Technical Note
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QS-339B-01
TECHNICAL
::: NOTE
Protocols for Laboratory
Verification of Performance of the
BioFire® FilmArray® Blood Culture
Identification (BCID) Panel
Laboratory Protocols for Use with a
ZeptoMetrix NATtrol™ Verification 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 of a verification procedure to assist your
laboratory in developing a protocol for the verification of BioFire BCID Panel
performance on BioFire® FilmArray® Systems as required by CLIA. Two possible
verification schemes, compatible with the BioFire BCID Panel, have been designed
using non-clinical specimens. Each verification scheme provides positive and
negative tests for each organism detected by the BioFire BCID Panel 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 of the performance of the BioFire BCID
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.
BioFire Intended Use
The BioFire BCID Panel is a qualitative multiplexed nucleic acid-based in vitro
diagnostic test intended for use with BioFire Systems. The BioFire BCID Panel is
capable of simultaneous detection and identification of multiple bacterial and yeast
nucleic acids and select genetic determinants of antimicrobial resistance. The
BioFire BCID Panel assay is performed directly on blood culture samples identified
as positive by a continuous monitoring blood culture system. Results are intended
to be interpreted in conjunction with Gram stain results.
The following gram-positive bacteria, gram-negative bacteria, and yeast are
identified using the BioFire BCID Panel: Enterococci, Listeria monocytogenes,
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Staphylococci (including specific differentiation of Staphylococcus aureus),
Streptococci (with specific differentiation of Streptococcus agalactiae,
Streptococcus pneumoniae, and Streptococcus pyogenes), Acinetobacter
baumannii, Enterobacteriaceae (including specific differentiation of the
Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella
pneumoniae, Proteus, and Serratia marcescens), Haemophilus influenzae,
Neisseria meningitidis (encapsulated), Pseudomonas aeruginosa, Candida
albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida
tropicalis.
The BioFire® FilmArray® Blood Culture Identification (BCID) Panel also contains
assays for the detection of genetic determinants of resistance to methicillin (mecA),
vancomycin (vanA and vanB), and carbapenems (bla
of potentially antimicrobial resistant organisms in positive blood culture samples.
The complete intended use statement and additional information about the use of
the BioFire® FilmArray® System can be found in the BioFire® FilmArray® Blood
Culture Identification (BCID) Panel Instruction Booklet.
Performance Verification: Overview
Two different examples of performance verification procedures are described: (1)
a Simple Protocol for the verification of BioFire BCID Panel performance and (2) a
Blood Culture Media Protocol that evaluates BioFire BCID Panel performance
when organisms are in a blood culture media sample matrix. These protocols are
examples of procedures to assist your laboratory in developing a protocol for the
verification of BioFire BCID Panel performance on BioFire Systems.
The performance verification protocols have been designed to take advantage of
the multiplex nature of the BioFire BCID Panel. Verification testing efficiency is
maximized by evaluating multiple target organisms in a single test run. Each
procedure described below will generate multiple positive and negative results for
each of the BioFire BCID Panel assays. The procedures were developed using a
BCID Verification Panel available from ZeptoMetrix™ Corporation, Buffalo, NY
(part number NATBCP-BIO).
A BioFire System is defined as all BioFire® 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 test replicates are evenly distributed among the
instruments or modules.
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 BioFire BCID Panel.
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) to aid in the identification
KPC
Technical Note
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TECHNICAL
::: NOTE
Verification
Protocol
Organisms
per Pool
Number
of
Sample
Pools
Replicates
per
Sample
Pool
Pouches
Required
Expected
Positive
Results
Expected
Negative
Results
Approximate
Days of
Testinga
Example 1:
Simple
protocol
5 or 6 4 4
16
4 per
organism
12 per
organism
4
Example 2:
Blood
Culture
media
protocol
5 or 6 4 4
16
4 per
organism
12 per
organism
4
Material
Part Number
BioFire® FilmArray® Blood Culture
Identification (BCID) Panel (30 tests per
kit)
BioFire Diagnostics, LLC RFIT-ASY-0126
Blood culture mediaa
BD BACTEC™ Plus Aerobic/F Medium
(with resin) BD, 442192 (or equivalent)
BioFire® FilmArray® Blood Culture
Identification (BCID) Panel Instruction
Booklet
BioFire Diagnostics, LLC (RFIT-PRT-0369)
BioFire® FilmArray® Blood Culture
Identification Panel Quick Guide
BioFire Diagnostics, LLC (RFIT-PRT-0370)
Control Organism
ZeptoMetrix NATBCP-BIOb
Table 1. Overview of Verification Protocols
a
The approximate number of days for testing assumes a BioFire® FilmArray® System configured with one
instrument/module.
