Alinity m Resp-4-Plex AMP Kit Instruction Manual

Alinity m Resp-4-Plex AMP Kit

INTRODUCTION

This Emergency Use Authorization (EUA) package insert must be read carefully prior to use. EUA package insert instructions must be followed accordingly. Reliability of EUA assay results cannot be guaranteed if there are any deviations from the instructions in this package insert.

NAME
Alinity m Resp-4-Plex AMP Kit

INTENDED USE
The Alinity m Resp-4-Plex assay is a multiplex real-time reverse transcription (RT) polymerase chain reaction (PCR) test intended for the simultaneous qualitative detection and differentiation of RNA from SARS-CoV-2, influenza A virus (flu A), influenza B virus (flu B), and/or Respiratory Syncytial Virus (RSV) in anterior nasal or nasopharyngeal swab specimens collected by a healthcare provider (HCP), or in anterior nasal swab specimens that are self- collected at a healthcare location, from individuals suspected by their HCP of respiratory viral infection consistent with COVID-19. Clinical signs and symptoms of respiratory viral infection due to SARS-CoV-2, influenza, and RSV can be similar. Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. § 263a, that meet requirements to perform moderate or high complexity tests. Results are for the identification and differentiation of RNA from influenza A, influenza B, RSV, and SARS-CoV-2. The Alinity m Resp-4-Plex assay is not intended to detect influenza C virus. RNA from influenza A, influenza B, RSV, and SARS-CoV-2 is generally detectable in respiratory specimens during the acute phase of infection. Positive results are indicative of active infection but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. Clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. The agent detected may not be the definite cause of disease. Laboratories within the United States and its territories are required to report all SARS-CoV-2 results to the appropriate public health authorities.
Negative results do not preclude influenza A, influenza B, RSV, or SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.
Testing with the Alinity m Resp-4-Plex assay is intended for use by trained operators who are proficient in performing tests using the Alinity m
system and in vitro diagnostic procedures. The Alinity m Resp-4-Plex assay is only for use under the Food and Drug Administration’s Emergency Use Authorization.

SUMMARY AND EXPLANATION OF THE TEST

The Alinity m Resp-4-Plex assay is a multiplex real-time reverse transcription (RT) polymerase chain reaction (PCR) test for use with the automated Alinity m System for the qualitative detection and differentiation of RNA from flu A, flu B, RSV, and SARS-CoV-2 in respiratory specimens from individuals suspected of a respiratory viral infection consistent with COVID-19.

BIOLOGICAL PRINCIPLES OF THE PROCEDURE

The Alinity m Resp-4-Plex assay consists of 2 reagent kits:

• Alinity m Resp-4-Plex AMP Kit
• Alinity m Resp-4-Plex CTRL Kit

The Alinity m Resp-4-Plex assay detects flu A, flu B, RSV, SARS-CoV-2, and internal control (IC) target sequences through the use of target-specific fluorescent-labeled oligonucleotide probes. The probes do not generate a detectable signal unless they are specifically bound to the amplified product. The 2 SARS-CoV-2-specific probes are labeled with the same fluorophore and the flu A, flu B, RSV, and IC-specific probes are each labeled with distinct fluorophores, thus allowing for simultaneous detection and differentiation of amplified products of all 4 viruses and IC in a single reaction vessel.
An RNA sequence that is unrelated to the flu A, flu B, RSV, and SARS-CoV-2 sequences is introduced into each sample at the beginning of sample preparation. This unrelated RNA sequence is simultaneously amplified by RT-PCR and serves as an IC to demonstrate that the process has proceeded correctly for each sample.
The Alinity m Resp-4-Plex assay is to be used with the Alinity m System, which performs sample preparation, RT-PCR assembly, amplification, detection, result analysis and reporting. All testing steps of the Alinity m Resp-4-Plex assay procedure are executed automatically by the Alinity m System.
The Alinity m System is a continuous random access analyzer that can perform the Alinity m Resp-4-Plex assay in parallel with other Alinity m assays on the same instrument.
Application parameters specific to Alinity m Resp-4-Plex assay are contained in an assay-specific application specification file, which is distributed electronically and loaded onto the Alinity m System.

