Technical Note
BioFire Diagnostics, LLC
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QS-339B-01
TECHNICAL
::: NOTE
Protocols for Laboratory
Verification of Performance of the
FilmArray® Respiratory Panel (RP)
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 RP performance on
FilmArray® 1.5, 2.0 and Torch Systems as required by CLIA. Two possible
verification schemes, compatible with the FilmArray RP, have been designed.
Each verification scheme provides positive and negative tests for each organism
detected by the FilmArray RP using non-clinical specimens 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 RP 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
The FilmArray RP is a multiplexed nucleic acid test intended for use with FilmArray
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 RP: adenovirus,
coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43,
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/enterovirus, respiratory syncytial virus, Bordetella 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 Instruction
Booklet.
Note: Chlamydia pneumoniae is sometimes referred to as Chlamydophila
pneumoniae.
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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
2 or 3 7 4
28
4 per organism
24 per organism
6
Example 2: Viral
Transport Medium
(VTM) protocol
1
19 4 76
4 per organism
72 per organism
14
Material
Part Number
FilmArray RP Kit (30 tests)
BioFire Diagnostics,LLC RFIT-ASY-0124
Control Organism
ZeptoMetrix NATRVP-IDIa
Transport Medium (e.g. Remel M4 Viral Transport
Media)
Various media are appropriate
Performance Verification: Overview
Each procedure described below will generate multiple positive and negative
results for each of the FilmArray® Respiratory Panel (RP) assays. The procedures
were developed using a Respiratory Verification Panel available from ZeptoMetrix
Corporation, Buffalo, NY (part number NATRVP-IDI).
Two different examples of performance verification procedures are described: (1)
a Simple Protocol for the verification of the FilmArray RP performance on any
FilmArray® System and (2) a Viral Transport Media (VTM) Protocol that evaluates
FilmArray RP performance when organisms are in a VTM sample matrix.
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 or
modules.
Performance verification protocols should be designed to take advantage of the
multiplex nature of the FilmArray RP. Verification testing efficiency is maximized
by evaluating multiple target organisms in a single test run.
Clinical/patient specimens may be used in place of or in addition to the verification
protocol examples described here in order to assess clinical sensitivity and sample
matrix effects in the performance verification of the FilmArray RP.
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.
Performance Verification: Materials
The following materials may be used 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 RP. However, when alternate organism sources are used (i.e. not the ZeptoMetrix NATRVPIDI material), the sample volumes or pooling schemes suggested in the examples below may need to
be adjusted.
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Technical Note
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QS-339B-01
TECHNICAL
::: NOTE
Organism
Approximate
volume
Approximate Final
Volume of Pool
Pool 1
Adenovirus
0.6 mL
1.2 mL
Influenza A subtype H1
0.6 mL
Pool 2
Influenza B
0.6 mL
1.8 mL
Parainfluenza virus 4
0.6 mL
Coronavirus OC43
0.6 mL
Pool 3
Human rhinovirus/Enterovirus
0.6 mL
1.8 mL
Influenza A subtype H3
0.6 mL
Coronavirus 229E
0.6 mL
Pool 4
Parainfluenza virus 1
0.6 mL
1.8 mL
Parainfluenza virus 2
0.6 mL
Mycoplasma pneumoniae
0.6 mL
Pool 5
Influenza A subtype H1-2009
0.6 mL
1.2 mL
Parainfluenza virus 3
0.6 mL
Pool 6
Respiratory Syncytial Virus
0.6 mL
1.8 mL
Coronavirus NL63
0.6 mL
Human Metapneumovirus
0.6 mL
Pool 7
Bordetella pertussis
0.6 mL
1.8 mL
Chlamydia pneumoniae
0.6 mL
Coronavirus HKU1
0.6 mL
Performance Verification: Protocols
Simple Protocol
The Simple protocol utilizes samples prepared by pooling together either 2 or 3
different organisms (ZeptoMetrix NATRVP-IDI control organism). 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 resourceefficient 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 28 pooled samples, providing
4 positive results and 24 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.
Table 3. Proposed Organism Pooling Scheme
An example Simple Protocol workflow is provided below.
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TECHNICAL
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Simple Protocol Example
The estimated total time for completion for this Simple Protocol verification
example is 6 days for a FilmArray® System configured with 1 instrument. A
proposed organism pooling scheme is presented above in Table 3.
Day 1
1. Prepare three sample pools (i.e. pools #1, #2, and #3 in Table 3 above) from
ZeptoMetrix NATRVP-IDI control material.
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 3 sample pools is
based on testing 6 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 System.
a. Use a transfer pipette to remove the entire contents of the ZeptoMetrix
organism vial (approximately 0.6 mL) and transfer to a new vial or tube.
b. Repeat with the second (and third) organism to combine the appropriate
organisms into a single vial or tube (approximately 1.2 mL total volume for
two organisms or 1.8 mL for three organisms).