Performance Verification: Materials
The following materials may be used to perform verification procedures:
Table 2. Recommended materials for verification protocol
a
See Table 77 in the BioFire® FilmArray® Blood Culture Identification (BCID) Panel Instruction Booklet
for other acceptable blood culture media/bottle types.
b
Any appropriate source of organism may be used for verification of any or all of the assays in the
BioFire
BCID Panel. However, when alternate organism sources are used (i.e. not the ZeptoMetrix
NATBCP-BIO material), the sample volumes or pooling schemes suggested in the examples below
may need to be adjusted.
Performance Verification Protocol
Simple Protocol
The Simple Protocol utilizes samples prepared by pooling either 5 or 6 different
organisms (ZeptoMetrix NATBCP-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 BCID Verification Panel organisms beyond levels
proposed in these guidelines may lead to inconsistent results and is not recommended.
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Organism
Approximate
Organism
Volume
Approximate
Final Volume
of Pool
Pool 1
Candida albicans
0.2 mL
1.0 mL
Neisseria meningitidis
0.2 mL
Staphylococcus aureus (MRSA)*
0.2 mL
Streptococcus agalactiae
0.2 mL
Streptococcus pyogenes
0.2 mL
Pool 2
Acinetobacter baumannii
0.2 mL
1.2 mL
Candida glabrata
0.2 mL
Candida krusei
0.2 mL
Enterobacter cloacae complex
0.2 mL
Listeria monocytogenes
0.2 mL
Staphylococcus epidermidis (MSSE)**
0.2 mL
Pool 3
Candida parapsilosis
0.2 mL
1.2 mL
Haemophilus influenzae
0.2 mL
Enterococcus faecalis (VRE)
0.2 mL
Klebsiella pneumoniae (KPC)
0.2 mL
Serratia marcescens
0.2 mL
Streptococcus pneumoniae
0.2 mL
Pool 4
Escherichia coli
0.2 mL
1.0 mL
Pseudomonas aeruginosa
0.2 mL
Klebsiella oxytoca
0.2 mL
Candida tropicalis
0.2 mL
Proteus
0.2 mL
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 BioFire® 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
*MRSA, methicillin resistant S. aureus
**MSSE, methicillin susceptible S. epidermidis
Simple Protocol Example
The estimated total time for completion for this Simple Protocol verification
example is 4 days for a BioFire System configured with 1 module. A proposed
organism pooling scheme is presented above in Table 3. Refer to Figure 1 for the
suggested workflow.
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 BioFire System.
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Day 1
1. Prepare two sample pools (i.e. pools #1 and #2, in Table 3 above) from
ZeptoMetrix NATBCP-BIO control material. Organism vials should be well
mixed prior to preparing each pool.
a. Use a transfer pipette to remove 0.2 mL material from the ZeptoMetrix
organism vial (draw material to the second 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 1.0 mL total
volume for five organisms or 1.2 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 BioFire® FilmArray® Blood
Culture Identification (BCID) Panel Instruction Booklet and BioFire® FilmArray® BCID
Panel 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.
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.
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TECHNICAL
::: NOTE
Figure 1. Workflow for Simple Protocol
Blood Culture Media Protocol Example
The Blood Culture Media Protocol evaluates BioFire® FilmArray® Blood Culture
Identification (BCID) Panel performance in a blood culture sample matrix.
Sample material is pooled and added to an equal volume of blood culture media
matrix.
The Blood Culture Media 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 BioFire® FilmArray®
System.
Blood culture media 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.
