Guidelines for Laboratory Verification of Performance of ... · 6 | P a g e Advisory Notice BioFire...

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Advisory Notice BioFire Diagnostics, LLC

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ADVISORY ::: NOTICE

Guidelines for Laboratory Verification of Performance of the FilmArray™ GI 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 examples of verification procedures to assist your laboratory in developing a protocol for the verification of the FilmArray Gastrointestinal (GI) Panel performance required by CLIA. Several possible verification schemes, compatible with the FilmArray GI Panel, have been designed. Each scheme provides positive and negative tests for each organism detected by the FilmArray GI Panel and may be easily modified or expanded to meet specific criteria. Each scheme was designed so that a trained laboratory technician could test at least 4 FilmArray pouches per day on each FilmArray instrument to be verified. 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, patient samples can be tested for verification or to evaluate matrix effects on the performance of the FilmArray GI Panel. 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 GI Panel is a multiplexed nucleic acid test intended for use with the FilmArray instrument for the simultaneous qualitative detection and identification of multiple gastrointestinal viral, parasitic and bacterial nucleic acid targets in stool in Cary Blair samples obtained from individuals suspected of gastrointestinal tract infections. The following organisms and subtypes are identified using the FilmArray GI Panel: Campylobacter (C. jejuni, C. coli, and C. upsaliensis), Clostridium difficile toxin A/B, Plesiomonas shigelloides, Salmonella, Vibrio (V. parahaemolyticus, V. vulnificus, and V. cholerae), Yersinia entercolitica, Enteraggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC), Entertoxigenic E. coli (ETEC) lt/st, Shiga-like toxin-producing E. coli (STEC) stx1/stx2, E. coli O157, Shigella/Enteroinvasive E. coli (EIEC), Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Adenovirus F 40/41, Astrovirus, Norovirus GI/GII, Rotavirus A, and Sapovirus (I, II, IV, and V).

The complete intended use statement and additional information about the use of the FilmArray system can be found in the FilmArray GI Panel Instruction Booklet.

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Performance Verification: Overview

Each procedure described below will generate multiple positive and negative results for each of the organisms targeted by the FilmArray GI Panel. The procedures were developed using a NATtrol™ GI Panel available from ZeptoMetrix Corporation, Buffalo, NY (part number NATGIP-BIO). Four different examples of performance verification procedures are described: (1) a simple protocol for the verification of a single FilmArray instrument in a synthetic background (Negative) provided with the NATtrol GI Panel, (2A) expanded verification of a single instrument in a synthetic background (Negative) provided with the NATtrol GI Panel, (2B) an expanded protocol for the verification of two FilmArray instruments in a synthetic background (Negative) provided with the NATtrol GI Panel, and (3) a simple Cary Blair Media protocol that evaluates the performance of each assay on a single FilmArray instrument with a stool in Cary Blair sample matrix. The Simple (1) and Expanded (2A, 2B) procedures have been designed to take advantage of the multiplex nature of the FilmArray GI Panel. Verification testing efficiency is maximized by evaluating multiple target organisms in a single test run. In addition to, or in place of, verification schemes described here, a laboratory may chose to test clinical/patient samples to assess clinical sensitivity and sample matrix effects in its performance verification of the FilmArray GI Panel. Table 1. Overview of Verification Protocols

Verification Protocol Organisms

per Poola

Number of Sample Pools

Replicates per Sample

Pool

Pouches Required

Expected Positive Results

Expected Negative Results

Approximate Days of Testing

Example 1: Simple protocol (one instrument)

5 or 6 4 4 16 4 per organism 12 per organism 4

Example 2A: Expanded protocol (one instrument)


Example 2B: Expanded protocol (two instruments)

5 or 6 4 4* 32 4 per organism* 12 per organism* 4

Example 3: Stool in Cary Blair protocol (one instrument)


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.

* Per instrument.

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Performance Verification: Materials

The following materials may be needed to perform verification procedures:

Material Part Number

FilmArray GI Panel Kit (30 tests) Biofire Diagnostics RFIT-ASY-0116

Control Organism ZeptoMetrix NATGIP-BIO a

Cary Blair Transport Media Thermo Scientific Part # 23-005-47 (or equivalent)

5mL sample tubes VWR Part # 89497-740 (or equivalent)

Transfer pipettes VWR Part # 13-711-43 (or equivalent)

aAny appropriate source of organism may be used for verification of any or all of the assays in the FilmArray GI system. However, when alternate organism sources are used (i.e. not the ZeptoMetrix material), the sample volumes or pooling schemes suggested in the examples below may need to be adjusted.

