Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument

Document ID: 62U3QES2XUJM-3-2794

Effective Date: 3/24/2020

INFLUENZA (FLU) AND RSV BY PCR (NAAT) ON THE LIAISON MDX

INSTRUMENT

SCOPE
 This procedure applies to the technical staff that have been authorized to perform this assay.

PURPOSE
This procedure provides instructions for analyzing and resulting Influenza A, B, and RSV using
the DiaSorin assay system.
Influenza, commonly called “the flu”, is an illness caused by viruses that infect the respiratory
tract. Compared with most other viral respiratory infections, such as the common cold, influenza
infection often causes more severe illness with a mortality rate of about 0.1% of people who are
infected with the virus.

PRINCIPLE
The DiaSorin Flu A/B & RSV assay system is a real-time RT-PCR system that enables the direct
amplification, detection and differentiation of human influenza A (Flu A) virus RNA, human
influenza B (Flu B) virus RNA and RSV RNA from unprocessed nasopharyngeal swabs that
have not undergone nucleic acid extraction. The system consists of the DiaSorin Flu A/B & RSV
assay, the Liaison MDX (with Liaison MDX Studio Software), the Direct Amplification Disc
and associated accessories.
In the DiaSorin Flu A/B & RSV assay, bi-functional fluorescent probe-primers are used together
with corresponding reverse primers to amplify Flu A, Flu B, RSV and internal control RNA. The
assay provides three results; conserved regions of influenza A viruses (matrix gene), influenza B
viruses (matrix gene) and RSV (M gene) are targeted to identify these viruses in the specimen.
An RNA internal control is used to detect RT-PCR failure and/or inhibition.

SPECIMENS
 Nasopharyngeal swab in one of the following media:
o M4RT multi-microbe transport medium (Remel)
o Universal Transport Media (UTM), (ThermoFisher Scientific)
o M6 transport medium (Remel)

Transport Condition Length of Time

2-8 °C refrigerated; transported on wet ice refrigerated upon collection; ≤3 days
frozen at -70 °C or colder; transported on dry ice >3 days

Storage Condition Length of Time

2- 8 °C (refrigerated) up to 3 days
frozen at ≤ -70 °C Long-term storage

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

 Sample Rejection Criteria

o Specimens on cotton or calcium alginate tipped swabs, swabs with wooden shafts,
specimens in transport media other than transport media recommended above.
o Room temperature specimens.
o Specimens from sites other than nasopharynx.
 For unacceptable specimens, follow the Reject, Redraw, Cancel and Credit procedure.

REAGENTS AND SUPPLIES

 DiaSorin Flu A/B & RSV Reaction Mix (DiaSorin Molecular, Catalog # MOL2650,
includes DADs)
o Store at -10 to -30 °C
o Use within 30 minutes of thawing
o Do not vortex, invert, or refreeze
 DiaSorin Flu A/B & RSV Direct Amplification Discs (DADs)
o Store at room temperature
 Viral Transport Medium (any of the following)
o Micro Test M4 Viral Transport Medium (Remel, Catalog # R12500)
o Universal Transport Medium (ThermoFisher Scientific, Catalog # 305C)
o M6 Viral Transport Medium (Remel, Catalog # R12533)
 Replacement Foil Wedges (DiaSorin Molecular, Catalog # REF MOL1550)
 50 µL pipette and sterile, nuclease-free disposable pipette tips with filters

EQUIPMENT

 Liaison MDX with Liaison MDX Studio Software version 4.2 or higher
 Mini-centrifuge
 Vortex

CALIBRATION

The spectral matrix must be installed in each Liaison by Diasorin and should not be changed
unless an updated QR code for the instrument is provided by Diasorin. The spectral matrix is
unique to each Liaison. If the matrix label will not scan or cannot be found, contact Diasorin
Molecular.

