Technical Note
BioFire Diagnostics, LLC
www.BioFireDX.com
QS-339B-01
TECHNICAL
::: NOTE
Protocols for Laboratory
Verification of Performance of the
FilmArray® Respiratory Panel 2 (RP2)
Purpose
The Clinical Laboratory Improvement Amendments (CLIA), passed in 1988,
establishes quality standards for all laboratory testing to ensure the accuracy and
reliability of patient test results, regardless of where the test is performed. The
CLIA regulations include a requirement for verifying the performance specifications
of unmodified, moderate complexity tests cleared or approved by the FDA.
This document provides examples of procedures to assist your laboratory in
developing a protocol for the verification of FilmArray RP2 performance on
FilmArray® 2.0 and Torch Systems as required by CLIA. Two possible verification
schemes, compatible with the FilmArray RP2, have been designed. Each
verification scheme provides positive and negative tests for each organism
detected by the FilmArray RP2 and may be easily modified or expanded to meet
specific criteria. Day-to-day variation is evaluated by testing each sample on two
separate days. To evaluate user-to-user variation, multiple laboratory technicians
may test the same sample. In addition, testing patient samples for verification or
to evaluate matrix effects on the performance of the FilmArray RP2 should be done
under the guidance of the Laboratory Director, but is not described here.
As per the CLIA regulation, the Laboratory Director is ultimately responsible for
ensuring that verification procedures meet the appropriate standards for CLIA and
applicable laboratory accrediting agencies.
FilmArray Intended Use
FilmArray RP2 is a multiplexed nucleic acid test intended for use with FilmArray
2.0 and Torch Systems for the simultaneous qualitative detection and identification
of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs
(NPS) obtained from individuals suspected of respiratory tract infections. The
following organisms and subtypes are identified using the FilmArray RP2:
adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus
OC43, enterovirus, human metapneumovirus, influenza A, influenza A subtype H1,
influenza A subtype H3, influenza A subtype 2009 H1, influenza B, parainfluenza
virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4,
rhinovirus, respiratory syncytial virus, Bordetella parapertussis, B. pertussis,
Chlamydia pneumoniae, and Mycoplasma pneumoniae.
The complete intended use statement and additional information about the use of
the FilmArray System can be found in the FilmArray Respiratory Panel 2
Instruction Booklet.
1 | P a g e FLM1-PRT-0232-01
Technical Note
BioFire Diagnostics, LLC
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QS-339B-01
TECHNICAL
::: NOTE
Verification
Protocol
Organisms
per Poola
Number
of
Sample
Pools
Replicates
per
Sample
Pool
Pouches
Required
Expected
Positive
Results
Expected
Negative
Results
Approximate
Days of
Testingb
Example 1:
Simple
protocol
5 or 6 4 4
16
4 per
organism
12 per
organism
4
Example 2:
Viral Transport
Medium (VTM)
protocol
5 or 6 4 4
16
4 per
organism
12 per
organism
4
Performance Verification: Overview
Each procedure described below will generate multiple positive and negative
results for each of the FilmArray® Respiratory Panel 2 (RP2) assays. The
procedures were developed using a Respiratory Verification Panel available from
ZeptoMetrix Corporation, Buffalo, NY (part number NATRVP2-BIO).
Two different examples of performance verification procedures are described: (1)
a Simple Protocol for the verification of FilmArray RP2 performance and (2) a Viral
Transport Media (VTM) Protocol that evaluates FilmArray RP2 performance when
organisms are in a VTM sample matrix. These protocols are examples of
procedures to assist your laboratory in developing a protocol for the verification of
FilmArray RP2 performance on FilmArray® 2.0 and Torch Systems.
A FilmArray System is defined as one or more FilmArray® Instruments or modules
connected to and controlled by a single computer system. If the laboratory director
chooses not to perform the verification protocol on each individual instrument, it is
advised that test replicates are evenly distributed among the instruments.
Performance verification protocols should be designed to take advantage of the
multiplex nature of the FilmArray RP2. Verification testing efficiency is maximized
by evaluating multiple target organisms in a single test run.
In addition to, or in place of, the verification protocol examples described here, a
laboratory may choose to test clinical/patient samples to assess clinical sensitivity
and sample matrix effects in its performance verification of the FilmArray RP2.
Table 1. Overview of Verification Protocols
a
Depending on the material used for verification, pooling of organisms may not be appropriate and the
values in the table may need to be modified.
b
The approximate number of days for testing assumes a system configured with one instrument.