Sample Preparation

The Alinity m System provides automated sample preparation using the Alinity m Sample Prep Kit 2, Alinity m Lysis Solution, and Alinity m Diluent Solution. The purpose of sample preparation is to extract and concentrate the target RNA molecules to make the target accessible for amplification, and to remove potential inhibitors of amplification from the extract. The Alinity m System employs magnetic microparticle technology to facilitate nucleic acid capture, wash, and elution. The Internal Control (IC) is introduced into each specimen at the beginning of the sample preparation process to demonstrate that the process was completed correctly for each specimen and control sample.
During the sample preparation protocol, flu A, flu B, RSV, and/or SARS-CoV-2 virions are disrupted by lysis reagent, nucleic acids are captured on the magnetic microparticles, and inhibitors and unbound sample components are removed by washing steps within the Integrated Reaction Unit (IRU). The resulting purified RNA is then combined with liquid unit-dose Alinity m Resp-4-Plex activation reagent and liquid unit-dose Alinity m Resp-4-Plex amplification/detection reagent and transferred into a reaction vessel. Alinity m Vapor Barrier Solution is then added to the reaction vessel which is then transferred to an amplification/detection unit for reverse transcription, PCR amplification, and real-time fluorescence detection.
A positive control and a negative control are processed in the same manner and tested at least once every 24 hours to help confirm that instrument and reagent performance remain satisfactory.

Amplification

During the amplification reaction, the target RNA is converted to cDNA by the reverse transcriptase. The flu A, flu B, RSV, and SARS-CoV-2 and IC reverse primers anneal to their respective targets and are extended during a prolonged incubation period. After a denaturation step, in which the temperature of the reaction is raised above the melting point of the double-stranded cDNA:RNA product, a second primer anneals to the cDNA strand and is extended by the DNA polymerase to create a double-stranded DNA product.
During each round of thermal cycling, amplification products dissociate to single strands at high temperature allowing primer annealing and extension as the temperature is lowered. Exponential amplification of the product is achieved through repeated cycling between high and low temperatures, resulting in exponential amplification of target sequences. Amplification of the 6 targets (flu A, flu B, RSV, SARS-CoV-2 RdRp gene,
SARS-CoV-2 N gene, and IC) takes place simultaneously in the same reaction mixture.
The target sequences for the Alinity m Resp-4-Plex assay are:

• the RdRp and N genes of the SARS-CoV-2 genome
• the Matrix gene of the flu A genome
• the Nonstructural 1 gene of the flu B genome
• the Matrix gene of the RSV genome

The selected target sequences are highly conserved and also specific to the target viruses.
The IC target sequence is derived from the hydroxypyruvate reductase gene from the pumpkin plant, Cucurbita pepo, and is delivered in an Armored RNA® particle that has been diluted in negative human plasma. A gene from the pumpkin plant was selected for the IC so that it is not competitive with any microorganism or human sequence of interest that may be in the specimen.

Detection

Fluorescent detection of amplification products occurs as the flu A, flu B, RSV, SARS-CoV-2, and IC probes anneal to their targets (real-time fluorescence detection). The probes have a fluorescent moiety that is covalently linked to the 5′ end and have a quencher molecule at the 3′ end. In the absence of target sequences, probe fluorescence is quenched. In the presence of target sequences, hybridization to complementary sequences separates the fluorophore and the quencher and allows fluorescent emission and detection.
The Alinity m Resp-4-Plex assay detects the flu A, flu B, RSV, SARS-CoV-2 and IC target sequences through the use of target-specific fluorescent-labeled oligonucleotide probes. The probes do not generate a detectable signal unless they are specifically bound to the amplified product. The 2 SARS- CoV-2-specific probes are labeled with the same fluorophore and the flu A, flu B, RSV, and IC-specific probes are each labeled with distinct fluorophores, thus allowing for simultaneous detection and differentiation of all 4 viruses and IC amplified products in a single reaction vessel.