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TECHNICAL
::: NOTE
Organism
Approximate
Organism
Volume
Volume of
Blood
Culture
Medium
Approximate
Final
Volume of
Pool
Pool 1
Candida albicans
0.2 mL
1.0 mL
2.0 mL
Neisseria meningitidis
0.2 mL
Staphylococcus aureus (MRSA)*
0.2 mL
Streptococcus agalactiae
0.2 mL
Streptococcus pyogenes
0.2 mL
Pool 2
Acinetobacter baumannii
0.2 mL
1.2 mL
2.4 mL
Candida glabrata
0.2 mL
Candida krusei
0.2 mL
Enterobacter cloacae complex
0.2 mL
Listeria monocytogenes
0.2 mL
Staphylococcus epidermidis
(MSSE)**
0.2 mL
Pool 3
Candida parapsilosis
0.2 mL
1.2 mL
2.4 mL
Haemophilus influenzae
0.2 mL
Enterococcus faecalis (VRE)
0.2 mL
Klebsiella pneumoniae (KPC)
0.2 mL
Serratia marcescens
0.2 mL
Streptococcus pneumoniae
0.2 mL
Pool 4
Escherichia coli
0.2 mL
1.0 mL
2.0 mL
Pseudomonas aeruginosa
0.2 mL
Klebsiella oxytoca
0.2 mL
Candida tropicalis
0.2 mL
Proteus
0.2 mL
Table 4. Proposed Blood Culture Media Sample Preparation Scheme
*MRSA, methicillin resistant S. aureus
**MSSE, methicillin susceptible S. epidermidis
Blood Culture Protocol Example
The estimated total time for completion for the Blood Culture Protocol verification
example is 4 days for a BioFire® FilmArray® System configured with 1 module.
A proposed organism pooling scheme is presented above in Table 4. Refer to
Figure 2 for the suggested workflow.
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TECHNICAL
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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 BioFire
®
FilmArray® System.
Day 1
1. Prepare two sample pools (i.e. pools #1 and #2, in Table 4 above) from
ZeptoMetrix NATBCP-BIO control material and blood culture media.
Organism vials should be well mixed prior to preparing each pool.
a. Pipet 1.0 or 1.2 mL of blood culture media (as described in Table 4) into
a sterile tube or vial.
b. Use a transfer pipette to remove 0.2 mL material from the ZeptoMetrix
organism vial (draw material to the second line of the transfer pipette) and
transfer to the tube containing blood culture media.
c. Repeat with the second (and subsequent) organisms to combine the
appropriate organisms into a single vial or tube (approximately 2.0 mL
total volume for five organisms or 2.4 mL for six organisms).
d. 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 BioFire® FilmArray® Blood
Culture Identification (BCID) Panel Instruction Booklet and BioFire® FilmArray® BCID
Panel 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.
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.
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Figure 2. Workflow for Blood Culture Media Protocol
.
Expanding the protocols
The protocol described above can be expanded by increasing the number of tests
from each of the organism pools. Each organism pool contains approximately 2
mL, which is enough material to complete many tests for each pool.
Verification of Loaner, Repaired, and Permanent Replacement
Instruments
If it becomes necessary to verify the performance of a loaner, repaired, or
permanent replacement 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 BioFire® FilmArray® Blood
Culture Identification (BCID) Panel. The Laboratory Director should determine
the appropriate number of samples to test. Proficiency samples should not be
pooled or diluted.
2. Test the selected specimens/samples on the loaner, repaired, or permanent
replacement instrument and document the results.
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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 BioFire 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
Organism
System
Serial #
Was the
Organism
Detected?
No.
Positive
No.
Negative
No. Days
Tested
No.
Users
Patient
Samples
Tested?
Acinetobacter baumannii
Yes
No
Candida albicans
Yes
No
Candida glabrata
Yes
No
Candida krusei
Yes
No
Candida parapsilosis
Yes
No
Candida tropicalis
Yes
No
Enterobacter cloacae
Yes
No
Enterococcus faecalis
(with vanA/B call)
Yes
No
Escherichia coli
Yes
No
Haemophilus influenzae
Yes
No
Klebsiella oxytoca
Yes
No
Klebsiella pneumoniae
(with KPC call)
Yes
No
Listeria monocytogenes
Yes
No
Neisseria meningitidis
Yes
No
Proteus mirabilis
Yes
No
Pseudomonas
aeruginosa
Yes
No
Serratia marcescens
Yes
No
Staphylococcus aureus
(MRSA, with mecA call)
Yes
No
Staphylococcus
epidermidis (MSSE, no
mecA call)
Yes
No
Streptococcus agalactiae
Yes
No
Streptococcus
pneumoniae
Yes
No
Streptococcus pyogenes
Yes
No
BioFire® FilmArray® Blood Culture Identification Verification
Record
System Serial # ___________________________
BioFire® FilmArray® Blood Culture Identification (BCID) Panel, Kit Part #:
__________________ Lot #: __________
Organism/Sample Source and Lot #: _______________________________
Reviewed by:
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Signature Date
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