QUALITY CONTROL

Quality control is required to be run every 30 days and upon receipt of new lots and/or shipments
of reagents. Process the following controls for QC testing:
 DiaSorin Flu A/B & RSV positive control
o In place of DiaSorin control, a pooled sample of previously positive patient
samples may be used
 Negative control
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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

Step Instructions for Prepared Controls:

Aliquots of pooled previously positive Flu A, Flu B, and RSV patient samples are
1.
stored in the -20 °C freezer in the Max room.
2. Thaw vial. Vortex and quick spin.
3. Set up as patient sample (See procedure below).
Step Instructions for Positive Pooled Patient Control Preparation:
Collect patient samples that are positive for each Samples from M4, UTM, or
1.
analyte (positive Flu A, B, and RSV). M6 media can be used.
2. Add 1 mL of each positive patient sample to a sterile tube (total 5 mL). Mix well.
3. Aliquot 75 µL of mixed pooled sample into 2 mL screw-topped tubes.
Label appropriately and store in the -20 °C freezer in the Max room. The
4.
expiration date is two years after the preparation date.
Step Instructions for Negative Control Preparation:
The negative control is an aliquot of M4, UTM, or Stored in the small refrigerator in
1.
M6 media. the Flu room.
2. Thaw vial. Vortex and quick spin.
3. Set up as patient sample (See procedure below).

QC testing results must be as follows:

Internal
QC Flu A Flu B RSV Control
Pooled Positive Detected Detected Detected Valid
Negative Control Not Detected Not Detected Not Detected Valid

 For New Lot Checks, document results on New Lot Notification form. Keep with kit until it is
put into use. When kit is in use, turn form in for review.
 Document invalid/failed runs on Instrument Troubleshooting and Occurrence Log.
 Perform regular contamination checks as indicated in Environmental Contamination Checks
procedure.
 All reagents should be discarded appropriately when their expiration date is exceeded.

PROCEDURE

Step Test Set Up

1. Perform all testing in a Biological Safety Cabinet (BSC).
Thaw one reaction-mix vial at room temperature NOTE: Must be used within 30
2.
for each sample or control to be tested. minutes after thawing.
3. Retrieve the specimen(s).

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

4. Create a worksheet (FLUNAT) and print plate labels.

5. Label a Liaison Universal Disc Map with a numbered plate label for each specimen.
6. Set the disc on one of the blue disc cold blocks in the BSC.
Quick spin the reaction mix vials once thawed (do not vortex or invert). Vortex patient
7.
samples.
One wedge at a time, peel the adhesive foil back to
Avoid touching the underside of
8. expose the Sample (SAMPLE) and Reaction (R)
the foil that will be in contact
wells without completely removing the adhesive
with the wells and disc surface.
foil cover (Fig. 1 & 2).

Figure 1 Figure 2

9. Transfer 50 μL of the reaction-mix into Reaction (R) well.

10. Transfer 50 μL of sample into Sample well (SAMPLE).
Cover the wedge sealing the wells with the peeled If the original foil is torn, replace
11. adhesive foil, pressing down firmly near the edge it with a Replacement Foil
of the wedge. Wedge.
12. Tear off the tab portion of the foil cover along the perforation.

13. Repeat steps 7 to 12 for the next sample(s).

14. Scan the DiaSorin Flu A/B & RSV Direct Reaction Mix vial or barcode card.
If the disc was used previously, it
Scan the disc barcode on the Direct Amplification
15. is helpful to scan it into the same
Disc (DAD).
computer.
Scan each specimen number. Make sure the scanned specimen number matches the
16.
intended wedge.
17. Load the sealed Direct Amplification Disc into the Liaison MDX and start the run.
Upon completion of the run, print the Result
The software automatically
18. Report. Include the Ct values and graphs by
interprets and displays results.
selecting the check boxes.
Fully used discs must be disposed of in a covered Discs can be reused if all
19.
waste container used to dispose of amplicon. wedges are not used.
Clean BSC with 0.1x bleach, 70% ethanol, RNAse Away, and nuclease-free water. Turn
20.
on UV light for 2 hours. Sign the Biosafety Hood Daily Cleaning Log.

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

MAINTENANCE

After Every Use At End of Shift

1. Wipe outer surfaces of the Liaison MDX with
1. Clean Cold Blocks with a SaniWipe
0.1x bleach followed by 70% ethanol.
2. Clean Computer Keyboards with a 2. Clean workbench area with 0.1x bleach
SaniWipe followed by 70% ethanol.
3. Clean Liaisons and counters with a
3. Document on DiaSorin Liaison Cleaning Log
SaniWipe

