for the concentrated master mix, 3.75 µl for the reverse primer, and 2.5 µl for the forward primer. Total master mix to be used for one sample was 10 µl.
When programmed this way, the volume calculation for the master mix to run only one sample will be wrong. It is correctly calculated if more than 1 sample is requested for the test.


Worklist_setup.xml and worklist_sample.xml mismatch on the PSU

When integrating samples from the purification archive, the following scenario leads to a worklist_setup.xml and worklist_sample.xml mismatch: 

  • One sample is requested from the purification archive (Purification Plate1). The sample is integrated into the next run with the status ON_HOLD_PSU (which means that the sample is added to the worklist_Setup.xml), once the next batch has reached the status PURIFICATION_STARTED.
  • Another sample is requested from the same purification archive plate (Purification Plate1) as the previous integrated sample before the MP96 is completed. Once the results from MP96 are available and interpreted, the most recently added sample will be added to the worklist_sample.xml file. Therefore, there will be a mismatch between the worklist_Setup.xml and worklist_sample.xml files.

Workaround: Do not add additional samples from a plate that is currently in the workflow when the status of a sample from that plate is already ON_HOLD_PSU.

The tblPCR_Target table is not updated properly for Internal Control linked target
When an Internal Control (IC) target is changed to a standard target, the linked target is not updated accordingly and will end up with a Control Data Missing call.
Check the target settings when a linked IC has been modified.
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The FLOW Process
 
The FLOW Software manages the entire workflow for data exchange between all modules involved in the FLOW process, as well as data exchange with the connected LIMS:

  • FLOW PCR Setup Instrument in primary sample handling mode with FLOW PCR Setup Instrument Software Version 2.0 and higher.
  • MagNA Pure 96 Instrument with MagNA Pure 96 Software Version 3.0 and higher.
  • FLOW PCR Setup Instrument with FLOW PCR Setup Instrument Software Version 2.0 and higher.
  • LC480 Instrument with LightCycler® 480 Software Version 1.5.1.
 
The FLOW Solution now offers several configuration possibilities:

  • FLOW Classic consists of five individual components: one FLOW PCR Setup System in primary sample handling mode, one MagNA Pure 96 System, one FLOW PCR Setup System in PCR Setup mode, one LC480 Instrument, and one FLOW Software. FLOW Classic can be extended to include up to three MagNA Pure 96 Systems and up to five LC480 Instruments for increased throughput.
  • In FLOW Flex, the FLOW PCR Setup System covers both primary sample handling and PCR setup workflow steps. FLOW Flex has a slightly lower throughput, but a smaller space requirement. FLOW Flex can be upgraded to FLOW Classic if necessary. As with FLOW Classic, FLOW Flex can also be extended to include up to 3 MagNA Pure 96 Systems and up to five LC480 Instruments.
  • The FLOW Fusion application can be used for parallel operation of two FLOW installations (FLOW Classic or FLOW Flex).
 

Sample processing workflow
 
1. On the FLOW PCR Setup Instrument in primary sample handling mode, samples are pipetted into the MagNA Pure 96 Processing Cartridge.
2. On the MagNA Pure 96 Instrument, the samples are purified, that is, RNA or DNA is extracted from the samples. The eluates are pipetted into the MagNA Pure 96 Output Plate.
3. On the FLOW PCR Setup Instrument in PCR setup mode, the reaction mix components and the eluates are transferred into a LightCycler® 480 Multiwell Plate, based on a predefined PCR pipetting setup.
4. The LightCycler® 480 Instrument performs the real-time, online PCR of the samples.
 
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Result Approval Workflow
The validation function for test results is located under Orders > Results tab. The function allows for the following two modes for validating test results: 
  • Single-approval mode: A result is approved by one user, after which it is valid and reported back to the LIS.
  • Dual-approval mode: A result must be approved by two users, after which it is valid and reported back to the LIS.
It is preferable to use the dual-approval mode. The approval mode is set during system installation. If you wish to change the mode, contact your local Roche representative.
 
For dual-approval mode, operators can have the following approval rights:
  • No authorization to perform an initial or final approval.
  • Authorization to perform final approval only.
  • Authorization to perform initial approval only.
  • Authorization to perform initial and final approval.

The operator can alter the status of a sample depending on the approval rights and the current status of the sample. Samples are displayed as read only when the operator currently has no rights to change the sample status. For detailed information about configuring approval rights, refer to the section ‘Security Access’ in the FLOW Solution Operator’s Manual.

How to perform a final approval for single-approval mode or initial approval for dual- approval mode 
  • In the result table, select the sample(s) that are ready to be approved. Click Approve to approve the results. A green check mark is displayed in the first column of the result table.
  • If a sample should be sent back for a retest on the FLOW PCR Setup Instrument in primary sample handling mode, click PSH retest. A black PSH icon is displayed in the first column of the result table.
  • If a sample should be sent back for a retest on the FLOW PCR Setup Instrument in PCR setup mode, click PSU retest. A yellow PSU icon is displayed in the first column of the results table.
If you have selected a sample for PSU retest, the software checks whether there is sufficient volume available in the elution plate to process the retest. If the available volume is not sufficient, FLOW changes the PSU retest to a PSH retest.  
  • Click Commit and Save. Enter your password and click Save.
  • In single-approval mode, the icons in the Status column change to either a green check mark (approved), PSH icon (PSH retest), or PSU icon (PSU retest).
  • In dual-approval mode, the icons in the Status column change to either a half-filled green check mark (approved), PSH icon (PSH retest), or PSU icon (PSU retest).

 How to perform a final approval of a result in dual-approval mode

The second approver logs into the software and repeats the procedure described above (4-eyes principle).

Result Reports
A Result Report contains a summary of the experiment for the selected sample or plate.
The summary contains information about the barcodes for the sample, PCR plate, and Internal Control, as well as the results obtained from the LC480 Instrument and the respective call from the FLOW Software based on the definitions of the control concept. For quantitative experiments, the concentration is listed in the defined units.
The report also indicates the PCR test and target names, as well as the date of the run.

FLOW result file and LIS configuration
The LIS order file (requested test or PCR target) has no effect on the FLOW result file.
The FLOW Host Interface Manual provides information about the result file structure, as well as examples of results files used within Aurora FLOW:
  • P: Patient number
  • O: the Order
  • R: Results.
According to the examples provided in the Host Interface Manual, FLOW sends back results at the PCR Target level, that is, multiple result lines linked to the PCR Target. This is also in agreement with the structure of the Results record.", "Country": "XG", "Code": "Results", "Name": "Results" } ] } } ] }

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