Performance Verification: Organism Pooling

The protocols described below utilize samples prepared by pooling together either 5 or 6 different organisms (ZeptoMetrix NATGIP-BIO control organism). The pooling scheme (Table 2) was designed so that both positive and negative target results can be obtained in a time and resource-efficient manner.

Note: To obtain the expected number of positive and negative results for each assay,

it is important to avoid grouping organisms that share assays in the same sample (for example, E. coli O157 and E. coli EPEC). The organism pooling schemes presented were

designed with this in mind.

Note: Dilution of ZeptoMetrix GI Verification Panel organisms beyond levels proposed

in these guidelines may lead to inconsistent results and is not recommended.

Note: When preparing the organism pools as described in this protocol, it is critical to

only prepare one pool at a time, and to decontaminate the work area thoroughly before working with organism from additional pools. Working with organism from multiple pools at the same time greatly increases the risk of contaminating the pools with organism(s) from other pools, which would result in erroneous results in the form of false positives detected.

Note: When using the optional protocol for testing organism using a true clinical stool

in Cary Blair background there is a probability that the background will contain a previously unknown GI pathogen that will result in a positive detection by the FilmArray GI Panel. It is highly recommended to confirm that the stool in Cary Blair background used is negative by testing the background with 1 or more pouches of the FilmArray GI Panel. The number of pouches used to confirm the negativity of the stool in Cary Blair background is at the discretion of the Laboratory Director.

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Table 2. Example Organism Pooling Scheme for Simple and Expanded Protocols

Note: All volumes are approximate.

Note: A synthetic stool diluant (Negative) is provided with the ZeptoMetrix

(NATGIP-BIO) Control Organism. Four 0.85mL aliquots are provided to allow the user to create all of the specified organism pools.

Organism Organism Volume

Negative Or

Stool in Cary-Blair Media

Approximate Final Volume of

Pool

Pool A Enteraggregative E. coli (EAEC) 0.25 mL

0.85mL 2.1mL

Adenovirus Type 41 0.25 mL

Cryptosporidium parvum 0.25 mL

Salmonella typhimurium 0.25 mL

Sapovirus 0.25 mL

Pool B Enteropathogenic E. coli (EPEC) 0.25 mL

0.85mL 2.35mL

Norovirus GI 0.25 mL

Norovirus GII 0.25 mL

Cyclospora cayetanensis 0.25 mL

Shigella sonnei 0.25 mL

Astrovirus 0.25 mL

Pool C Entertoxigenic E. coli (ETEC) lt/st 0.25 mL

0.85mL 2.35mL

Entamoeba histolytica 0.25 mL

Clostridium difficile 0.25 mL

Vibrio cholerae 0.25 mL

Campylobacter jejuni 0.25 mL

Campylobacter coli 0.25 mL

Pool D E. coli O157 0.25 mL

0.85mL 2.1mL

Rotavirus 0.25 mL

Giardia lamblia 0.25 mL

Plesiomonas shigelloides 0.25 mL

Yersinia entercolitica 0.25 mL

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Example 1: Simple Protocol for Verification of One Instrument

In this example, a total of 16 pouches are tested, providing 4 positive results and 12 negative results per organism. This example assumes 4 tests will be completed per day on an instrument. The actual number should be determined by the individual laboratory. The estimated total time to completion for this verification example is 4 days.

Figure 1. Simple protocol workflow. Each sample pool has enough material for 4 tests (1-4) to assess variability from day-to-day and user-to-user. Workflow can be repeated for each sample pool until all testing is complete.

Day 1

1. Prepare one sample pool (i.e. pool A) from ZeptoMetrix NATGIP-BIO control material. An example organism pooling scheme is presented above in Table 2.

a. Use a transfer pipette to remove entire contents of a vial of ZeptoMetrix Negative (approximately 0.85mL) and transfer it to a tube large enough (at least 3mL) to hold the entire organism pool volume.

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b. Use a transfer pipette to remove the entire contents of the ZeptoMetrix organism vial (approximately 0.25mL) and transfer to the larger tube containing the Negative diluent.

c. Repeat step b. for each of the remaining organisms to combine the appropriate organisms for each pool into a single vial or tube.

d. Ensure the pooled sample is effectively mixed by vortexing prior to removing a sample for testing.

