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Technical Note
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TECHNICAL
::: NOTE
Protocols for Laboratory
Verification of Performance of the
FilmArray® Meningitis/Encephalitis
(ME) Panel
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 an example verification procedure to assist your
laboratory in developing a protocol for the verification of the FilmArray ME Panel
performance on FilmArray® 1.5, 2.0 and Torch Systems as required by CLIA. This
FilmArray ME Panel verification scheme has been designed to generate positive
and negative tests for each organism detected by the FilmArray ME Panel using
non-clinical specimens and may be easily modified or expanded to meet specific
criteria. The procedure includes an evaluation of day-to-day and user-to-user
variation when repeatedly testing the same sample. In addition, testing patient
samples for verification or to evaluate matrix effects on the performance of the
FilmArray ME Panel 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 ME Panel is a multiplexed nucleic acid test intended for use with
FilmArray Systems for the simultaneous qualitative detection and identification of
multiple viral, yeast, and bacterial nucleic acid targets in cerebrospinal fluid (CSF)
samples obtained from individuals suspected of meningitis and/or encephalitis.
The following are identified using the FilmArray ME Panel: Escherichia coli K1,
Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis,
Streptococcus agalactiae, Streptococcus pneumoniae, Cryptococcus
gattii/neoformans, cytomegalovirus, herpes simplex virus 1, herpes simplex virus
2, human herpesvirus 6, enterovirus, human parechovirus, varicella zoster virus.
The complete intended use statement and additional information about the use of
the FilmArray System can be found in the FilmArray Meningitis/Encephalitis (ME)
Panel Instruction Booklet.
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Technical Note
BioFire Diagnostics, LLC
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TECHNICAL
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Organisms
per Poola
Number of
Sample
Pools
Replicates
per Sample
Pool
Pouches
Required
Expected
Positive
Results
Expected
Negative
Results
Approximate
Days of
Testingb
4 or 5 3 4
12
4 per organism
8 per organism
4
Material
Part Number
FilmArray ME Panel Kit (30 tests)
Biofire Diagnostics, LLC RFIT-ASY-0118
Control Organism
ZeptoMetrix NATMEP-BIO
a
5mL sample tubes
VWR Part # 89497-740 (or equivalent)
Transfer pipettes
VWR Part # 13-711-43 (or equivalent)
Artificial cerebrospinal fluid (aCSF)
Tocris Biosciences #3525
Performance Verification: Overview
The procedure described below will generate multiple positive and negative results
for each of the organisms targeted by the FilmArray® Meningitis/Encephalitis (ME)
Panel. The procedures were developed using a panel available from ZeptoMetrix
Corporation, Buffalo, NY (NATMEP-BIO).
An example procedure for performance verification is described below. The
procedure can be performed without any further dilution of the reference material.
The procedures can also be performed using samples prepared in an artificial
cerebrospinal fluid (aCSF) background (published recipes or commercially
available) or with cerebrospinal fluid (CSF) that has been verified as negative for
ME Panel targets.
A FilmArray® System is defined as all FilmArray® Instruments or modules that are
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 the test replicates are evenly distributed among the instruments and
modules within a single instrument.
The procedures have been designed to take advantage of the multiplex nature of
the FilmArray ME Panel. Verification testing efficiency is maximized by evaluating
multiple target organisms in a single test run.
Clinical/patient specimens may be used in addition to, or in place of the verification
schemes described here, in order to assess clinical sensitivity and sample matrix
effects for verification of the FilmArray ME Panel.
Table 1. Overview of Verification Protocol
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 protocol
a
Any appropriate source of organism may be used for verification of any or all of the assays in the
FilmArray ME Panel. However, when alternate organism sources are used, the sample volumes or
pooling schemes suggested in the examples below may need to be adjusted.
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TECHNICAL
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Control
Organism
Control
Organism
Volume
Approximate
Final
Volume
of Pool
Optional Addition of
aCSF or CSF
Volume of
aCSF or CSF
Approximate
Final Volume
of Pool
Pool 1
Escherichia coli K1
0.30 mL
1.5 mL
0.9 mL
2.4 mL
Cytomegalovirus
(CMV)
0.30 mL
Echovirus type 11
0.30 mL
Streptococcus
pneumoniae
0.30 mL
Human herpesvirus
6 (HHV-6)
0.30 mL
Pool 2
Herpes simplex
virus 1 (HSV-1)
0.30 mL
1.2 mL
0.9 mL
2.1 mL
Neisseria
meningitidis
0.30 mL
Streptococcus
agalactiae
0.30 mL
Cryptococcus gattii
0.30 mL
Pool 3
Haemophilus
influenzae
0.30 mL
1.5 mL
0.9 mL
2.4 mL
Herpes simplex
virus 2 (HSV-2)
0.30 mL
Varicella zoster
virus (VZV)
0.30 mL
Listeria
monocytogenes
0.30 mL
Human
parechovirus
(HPeV)
0.30 mL
Performance Verification: Protocol
The protocol can be followed to test a total of 12 pouches, providing 4 positive
results and 8 negative results per organism. The number of samples tested per
day should be determined by the individual laboratory. This testing scheme can be
modified to run more samples per day based on the number of instruments in the
FilmArray® System.