REPORTING AND INTERPRETATION OF RESULTS

Step Interpreting Results

For each Sample ID entered, the software displays a result (“Detected”, “Not Detected”
1.
or “Invalid”) for Flu A, Flu B and RSV.
 Detected: indicates the presence of Flu A and/or Flu B and/or RSV RNA in the
patient sample.
2.
 Not Detected: indicates the absence of Flu A and/or Flu B and/or RSV RNA in the
patient sample.
Step Result Reporting
 NOTE: RSV tests that are
NOT ordered are NOT called
or reported (Positive or
Negative), unless patient is 1
year old or younger. For these
patients, contact the provider.
Enter results using LIS. Enter all the positive
1. patients by typing “pos” or “POS” in the value  NOTE: Positive RSVs on
field. hospitalized patients are
called to the hospital staff as a
courtesy only for the first time
the patient is tested.
 NOTE: Positive RSVs are not
critical values.
The comment “Result is
indeterminate due to a PCR
Enter all Indeterminate patient results by typing
inhibitor present in the specimen.
2. “ind” or “IND in the value field for Influenza A,
Consider repeat specimen if
Influenza B, and RSV.
clinically indicated” will be added
automatically.
Enter all negative patients by typing “neg” or “NEG” in the value field for Influenza A,
3.
Influenza B and then for RSV.

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

4. Check that all results are as entered correctly. Finalize the results.
 Another technologist will
verify the results, check for
errors, and electronically sign
Assemble all the DiaSorin Detail Reports and
5. in Worksheet Manager.
the disc map(s). Place in the “Finalized” bin.
 The worksheet is then placed
in the Section Head’s
mailbox for review.
If the assay was positive for any of the viruses, write an A, B, or R (standing for Flu
6.
A, Flu B, and RSV, respectively) on the top of the tube’s cap.

At the end of the shift, track specimens into the

Periodically the specimens will
positive Flu/RSV rack or the negative Flu/RSV
7. be transferred to long term
rack as appropriate. The racks are stored in the
storage in the -80 °C freezer.
Area 2 -20 °C freezer.
8. Dual or triple positive results must be repeated for confirmation.

TROUBLESHOOTING

Errors and Invalid Results

Invalid: indicates inability to conclusively
determine presence or absence of Flu A and/or Flu
B and/or RSV RNA in the patient sample. This result may be due to:
a. Internal Control (RNA IC)
The sample needs to be diluted and retested. failure OR
1. Make a 1:2 dilution using media. b. Failure to detect sufficient
2. For example: 100 µL of sample with 100 µL specimen.
media, mix, use 50 µL of this dilution.
3. If the problem is unresolved, report as
Indeterminate.
An EC500 error in absence of another positive
analyte result An EC500 error indicates a data
1. Refer to Figures 3 and 4 below. Follow the quality error for the particular
dilution protocol used for an Invalid test (see viral analyte(s). The software was
above) before reporting. unable to determine a valid
2. If a sample is invalid or has an error after amplification for that analyte(s).
repeating the assay, report all valid results and
enter invalid/error results as Indeterminate.

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

Figure 3

EC500 Error with “rainbow”

curves must be repeated with
1:2 dilution

Figure 4

An EC500 error in conjunction with another An EC500 error indicates a data

positive analyte result quality error for the particular
 If a sample is positive for one virus, and has an viral analyte(s). The software
EC500 error on another, refer to the graph of was unable to determine a valid
the crossing threshold. See Fig. 5. amplification for that analyte(s).

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

 If the analyte with the EC500 error shows no

amplification, the result may be called positive
for the first analyte and negative for the analyte
with the error.

Figure 5

Can be reported as A pos,

B neg without repeating.

Internal
Control

Flu A

Flu B: no
amplification

PROCEDURAL NOTES

 Avoid touching the underside of the foil that will be in contact with the wells and disc
surface.
 Do not attempt to remove adhesive foil cover from wedges that have been used or attempt to
re-use Sample and Reaction ports that have been used in previous runs.
 Discs may be reused until all 8 wedges have been used. Dispose of used discs without
detaching foil cover in a covered waste container used to dispose of amplicon.
 After each use store discs flat with the numbered foil side up.

LIMITATIONS

 As with other tests, negative results do not rule out Flu A, Flu B or RSV infections and
should not be used as the sole basis for treatment or other patient management decisions.
 False-negative results may occur if the virus has genomic mutations, insertions, deletions, or
rearrangements or if performed very early in the course of illness.

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

 Information on the kit barcode can only be transferred into the Liaison MDX Studio through
a bar-code scanner. If the scanner is not working, or if you are unable to transfer the
information for any reason, contact DiaSorin Diagnostics Technical Services.