Note: Pools 1 and 5 contain 1.2 mL and will be used to test four pouches
(approximately 0.3 mL for each pouch). If the volume of the sample is not
sufficient to reach the 3rd line of the Transfer Pipette when loading sample into
your pouch, note the volume shortage and proceed with testing.
c. Ensure the pooled sample is effectively mixed by vortexing 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 Quick Guide for pouch
preparation, pouch hydration, sample loading, and sample testing.
3. Repeat Step 2 for the remaining sample pools (i.e. pools #2 and #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.
®
Respiratory Panel
Day 2
To evaluate day-to-day variation, test the remaining volume of the sample pools
prepared on Day 1 by repeating Step 2 above.
Day 3
Prepare 3 new sample pools (i.e. pools #4, #5, and #6) as described in Step 1.
Test samples according to Step 2.
Day 4
To evaluate day-to-day variation, test the samples prepared on Day 3 by repeating
Step 2.
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Technical Note
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QS-339B-01
TECHNICAL
::: NOTE
Day 5-6
Repeat Steps 1–2 as above for any remaining sample pools (e.g. pool #7).
Figure 1. Workflow for Simple Protocol Example
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TECHNICAL
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VTM Sample
Approximate volume
ZeptoMetrix NATRVP-IDI Control Organism
0.6 mL
VTM
0.6 mL
VTM Protocol
The VTM Protocol utilizes samples prepared by adding a single control organism
(ZeptoMetrix NATRVP-IDI control organism) to an equal volume of Viral Transport
Medium (VTM) sample matrix.
The VTM Protocol can be followed to test a total of 76 samples, providing 4 positive
results and 72 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 or
modules 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.
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 14 days for a FilmArray System configured with 1 instrument.
Day 1
1. Prepare three VTM Samples (i.e. sample #1, #2, #3) by mixing ZeptoMetrix
NATRVP-IDI control material with VTM as proposed in Table 4.
Note: It is important to prepare only the number of samples that will be tested
within 3 days of preparation. The suggestion to prepare 3 samples is based on testing
6 pouches per day. The number of samples prepared may be increased or decreased
based on the laboratory’s work schedule and number of instruments configured on
the FilmArray System.
a. Pipet 0.6 mL of VTM into a sterile tube or vial.
b. Use a transfer pipette to remove the entire contents of the ZeptoMetrix
organism vial (approximately 0.6 mL) and add organism to the tube or vial
containing VTM (total sample volume for one organism in VTM will be
approximately 1.2 mL).
c. Ensure the VTM sample is effectively mixed by vortexing prior to testing.
2. Test two samples (pouches) from a single VTM sample. 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 Quick Guide for pouch
preparation, pouch hydration, sample loading, and sample testing.
®
Respiratory Panel
6 | P a g e FLM1-PRT-0060-08
Technical Note
BioFire Diagnostics, LLC
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QS-339B-01
TECHNICAL
::: NOTE
3. Repeat Step 2 for the remaining samples to be tested that day.
Note: The approximate volume of VTM samples is 1.2 mL, which should be
sufficient to test four pouches per sample (approximately 0.3 mL for each pouch). If
the volume of the sample is not sufficient to reach the 3rd line of the Transfer Pipette
when loading sample into your pouch, note the volume shortage and proceed with
testing.
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.
Day 3–4
Repeat Steps 1–4 for 3 additional samples (i.e. #4, #5, and #6).
Day 5–6
Repeat Steps 1–4 for 3 additional samples (i.e. #7, #8, and #9).
Day 7–8
Repeat Steps 1–4 for 2 additional samples (i.e. #10, #11, and #12).
Day 9–10
Repeat steps 1–4 for the remaining samples (i.e. #13, #14 and #15).
Day 11–12
Repeat steps 1–4 for the remaining samples (i.e. #16, #17, and #18).
Day 13–14
Repeat steps 1–4 for the remaining samples (i.e. #19).
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TECHNICAL
::: NOTE
Figure 2. Workflow for VTM Protocol Example
8 | P a g e FLM1-PRT-0060-08
Technical Note
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TECHNICAL
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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, but should be verified
by the Laboratory Director.
1. Select a few specimens and/or proficiency samples (any combination of
positives and negatives) previously tested on the FilmArray® Respiratory
Panel (RP). 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.
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 Technical Support team for assistance.
BioFire Technical Support
Email: support@biofiredx.com
Phone: +1-801-736-6354, select Option 5
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Technical Note
BioFire Diagnostics, LLC
www.BioFireDX.com
QS-339B-01
TECHNICAL
::: NOTE
Organism
Instrument
Serial #
Was the
Organism
Detected?
No.
Positive
No.
Negative
No. Days
Tested
No.
Users
Patient
Samples
Tested?
Adenovirus
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
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
Computer System Serial Number #: _____________________
FilmArray RP Kit Part #: __________________ Lot #: _________
Organism/Sample Source and Lot #: ___________________________________
________________________________________________________________
FilmArray® Respiratory Panel (RP) Verification Record
Reviewed by: __________________________________________________
Signature Date
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