PREVENTION OF NUCLEIC ACID CONTAMINATION

The possibility of nucleic acid contamination on the Alinity m System is minimized because:

• Aerosol barrier pipette tips are used for all pipetting. The pipette tips are discarded after use.
• PCR amplification and detection are carried out automatically in a sealed reaction vessel.
• Disposal of the reaction vessel into the waste container is performed automatically by the Alinity m System.

For additional information on system and assay technology, refer to the Alinity m System Operations Manual, Section 3.

REAGENTS

Alinity m Resp-4-Plex AMP Kit (List No. 09N79-096)

• Alinity m Resp-4-Plex AMP Kit (List No. 09N79-096) is comprised of 2 types of multi-well trays: Alinity m Resp-4-Plex AMP TRAY 1 and Alinity m Resp-4-Plex ACT TRAY 2.
• Each Alinity m Resp-4-Plex AMP TRAY 1 (individually packed in a foil pouch) contains 48 unit-dose liquid amplification reagent wells and 48 unit-dose liquid IC wells. One well of each is used per test. Amplification reagent wells consist of synthetic oligonucleotides, DNA polymerase, reverse transcriptase, dNTPs and 0.15 % ProClin® 950 in a buffered solution. Internal control (IC) wells consist of noninfectious Armored RNA® with unrelated IC sequences in negative human plasma. Negative human plasma was tested and found to be nonreactive for HBsAg, HIV-1 antigen, Syphilis, HIV-1 RNA, HCV RNA, HBV DNA, antibody to HCV, antibody to HIV-1, and antibody to HIV-2. Preservative: 0.15% ProClin 950.
• Each Alinity m Resp-4-Plex ACT TRAY 2 (individually packed in a foil pouch) contains 48 unit-dose liquid activation reagent wells. One reagent well is used per test. Activation reagent wells consist of magnesium chloride and tetramethylammonium chloride. Preservative: 0.15% ProClin 950.

WARNINGS AND PRECAUTIONS

• For In Vitro Diagnostic Use Under the FDA Emergency Use Authorization.
• For use under an Emergency Use Authorization.
• Do not use beyond expiration date.
• For Prescription Use Only.
• This product has not been FDA cleared or approved, but has been authorized for emergency use by FDA under an EUA for use by authorized laboratories.
• This product has been authorized by FDA under an EUA for use by laboratories certified under CLIA to perform moderate or high complexity tests.
• This product has been authorized only for the detection and differentiation of nucleic acid from influenza A, influenza B, Respiratory Syncytial Virus and SARS-CoV-2, not for any other viruses or pathogens; and
• The emergency use of this product is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of COVID 19 under Section 564(b)(1) of the Federal Food, Drug and Cosmetic Act, 21 U.S.C. § 360bbb-3(b)(1), unless the declaration is terminated or the authorization is revoked sooner.
• Quality control requirements must be performed in conformance with local, state, and/or federal regulations or accreditation requirements and your laboratory’s standard quality control procedures.

CAUTION: This preparation contains human sourced and/or potentially infectious components. Components sourced from human blood have been tested and found to be nonreactive by appropriate FDA-licensed, approved, or cleared tests for antibody to HCV, antibody to HIV-1, antibody to HIV-2, HIV-1 Ag, HBsAg, and Syphilis. The material is also tested and found to be negative by appropriate FDA-licensed, approved, or cleared PCR methods for HIV-1 RNA, HCV RNA, and HBV DNA. No known test method can offer complete assurance that products derived from human sources or inactivated microorganisms will not transmit infection. These reagents and human specimens should be handled as if infectious using laboratory safety procedures, such as those outlined in Biosafety in Microbiological and Biomedical Laboratories,1 OSHA Standards on Bloodborne Pathogens,2 CLSI Document M29-A4,3 and other appropriate biosafety practices.4 Therefore all human sourced materials should be considered infectious.
These precautions include, but are not limited to, the following:

• Wear gloves when handling specimens or reagents.
• Do not pipette by mouth.
• Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in areas where these materials are handled.
• Clean and disinfect spills of specimens by including the use of a tuberculocidal disinfectant such as 1.0% sodium hypochlorite or other suitable disinfectant.1
• Decontaminate and dispose of all potentially infectious materials in accordance with local, state, and federal regulations.4

The following warnings and precautions apply to:
Alinity m Resp-4-Plex ACT TRAY 2.

• DANGER: Contains Tetramethylammonium chloride, and 2-Methyl-4-isothiazolin-3-one
  • H302: Harmful if swallowed.
  • H316: Causes mild skin irritation.
  • H317: May cause an allergic skin reaction.
  • H370: Causes damage to organs.
  • H412: Harmful to aquatic life with long lasting effects.
• Prevention
  • P260: Do not breathe mist / vapors / spray.
  • P264: Wash hands thoroughly after handling.
  • P272: Contaminated work clothing should not be allowed out of the workplace.
  • P273: Avoid release to the environment.
  • P280: Wear protective gloves / protective clothing / eye protection.
• Response
  • P301+P312: IF SWALLOWED: Call a POISON CENTER/doctor if you feel unwell
  • P302+P352: IF ON SKIN: Wash with plenty of water
  • P308+P311: IF exposed or concerned: Call a POISON CENTER / doctor.
  • P333+P313: If skin irritation or rash occurs: Get medical advice / attention.
  • P362+P364: Take off contaminated clothing and wash it before reuse.
• Disposal
  • P501: Dispose of contents / container in accordance with local regulations.

Reagent Storage

In order to minimize damage to foil pouches, it is recommended that the Alinity m Resp-4-Plex AMP TRAY 1 (AMP TRAY 1) and Alinity m Resp-4-Plex ACT TRAY 2 (ACT TRAY 2) are stored in the original kit packaging. Thaw reagent trays and open the foil pouch for the reagent trays just prior to loading on the Alinity m System. Onboard storage time begins when reagents are thawed and immediately loaded on the Alinity m System.

Reagent Handling

• Do not use reagents that have been damaged.

• IMPORTANT: Immediately prior to use on the Alinity m System, thaw amplification reagents at 15 to 30°C or at 2 to 8°C. Onboard storage time begins immediately after thaw. See ASSAY PROCEDURE section for additional instructions.

• Minimize contact with the surface of reagent trays during handling.

• Up to 2 lots of assay trays (AMP TRAY 1 and ACT TRAY 2) can be loaded on each Alinity m Assay Tray Carrier, as long as the AMP TRAY 1 and ACT TRAY 2 from the same AMP kit lot are included together as a set.

• The Alinity m System will track the onboard storage time of AMP TRAY 1 and ACT TRAY 2 while on the Alinity m System. The Alinity m System will not allow the use of AMP TRAY 1 and ACT TRAY 2 if the maximum onboard storage time has been exceeded.
  IMPORTANT: The maximal allowable onboard storage for the Alinity m Resp-4-Plex AMP TRAY 1 and ACT TRAY 2 is 96 hours from thaw/onboarding.

• For a detailed discussion of reagent handling precautions during system operation, refer to the Alinity m System Operations Manual, Section 8.

SPECIAL PRECAUTIONS

As with any test procedure, good laboratory practice is essential to the proper performance of this assay. Due to the high sensitivity of this test, care should be taken to keep reagents and amplification mixtures free of contamination.