2 | P a g e FLM1-PRT-0232-01
Technical Note
BioFire Diagnostics, LLC
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QS-339B-01
TECHNICAL
::: NOTE
Material
Part Number
FilmArray® Respiratory Panel 2 (RP2) Kit
(30 tests)
BioFire Diagnostics, LLC (RFIT-ASY-129)
FilmArray® Respiratory Panel 2 (RP2)
Instruction for Use
BioFire Diagnostics, LLC (RFIT-PRT-0522)
FilmArray® Respiratory Panel 2 (RP2)
Quick Guide
BioFire Diagnostics, LLC (RFIT-PRT-0541)
Control Organism
ZeptoMetrix NATRVP2-BIO
Transport Medium (e.g. Remel M4 Viral
Transport Media)
Various media are appropriate
5mL sample tubes
VWR Part # 89497-740 (or similar)
Transfer pipettes
VWR Part # 13-711-43 (or similar)
Performance Verification: Materials
The following materials may be needed to perform verification procedures:
Table 2. Materials needed for recommended verification protocols
a
Any appropriate source of organism may be used for verification of any or all of the assays in the
FilmArray® RP2. However, when alternate organism sources are used (i.e. not the ZeptoMetrix TBD
material), the sample volumes or pooling schemes suggested in the examples below may need to be
adjusted.
Performance Verification: Protocols
Simple Protocol
The Simple Protocol utilizes samples prepared by pooling together either 5 or 6
different organisms (ZeptoMetrix NATRVP2-BIO). The proposed organism pooling
scheme (Table 3) should be followed to obtain the expected number of positive
and negative results for each assay in a time and resource-efficient manner.
Note: Dilution of ZeptoMetrix Respiratory Verification Panel organisms beyond levels
proposed in these guidelines may lead to inconsistent results and is not recommended.
The Simple Protocol can be followed to test a total of 16 pooled samples, providing
4 positive results and 12 negative results per assay. The number of samples tested
per day should be determined by the individual laboratory. The testing scheme can
be modified to run more samples per day based on the number of instruments
configured on the FilmArray® System.
Pooled samples can be stored overnight (or up to 3 days) at refrigeration
temperature (2–8°C) for subsequent testing to evaluate day-to-day variation.
3 | P a g e FLM1-PRT-0232-01
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TECHNICAL
::: NOTE
Organism
Approximate
organism
volume
Approximate
Final Volume
of Pool
Pool 1
Adenovirus Type 3
0.3 mL
1.5 mL
Influenza A 2009 H1N1
0.3 mL
Influenza B
0.3 mL
Parainfluenza virus Type 4
0.3 mL
Coronavirus OC43
0.3 mL
Pool 2
Rhinovirus 1 A
0.3 mL
1.5 mL
Influenza A subtype H3
0.3 mL
Coronavirus 229E
0.3 mL
Parainfluenza virus Type 1
0.3 mL
Parainfluenza virus Type 2
0.3 mL
Pool 3
Adenovirus Type 1
0.3 mL
1.8 mL
Influenza A H1N1
0.3 mL
Parainfluenza virus Type 3
0.3 mL
Respiratory Syncytial Virus A
0.3 mL
Coronavirus NL63
0.3 mL
Bordatella parapertussis
0.3 mL
Pool 4
Adenovirus Type 31
0.3 mL
1.8 mL
Bordetella pertussis
0.3 mL
Chlamydia pneumoniae
0.3 mL
Mycoplasma pneumoniae
0.3 mL
Coronavirus HKU1
0.3 mL
Human Metapneumovirus 8
0.3 mL
Table 3. Proposed Organism Pooling Scheme
An example Simple Protocol workflow is provided below.
Simple Protocol Example
The estimated total time for completion for this Simple Protocol verification
example is 4 days for a FilmArray® 2.0 or Torch System configured with 1 module.
A proposed organism pooling scheme is presented above in Table 3.
Day 1
1. Prepare two sample pools (i.e. pools #1 and #2, in Table 3 above) from
ZeptoMetrix NATRVP2-BIO control material.
4 | P a g e FLM1-PRT-0232-01
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QS-339B-01
TECHNICAL
::: NOTE
Note: It is important to prepare only the number of sample pools that will be
tested within 3 days of preparation. The suggestion to prepare 2 sample pools is
based on testing 4 pouches per day. The number of samples prepared may be
increased or decreased based on the laboratory’s work schedule and number of
instruments connected within a FilmArray
a. Use a transfer pipette to remove 0.3 mL material from the ZeptoMetrix
organism vial (draw material to the third line of the transfer pipette) and
transfer to a new vial or tube.
b. Repeat with the second (and subsequent) organisms to combine the
appropriate organisms into a single vial or tube (approximately 1.5 mL total
volume for five organisms or 1.8 mL for six organisms).
c. Cap and vortex prior to testing.