RELATED DOCUMENTS

 Liaison Universal Disc Map

 DiaSorin Liaison Cleaning Log

REFERENCES

 DiaSorin Flu A/B & RSV package insert

 Liaison MDX Hardware Manual

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

HISTORY RECORD

INFLUENZA (FLU) AND RSV BY PCR (NAAT) ON THE LIAISON MDX

INSTRUMENT

Version
Revision Description
No.
1.0 New Document
 DiaSorin Flu A/B & RSV Direct Reaction Mix store at -10 to -80 °C
2.0  Thaw reaction-mix vials in cold block rather than room temp

 Changed scope to read “This procedure applies to the Molecular Pathology technical
3.0 staff that has been authorized to perform this assay”
 Added Reference guide as an Attachment

 Under Reporting Results, added comment regarding positive RSV result for
patients less than a year old. It is a Critical Value and must be promptly reported
to the ordering provider. The following information must be included: Patient’s
full name, Medical Record Number, Date and time of collection, Test name and
4.0
patient result.
 If a RSV test is positive but is NOT test requested, print a patient inquiry and give
to supervisor to contact provider/account
 Quality Control= more detailed instructions
 Instructions of handling an “Invalid” result added.
 ‘Do not call Positive RSVs that have not been ordered, unless first time run
hospital patient’ and ‘Positive RSV’s are not considered a critical value’
comments were added
 The Test Set Up instructions were modified to accommodate flu volume changes
5.0
 M6 media now an acceptable transport media
 Monthly QC needs to be done every 30 days--comment added to Quality Control
 Include CT values and graphs in Final Result Report
 Reporting Results: no longer need to include contact information for positive
patients
 Removed “When entering results: Choose the Preliminary option instead of
Finalize”
 10% bleach to 0.1x
6.0  Added reporting instructions for EC500 errors; which can be reported and which
to repeat
 Switched procedure to reflect move to Flu Room
 Added that a pooled pos OR Simplexa controls can be used
7.0  Changed QC frequency to daily
 Removed references to Nanosphere
8.0
 Removed references to Simplexa positive controls. Use control made in-house.

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

No changes to content. Approval workflow initiated for new lab director, Dr. Thomas
9.0
Fritsche, MD
10.0 Changed QC to every 30 days

 Added “RSV tests that are NOT ordered are NOT called or reported (Positive or
Negative), unless patient is 1 year old or younger. For these patients, contact the
11.0
provider.”
 Added “Dual and triple positives must be repeated for confirmation.”
 Removed “Thaw reaction-mix vials in designated cold block.”
 Added: Thaw at room temperature
12.0
 Added “: If a patient’s RSV-AG result does not correlate with the PCR result, notify
the account”

 Added Eswabs with Amies transport medium as an acceptable specimen type

 Changed Simplexa to DiaSorin
 Changed Integrated Cycler to Liaison MDX
 Removed: a week’s worth of positive and negative controls are kept in the
refrigerator
13.0
 Added: Catalog number for kit
 Added: NOTE: If a sample is invalid or has an error after repeating the assay,
report all valid results and enter invalid /error results as Indeterminate.
 Changed to accept samples frozen if older than 72 hrs
 Added Attachment 2: Disc Map
 Corrected numbering on page 7
 Added: If a patient has a RSV-AG assay performed at a MCHS Regional Center
or another laboratory and the result that does not correlate with the PCR result,
notify the account. If the provider would like the result put in the patient’s chart,
ask the provider to order a RSVNAT as a U-HAVE. Once the RSVNAT order is
14.0 arrived, create a worksheet for it and attach a copy of the original Results
Report. Label the amplification curve with both the original ABNAT label and
the RSVNAT label. Result the RSVNAT in LIS.
 Added: Another technologist will verify the specimen number on the amplification
curve matches the specimen number on the patient label before electronically
signing the worksheet.
15.0  Removed disc map as attachment
 Changed formatting to table
16.0
 Changed DAB to BSC
 Updated location of controls
17.0
 Removed Quick Reference

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 Influenza (Flu) and RSV by PCR (NAAT) on the Liaison MDX Instrument
Document ID: 62U3QES2XUJM-3-2794
Effective Date: 3/24/2020

 Removed E-swab as a specimen

 Removed “print worksheet”
 Removed reference to manager
 Added catalog numbers
18.0  Removed instructions regarding positive RSV-AG from Regional Centers
 Added instructions for storage
 Added reference to contamination checks
 Corrected storage temp for kit
 Corrected name of worksheet

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