• For in vitro diagnostic use under Emergency Use Authorization only.
• Positive results are indicative of the presence of flu A, flu B, RSV, and/or SARS-CoV-2 RNA.
• Laboratories within the United States and its territories are required to report all SARS-CoV-2 results to the appropriate public health authorities.
• All patient samples should be handled as if infectious, using good laboratory procedures as outlined in Biosafety in Microbiological and Biomedical Laboratories1 and in the CLSI Document M29-A4.3 Only personnel proficient in handling infectious materials and the use of the Alinity m
  Resp-4-Plex assay and the Alinity m System should perform this procedure.

Handling Precautions for Specimens

• The Alinity m Resp-4-Plex assay is only for use with anterior nasal and nasopharyngeal swab specimens that have been handled and stored as described in the SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST SITE section.
• Inadequate or inappropriate specimen collection, storage, and transport are likely to yield false test results. Training in specimen collection is highly recommended due to the importance of specimen quality. Refer to CLSI MM13-A 5 as an appropriate resource.
• During preparation of samples, compliance with good laboratory practices is essential to minimize the risk of cross-contamination between samples and the inadvertent introduction of ribonucleases (RNases) into samples during and after the extraction procedure.
• Proper aseptic technique should always be used when working with nucleic acid amplification.
• Amplification technologies, such as PCR, are sensitive to accidental introduction of product from previous amplification reactions. Incorrect results could occur if either the clinical specimen or the reagents used become contaminated by accidental introduction of even a small amount of amplification product. Measures to reduce the risk of contamination in the laboratory include physically separating the activities involved in the handling of contaminated waste in compliance with good laboratory practices.

INDICATION OF INSTABILITY OR DETERIORATION OF REAGENTS

• Deterioration of the reagents may be indicated when a control error occurs or controls are repeatedly out of the specified ranges.
• Reagents are shipped on dry ice and are stored at -25 to -15°C upon arrival. If reagents arrive in a condition contrary to this recommendation or are damaged, immediately contact your Abbott Representative.
• For troubleshooting information, refer to the Alinity m System Operations Manual, Section 10.

INSTRUMENT PROCEDURE

The Alinity m Resp-4-Plex application specification file must be installed on the Alinity m System prior to performing the assay.
For a detailed description of system operating instructions, refer to the Alinity m System Operations Manual, Section 5.

SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST SITE

Human anterior nasal and nasopharyngeal swab specimens can be used with the Alinity m Resp-4-Plex assay on the Alinity m System. Refer to the CDC Interim Guidelines for Collecting, Handling, and Testing for Patients with Suspected Novel Influenza A (H1N1) Virus Infection6
(https://www.cdc.gov/h1n1flu/specimencollection.htm), CDC Influenza Specimen Collection guidelines (https://www.cdc.gov/flu/pdf/professionals/flu- specimen-collection-poster.pdf) and the CDC Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons Under Investigation (PUIs) for Coronavirus Disease 2019 (COVID-19)7
(https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelines-clinical- specimens.html) or the FDA FAQs on Diagnostic Testing for SARS-CoV-2
(https://www.fda.gov/medical-devices/emergency-situations-medical-devices /faqs-testing-sars-cov-2).
Specimens in VTM (and its equivalents) can be stored at 5°C ± 3°C for a maximum of 80 hours prior to testing, or stored at -70°C or colder for a maximum of 7 days (with a maximum of 2 freeze/thaws) prior to testing. Package and label specimens in compliance with applicable state, federal, and international regulations covering the transport of clinical, diagnostic, or biological specimens.
For domestic and international shipments, specimens must be packaged, and transported according to the current edition of the International Air Transport Association (IATA) Dangerous Goods Regulation. Follow shipping regulations for UN 3373 Biological Substance, Category B when sending potential SARS-CoV-2 specimens.