Note: Store samples at refrigeration temperature (2–8°C) for up to 3 days for the

evaluation of day-to-day variation. 2. Prepare and test 2 samples (i.e. 1 and 2, see Figure 1) from a single sample

pool (i.e. pool A). The duplicate samples should be prepared consecutively and tested in a single day. For each sample:

a. Follow instructions in the FilmArray GI Panel Instruction Booklet or GI Panel Quick Guide for pouch preparation.

b. Prepare Sample Mix: Squeeze the contents of one sample buffer ampoule into a Sample Injection Vial. Using a transfer pipette, aspirate the pooled organism mixture to the 2nd line (~0.2mL), and dispense into the Sample Injection Vial. Close the lid of the Sample Injection Vial tightly and invert 3-5 times to completely mix the sample and buffer, then replace the Sample Injection Vial in the loading station.

c. Follow instructions in the FilmArray GI Panel Instruction Booklet or GI Panel Quick Guide for sample loading and FilmArray GI testing.

3. Repeat steps 1 and 2 for the remaining sample pool (i.e. pool B) to be tested

that day.

Note: It is important to prepare only the number of organism sample pools that will be

tested within 2 days of preparation. The suggestion to prepare 2 sample pools is based on ensuring day-to-day and user-to-user variability for each sample pool, testing 4 pouches per day using one instrument. The number of samples prepared may be increased or decreased based on the laboratory’s work schedule.

Day 2

To evaluate day-to-day variation, test the remaining samples (i.e. samples 3 and 4) from the same sample pools prepared on Day 1 by repeating Step 2 above.

Day 3

Prepare 2 new sample pools (i.e. pools C and D) as described in Step 1. Test samples according to Step 2 (i.e. samples 1 and 2) for each pool.

Day 4

To evaluate day-to-day variation, test the samples prepared on Day 3 by repeating Step 2 (i.e. samples 3 and 4).

Note: Please see Addendum 1 for a detailed day-to-day protocol for Example 1:

Simple protocol for verification of one instrument.

Note: If any FilmArray pouch control or instrument failures are observed during

testing there is enough remaining volume in each sample pool to run an additional pouch. Test the sample again and proceed as outlined.

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Example 2A and 2B: Expanded Protocol for Verification of One Instrument (2A) or Two Instruments (2B)

The simple protocol testing scheme (Example 1 above) is easily expanded by preparing more pouches from each vial of pooled organism mix. The following expanded scheme can be used to double the number of verification samples tested on a single instrument or it can be used to verify two instruments simultaneously. In this expanded protocol, a total of 32 pouches are tested. When all pouches are tested on a single instrument, this scheme provides 8 positive results and 24 negative results per organism with an estimated total time to completion of 8 days. When this scheme is used to verify 2 instruments, the scheme provides 4 positive results and 12 negative results per organism per instrument. The estimated time to complete this verification scheme for 2 instruments is 4 days. An example of the workflow is shown in Figure 2.

Figure 2. Expanded protocol workflow. Each sample pool has enough material for 8 tests, to assess variability from day-to-day and user-to-user. Workflow can be repeated for each sample pool until all testing is complete.

Day 1

1. If this testing scheme will be used for the verification of one instrument, prepare one organism sample pool (i.e. Pool A) from ZeptoMetrix NATGIP-BIO control material. If the testing scheme will be used for the verification of 2 instruments, 2 sample pools will be prepared on this day (i.e. pools A and B) from ZeptoMetrix NATGIP-BIO control material. It is important to prepare only one sample pool at a time to avoid the risk of sample pool

Sample 1

Sample 2

Sample 3

Sample 4

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contamination. An example organism pooling scheme is presented above in Table 2.

a. Use a transfer pipette to remove entire contents of a vial of ZeptoMetrix Negative (approximately 0.85mL) and transfer it to a tube large enough (at least 3mL) to hold the entire organism pool volume.

b. Use a transfer pipette to remove the entire contents of the ZeptoMetrix organism vial (approximately 0.25mL) and transfer to the larger tube containing the Negative diluent.

c. Repeat with each of the remaining organisms to combine the appropriate organisms for each pool into a single vial or tube.