2. Test two samples from a single sample pool. The duplicate samples should be
tested in a single day by different users.
Note: For each sample, follow instructions in the FilmArray
Instruction Booklet and FilmArray® Respiratory Panel 2 Quick Guide for pouch
preparation, pouch hydration, sample loading, and sample testing.
3. Repeat Step 2 for the remaining sample pools (i.e. pool #2) to be tested that
day.
4. Refrigerate samples (2–8°C) for up to 3 days for the evaluation of day-to-day
variation.
®
System.
®
Respiratory Panel 2
Day 2
To evaluate day-to-day variation, test the remaining volume of the sample pools
prepared on Day 1 by repeating Step 2 and 3 above.
Day 3
Prepare 2 new sample pools (i.e. pools #3 and #4) as described in Step 1. Test
samples according to Step 2 and 3 above.
Day 4
To evaluate day-to-day variation, test the samples prepared on Day 3 by repeating
Step 2 and 3 above.
Note: The remaining material from Zeptometrix NATRVP2-BIO may be stored
according to manufacturer’s instructions for use at a later date.
5 | P a g e FLM1-PRT-0232-01
Technical Note
BioFire Diagnostics, LLC
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QS-339B-01
TECHNICAL
::: NOTE
Figure 1. Workflow for Simple Protocol
VTM Protocol
The VTM Protocol evaluates FilmArray® Respiratory Panel 2 (RP2) performance
in a VTM sample matrix. Sample material is pooled and added to an equal volume
of Viral Transport Medium (VTM) matrix.
The VTM Protocol can be followed to test a total of 16 pooled samples, providing
4 positive results and 12 negative results per assay. The number of samples
tested per day should be determined by the individual laboratory. The testing
scheme can be modified to run more samples per day based on the number of
instruments configured on the FilmArray® System.
VTM samples can be stored overnight (or up to 3 days) at refrigeration temperature
(2–8°C) for subsequent testing to evaluate day-to-day variation. To evaluate user-
to-user variation, multiple laboratory technicians may perform testing.
6 | P a g e FLM1-PRT-0232-01
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QS-339B-01
TECHNICAL
::: NOTE
Organism
Approximate
organism
volume
Volume VTM
Approximate
Final Volume
of Pool
Pool 1
Adenovirus Type 3
0.3 mL
1.5 mL
3.0 mL
Influenza A 2009 H1N1
0.3 mL
Influenza B
0.3 mL
Parainfluenza virus Type 4
0.3 mL
Coronavirus OC43
0.3 mL
Pool 2
Rhinovirus 1 A
0.3 mL
1.5 mL
3.0 mL
Influenza A subtype H3
0.3 mL
Coronavirus 229E
0.3 mL
Parainfluenza virus Type 1
0.3 mL
Parainfluenza virus Type 2
0.3 mL
Pool 3
Adenovirus Type 1
0.3 mL
1.8 mL
3.6 mL
Influenza A H1N1
0.3 mL
Parainfluenza virus Type 3
0.3 mL
Respiratory Syncytial Virus A
0.3 mL
Coronavirus NL63
0.3 mL
Bordetella parapertussis
0.3 mL
Pool 4
Adenovirus Type 31
0.3 mL
1.8 mL
3.6 mL
Bordetella pertussis
0.3 mL
Chlamydia pneumoniae
0.3 mL
Mycoplasma pneumoniae
0.3 mL
Coronavirus HKU1
0.3 mL
Human Metapneumovirus 8
0.3 mL
Table 4. Proposed VTM Sample Preparation Scheme
An example VTM Protocol workflow is provided below.
VTM Protocol Example
The estimated total time to completion for this VTM Protocol verification example
is 4 days for a FilmArray® 2.0 or Torch System configured with 1 module. Refer to
Figure 2 for the suggested workflow.
Day 1
1. Prepare two sample pools (i.e. pools #1 and #2, in Table 4 above) from
ZeptoMetrix NATRVP2-BIO control material and VTM.
7 | P a g e FLM1-PRT-0232-01
Technical Note
BioFire Diagnostics, LLC
www.BioFireDX.com
QS-339B-01
TECHNICAL
::: NOTE
Note: It is important to prepare only the number of samples that will be tested
within 3 days of preparation. The suggestion to prepare 2 sample pools is based on
testing 4 pouches per day. The number of samples prepared may be increased or
decreased based on the laboratory’s work schedule and number of modules
configured on the FilmArray® System.
a. Pipet 1.5 mL or 1.8 mL of VTM (as described in Table 4) into a sterile tube
or vial.
b. Use a transfer pipette to remove 0.3 mL material from the ZeptoMetrix
organism vial (draw material to the third line of the transfer pipette) and
transfer to the tube containing VTM.
c. Repeat with the second (and subsequent) organisms to combine the
appropriate organisms into a single vial or tube (approximately 3.0 mL total
volume for five organisms or 3.6 mL for six organisms).
d. Cap and vortex prior to testing.