The recommended protocol requires the preparation of 3 organism pools for
testing, each containing up to 5 different control organisms (ZeptoMetrix NATMEPBIO). If a larger volume of each pool is desired, organisms can be combined with
an additional volume of artificial cerebrospinal fluid (aCSF) background (Tocris
Biosciences Part # 3525 or equivalent) or follow published recipes for aCSF
preparation) or with residual clinical CSF that has been verified as negative for ME
Panel targets.
The proposed pooling scheme (Table 3) should be followed to obtain the expected
positive and negative results for each assay in a time and resource-efficient
manner.
Note: Dilution of ZeptoMetrix ME Verification Panel organisms beyond levels
proposed in these guidelines may lead to inconsistent results and is not recommended.
Table 3. Recommended Organism Pooling Scheme
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TECHNICAL
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Protocol Example
The estimated total time to completion for this verification example is 4 days for
systems configured with one instrument (Figure 1).
Day 1
1. Prepare one organism pool (e.g. pool 1) from ZeptoMetrix NATMEP-BIO
control material. An example organism pooling scheme is presented in
Table 3.
Note: It is important to prepare only the number of organism pools that will be
tested within 3 days of preparation. The number of organism pools prepared and
samples tested may be increased or decreased based on the laboratory’s work
schedule and the number of instruments connected within a FilmArray
a. Transfer 0.3 mL of the ZeptoMetrix organism to a tube large enough (at
least 3 mL) to hold the entire organism pool volume.
b. Repeat step a. for each of the remaining organisms to combine the
appropriate organisms for each pool into a single vial or tube
(approximately 1.5 mL total volume). Vortex to mix well.
c. Optional: transfer 0.9 mL of aCSF or CSF to the organism pool
(approximately 2.4 mL total volume) and vortex to mix well.
d. Proceed to Step 2 for testing. The organism pool may be stored
refrigerated (2–8°C) for up to 3 days for the evaluation of day-to-day
variation.
2. Test 2 samples (e.g. samples A and B) from a single organism pool (e.g. pool
1). The duplicate samples should be tested in a single day by the same
operator to evaluate run-to-run variation or different operators to evaluate
operator-to-operator variation.
Note: Follow instructions in the FilmArray
Instruction Booklet or FilmArray Meningitis/Encephalitis Panel Quick Guide for pouch
preparation, pouch hydration, sample loading, and sample testing.
3. Repeat steps 1 and 2 for another organism pool (e.g. pool 2) to be tested that
day.
®
Meningitis/Encephalitis (ME) Panel
®
System.
Day 2
To evaluate day-to-day variation, test the remaining samples (e.g. samples
C and D) from the same organism pool(s) prepared on Day 1 by repeating Step 2
above.
Day 3
Prepare a new organism pool (e.g. pool 3) as described in Step 1. Test samples
according to Step 2 (e.g. samples A and B) for the pool.
Day 4
To evaluate day-to-day variation, test the remaining samples (e.g. samples C and
D) from the organism pool(s) prepared on Day 3 by repeating Step 2 above.
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Technical Note
BioFire Diagnostics, LLC
www.BioFireDX.com
QS-339B-01
TECHNICAL
::: NOTE
Figure 1. Protocol workflow
Expanding the protocols
The protocols described above can be expanded to increase the number of
samples or tests for each of the organism pools. Additional testing (up to 11 tests
per pool when using the additional volume of aCSF or CSF described in Table 3)
may include more replicates per pool, more replicates per day (no more than 3
days per pool), more replicates per operator, and/or more instruments per
FilmArray® System.
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®
Meningitis/Encephalitis (ME) Panel. The Laboratory Director should determine
the appropriate number of samples to test. Three to six samples may be
sufficient.
Note: Proficiency samples or other stored samples should not be pooled or
diluted prior to testing.
2. Test the selected samples on the loaner or repaired instrument and document
the results.
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Technical Note
BioFire Diagnostics, LLC
www.BioFireDX.com
QS-339B-01
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: [email protected]
Phone: +1-801-736-6354, select Option 5
®
Technical Support team for assistance.
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Technical Note
BioFire Diagnostics, LLC
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QS-339B-01
TECHNICAL
::: NOTE
Organism
Instrument
Serial #
Was the
Organism
Detected?
No.
Positive
No.
Negative
No. Days
Tested
No.
Users
Patient
Samples
Tested?
Escherichia coli K1
Yes
No
Cytomegalovirus (CMV)
Yes
No
Enterovirus (EV)
Yes
No
Streptococcus pneumoniae
Yes
No
Human herpesvirus 6 (HHV-6)
Yes
No
Herpes simplex virus 1 (HSV-1)
Yes
No
Neisseria meningitidis
Yes
No
Streptococcus agalactiae
Yes
No
Cryptococcus gattii
Yes
No
Haemophilus influenzae
Yes
No
Herpes simplex virus 2 (HSV-2)
Yes
No
Varicella zoster virus (VZV)
Yes
No
Listeria monocytogenes
Yes
No
Human parechovirus (HPeV)
Yes
No
Computer System Serial #: ____________________
FilmArray ME Panel kit Part #: __________________ Lot #: __________
Organism/sample Source and Lot #: ____________________________________
FilmArray® Meningitis/Encephalitis (ME) Panel Verification Record
Reviewed by:
Signature Date
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