Note: It is important to prepare only the number of sample pools that will be tested

within 2 days of preparation. The suggestion to prepare 2 sample pools is based on ensuring day-to-day and user-to-user variability for each sample pool, testing 4 pouches per day using one instrument. The number of samples prepared may be increased or decreased based on the laboratory’s work schedule.

Note: The sample pools can be tested in duplicate on the same day and refrigerated

(2–8°C) for up to 3 days for the evaluation of day-to-day variation.

2. Prepare and test 2 samples (i.e. 1 and 2, see Figure 2) from a single sample

pool (i.e. pool A). The duplicate samples should be prepared consecutively and tested in a single day. For each sample:

a. Follow instructions in the FilmArray GI Panel Instruction Booklet or GI Panel Quick Guide for pouch preparation.

b. Prepare Sample Mix: Squeeze the contents of one sample buffer ampoule into a Sample Injection Vial. Using a transfer pipette, aspirate the pooled organism mixture to the 2nd line (~0.2mL), and dispense into the Sample Injection Vial. Close the lid of the Sample Injection Vial tightly and invert 3-5 times to completely mix the sample and buffer, then replace the Sample Injection Vial in the loading station.


4. If this testing scheme will be used for the verification of one instrument,

repeat Step 2 using the same sample pool (i.e. pool A) that was tested that day. If the testing scheme will be used for the verification of two instruments, repeat Step 1 and 2 for the remaining sample pool (i.e. pool B) to be tested that day.

Note: If you will be testing samples for the verification of 2 instruments (Example 2B),

prepare two pouches from the same organism pool and proceed with testing. If you will be testing two samples from the same organism pool on the same instrument (Example 2A), prepare and test one pouch at a time and store the remaining pooled organism at refrigeration temperature (2–8°C) until ready to test the second pouch.

Day 2

To examine day-to-day variation, test the samples prepared on Day 1 by repeating Steps 2–3 of the expanded protocol. For the verification of one instrument the remaining samples from the organism pool tested on Day 1 will be used (i.e. Pool A). For the verification of two instruments, the remaining samples from two organism pools will be tested (i.e. pools A and B).

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Day 3-4

Repeat Steps 1–3 for pooled samples. By the end of day 4 all samples should be completed for the verification of two instruments.

Day 5-8

Repeat Steps 1–3 of the expanded protocol as above for any remaining sample pools for the verification of one instrument.

Note: Please see Addendum 2 for a detailed day-to-day protocol for Example 2B:

Expanded protocol for the verification of two instruments.



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Example 3: Stool in Cary Blair Media Protocol for Verification of One Instrument

An example organism pooling scheme is presented above in Table 2. For this testing scheme, a stool sample in Cary Blair media will be used as the organism pool background rather than synthetic stool (Negative). It is the responsibility of the individual laboratory to obtain a stool sample that is negative for GI pathogens to be used with this protocol. These 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. In this example, a total of 16 pouches are tested, providing 4 positive results and 12 negative results per organism. This example assumes 4 tests will be completed per day on an instrument. The actual number should be determined by the individual laboratory. The estimated total time to completion for this verification example is 4 days.

Figure 3. Stool in Cary Blair protocol workflow. Each sample pool has enough material for 4 tests (1-4) to assess variability from day to day and user to user. Workflow can be repeated for each sample pool until all testing is complete.

Note: When using this protocol, there is potential for unexpected positive organism

calls to occur due to the use of a true clinical stool sample which may or may not contain previously undetected GI pathogens. It is advised to use a stool background that is negative for GI pathogens. It is recommended to use additional FilmArray GI pouches to confirm the negativity of the stool sample used. The number of additional pouches used to confirm negativity is at the discretion of the Laboratory Director.

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Day 1 1. Prepare one sample pool (i.e. pool A) by mixing ZeptoMetrix NATGIP-BIO

control material with a freshly prepared stool sample in Cary Blair transport media. An example organism pooling scheme is presented above in Table 2. a. Use a transfer pipette to transfer approximately 0.85mL of a prepared

stool in Cary Blair sample to a tube large enough (at least 3mL) to hold the entire organism pool volume.

b. Use a transfer pipette to remove the entire contents of the ZeptoMetrix organism vial (approximately 0.25mL) and transfer to the larger tube containing the stool sample.

c. Repeat with each of the remaining organisms to combine the appropriate organisms for each pool into a single vial or tube.


Note: Store samples at refrigeration temperature (2–8°C) for up to 3 days for the

evaluation of day-to-day variation.