2. Test two samples (pouches) from a single VTM sample pool. The duplicate
samples should be tested in a single day by different users.
Note: For each sample, follow instructions in the FilmArray
Instruction Booklet and FilmArray® Respiratory Panel 2 Quick Guide for pouch
preparation, pouch hydration, sample loading, and sample testing.
®
Respiratory Panel 2
3. Repeat Step 2 for the second sample pool (Pool #2 from Table 3) to be tested
that day.
4. Refrigerate samples (2–8°C) for up to 3 days for the evaluation of day-to-day
variation.
Day 2
To examine day-to-day variation, test the samples prepared on Day 1 by repeating
Step 2 and 3 above.
Day 3
Prepare 2 new sample pools (i.e. pools #3 and #4) as described in Step 1. Test
samples according to Step 2 and 3 above.
Day 4
To evaluate day-to-day variation, test the samples prepared on Day 3 by repeating
Step 2 and 3 above.
Note: The remaining material from Zeptometrix NATRVP2-BIO may be stored
according to manufacturer’s instructions for use at a later date.
8 | P a g e FLM1-PRT-0232-01
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TECHNICAL
::: NOTE
Figure 2. Workflow for VTM Protocol Example
Verification of loaner and repaired instruments
If it becomes necessary to verify the performance of a loaner or repaired
instrument, the following protocol may serve as a guideline.
1. Select a few specimens and/or proficiency samples (any combination of
positives and negatives) previously tested on the FilmArray® Respiratory
Panel 2 (RP2). The Laboratory Director should determine the appropriate
number of samples to test. Two to six samples may be sufficient. Proficiency
samples should not be pooled or diluted.
2. Test the selected specimens/samples on the loaner or repaired instrument and
document the results.
9 | P a g e FLM1-PRT-0232-01
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BioFire Diagnostics, LLC
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TECHNICAL
::: NOTE
Technical Support Contact Information
BioFire is dedicated to providing the best customer support available. If
you have any questions or concerns about this process, please contact
the FilmArray
BioFire Technical Support
Email: support@biofiredx.com
Phone: +1-801-736-6354, select Option 5
®
Technical Support team for assistance.
10 | P a g e FLM1-PRT-0232-01
Technical Note
BioFire Diagnostics, LLC
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QS-339B-01
TECHNICAL
::: NOTE
Organism
Detection
Representative
strains
Instrument
Serial #
Was the
Organism
Detected?
No.
Positive
No.
Negative
No.
Days
Tested
No.
Users
Patient
Samples
Tested?
Adenovirus
Type 1 C
□ Yes
□ No
Type 3 B
□ Yes
□ No
Type 31 A
□ Yes
□ No
Bordetella parapertussis
□ Yes
□ No
Bordetella pertussis
□ Yes
□ No
Chlamydia pneumoniae
□ Yes
□ No
Coronavirus 229E
□ Yes
□ No
Coronavirus NL63
□ Yes
□ No
Coronavirus OC43
□ Yes
□ No
Coronavirus HKU1
□ Yes
□ No
Human Metapneumovirus
□ Yes
□ No
Human Rhinovirus/Enterovirus
□ Yes
□ No
Influenza A subtype H1
□ Yes
□ No
Influenza A subtype H1-2009
□ Yes
□ No
Computer System Serial Number #: _____________________
FilmArray Respiratory Panel Kit Part #: __________________ Lot #: _________
Organism/Sample Source and Lot #: ___________________________________
FilmArray® Respiratory Panel2 Verification Record
11 | P a g e FLM1-PRT-0232-01
Technical Note
BioFire Diagnostics, LLC
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TECHNICAL
::: NOTE
Organism
Detection
Representative
strains
Instrument
Serial #
Was the
Organism
Detected?
No.
Positive
No.
Negative
No.
Days
Tested
No.
Users
Patient
Samples
Tested?
Influenza A subtype H3
□ Yes
□ No
Influenza B
□ Yes
□ No
Mycoplasma pneumoniae
□ Yes
□ No
Parainfluenza virus 1
□ Yes
□ No
Parainfluenza virus 2
□ Yes
□ No
Parainfluenza virus 3
□ Yes
□ No
Parainfluenza virus 4
□ Yes
□ No
Respiratory Syncytial Virus
□ Yes
□ No
Reviewed by: __________________________________________________
12 | P a g e FLM1-PRT-0232-01
Signature Date
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