2. Prepare and test two samples (i.e. 1 and 2, see Figure 3) from a single

sample pool (i.e. pool A). The duplicate samples should be prepared consecutively and tested in a single day. For each sample: a. Follow instructions in the FilmArray GI Panel Instruction Booklet or GI

Panel Quick Guide for pouch preparation and pouch hydration. b. Prepare Sample Mix: Squeeze the contents of one sample buffer

ampoule into a Sample Injection Vial. Using a transfer pipette, aspirate the pooled organism mixture to the 2nd line (~0.2mL), and dispense into the Sample Injection Vial. Close the lid of the Sample Injection Vial tightly and invert 3-5 times to completely mix the sample and buffer, then replace the Sample Injection Vial in the loading station.


3. Repeat Step 1 and 2 for the remaining sample pool (i.e. pool B) to be tested

that day.

Note: It is important to prepare only the number of organism sample pools that will be

tested within 2 days of preparation. The suggestion to prepare 2 sample pools is based on ensuring day-to-day and user-to-user variability for each sample pool, testing 4 pouches per day using one instrument. The number of samples prepared may be increased or decreased based on the laboratory’s work schedule.

Day 2

To evaluate day-to-day variation, test the remaining samples (i.e. samples 3 and 4) from the same sample pools prepared on Day 1 by repeating Step 2 above.

Day 3

Prepare 2 new sample pools (i.e. pools C and D) as described in Step 1. Test samples according to Step 2 (i.e. samples 1 and 2) for each pool.

Day 4

To evaluate day-to-day variation, test the samples prepared on Day 3 by repeating Step 2 (i.e. samples 3 and 4).

Note: Please see Addendum 1 for a detailed day-to-day protocol for Example 3: Stool

in Cary Blair media protocol for the verification of one instrument.

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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. 1. Select a few specimens and/or proficiency samples (any combination of

positives and negatives) previously tested on the FilmArray GI Panel. The Laboratory Director should determine the appropriate number of samples to test. Three to 6 samples may be sufficient. Proficiency samples should not be pooled or diluted.

2. Test the selected samples on the loaner or repaired instrument and document the results.

For additional information on this technology and other BioFire Diagnostics products, please visit us at www.BioFireDX.com or call 1-801-736-6354.

FLM1-PRT-0151-01

http://www.idahotech.com/

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Addendum 1: Day-to-day Workflow for Example Protocols 1 and 3 Example 1: Simple protocol for verification of one instrument. Example 3: Stool in Cary Blair media protocol for the verification of one instrument

Pool A

1 2

3 4

Pool B

1 2

3 4

Pool C

1 2

3 4

Pool D

1 2

3 4

Day 1

Day 2

Day 3

Day 4

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Addendum 2: Day-to-day Workflow for Example Protocol 2 Example 2B: Expanded protocol for the verification of two instruments

FilmArray™ Instrument Verification Record

Organism Was the

Organism Detected?

No. Positive

No. Negative

No. Days Tested

No. Users

Patient Samples Tested?

Enteroaggregative E. coli (EAEC) Yes

No

Adenovirus F 40/41 Yes

No

Cryptosporidium Yes

No

Salmonella Yes

No

Sapovirus (I, II, IV, and V) Yes

No

Enteropathogenic E. coli (EPEC) Yes

No

Norovirus GI/GII Yes

No

Cyclospora cayetanensis Yes

No

Shigella/ Enteroinvasive E. coli (EIEC) Yes

No

Astrovirus Yes

No

Entertoxigenic E. coli (ETEC) lt/st Yes

No

Entamoeba histolytica Yes

No

Clostridium difficile Toxin A/B Yes

No

Vibrio (V. parahaemolyticus, V. vulnificus, and V. cholerae)

Yes

No

Campylobacter (C. jejuni, C. coli, and C. upsaliensis)

Yes

No

Shiga-like toxin-producing E. coli (STEC) stx1/stx2

Yes

No

E. coli O157 Yes

No

Rotavirus A Yes

No

Giardia lamblia Yes

No

Plesiomonas shigelloides Yes

No

Yersinia entercolitica Yes

No

Reviewed by: __________________________________________ Signature Date

Instrument serial #: _____________________________

FilmArray GI Panel kit Part #: __________________ Lot #: __________

Organism/sample Source and Lot #: ____________________________________

_________________________________________________________________

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