Preparation for Analysis

Frozen specimen is thawed at 15 to 30°C or at 2 to 8°C.
Prior to processing, each specimen is vortexed 3 times for 2 to 3 seconds.
If needed, centrifuge specimens at 2000 g for 5 minutes before loading on the Alinity m System. Specimen can be transferred into an Alinity m Transport Tube or an Alinity m Aliquot Tube before loading onto the Alinity m System.
IMPORTANT: If present, swab and cap should be removed from the specimens before loading onto the Alinity m System.
Primary collection tubes may be used directly, if they meet the requirements listed in the tube type table in the Assay Procedure section below. All specimen tubes must be labeled with specimen ID barcodes or must be identified with a specimen ID, rack ID, and position in the rack. Refer to the Assay Procedure section of this package insert for tube sizes and requirements for minimum sample volume and use of caps. Avoid touching the inside of the cap when opening tubes.

PROCEDURE

Materials Provided

• Alinity m Resp-4-Plex AMP Kit (List No. 09N79-096)

Materials Required But Not Provided

• 08N53-002 Alinity m System with software version 1.5.2 or higher
• 09N79-086 Alinity m Resp-4-Plex CTRL Kit
• 09N12-001 Alinity m Sample Prep Kit 2
• 09N20-001 Alinity m Lysis Solution
• 09N20-003 Alinity m Diluent Solution
• 09N20-004 Alinity m Vapor Barrier Solution
• 09N79-05A (or higher) Alinity m Resp-4-Plex Application Specification File
• Vortex mixer
• Centrifuge capable of 2000 g
• Plate adapter for 384 well plates (eg, Eppendorf Catalog No. 022638955)
• Centrifuge with swing plate rotor capable of accommodating the plate adapter and capable of ≥ 100 g

For information on materials required for operation of the Alinity m System, refer to the Alinity m System Operations Manual, Section 1.

Other Optional Materials

• 09N49-010 Alinity m Transport Tube Pierceable Capped
• 09N49-011 Alinity m Transport Tube
• 09N49-013 Alinity m Aliquot Tube
• Viral transport medium (UVT/UTM)
• Sealable plastic bags

Procedural Precautions

• Read the instructions in this package insert carefully before processing samples.
• Use aerosol barrier pipette tips or disposable pipettes only 1 time when pipetting specimens. To prevent contamination to the pipette barrel while pipetting, care should be taken to avoid touching the pipette barrel to the inside of the sample tube or container. The use of extended aerosol barrier pipette tips is recommended.
• Work area and instrument platforms must be considered potential sources of contamination.
• Ensure the Alinity m Resp-4-Plex AMP TRAY 1 and ACT TRAY 2 are centrifuged prior to loading on the Alinity m System per instructions in Assay Procedure section.
• Monitoring procedures for the presence of amplification target contamination can be found in the Alinity m System Operations Manual, Section 9.
• To reduce the risk of nucleic acid contamination, clean and disinfect spills of specimens by including the use of a tuberculocidal disinfectant such as 1.0% sodium hypochlorite or other suitable disinfectant.
• To prevent contamination, change to new gloves before handling the Alinity m Sample Prep Kit 2, assay trays, system solutions, Integrated Reaction Unit (IRU) sleeves, and pipette tips. Also change to new gloves whenever they are contaminated by a specimen, a control, or a reagent. Always use powder-free gloves.
• The use of the Alinity m Resp-4-Plex CTRL Kit is integral to the performance of the Alinity m Resp-4-Plex assay. Refer to the QUALITY CONTROL PROCEDURES section of this package insert for details. Refer to the Alinity m Resp-4-Plex CTRL Kit package insert for preparation and usage.
• The Alinity m Resp-4-Plex control reagents are contained in single-use tubes with solid caps. Remove caps from the tube prior to use. Discard tubes after use.

ASSAY PROCEDURE

Thaw AMP TRAY 1 and ACT TRAY 2 at 15 to 30°C or at 2 to 8°C immediately prior to use.
Prior to loading on the Alinity m System, the AMP TRAY 1 and ACT TRAY 2 must be centrifuged as follows:

1. Load the trays onto the plate adapter (eg, Eppendorf Catalog No. 022638955).
2. Load the plate adapter (with the trays) on a swing plate centrifuge capable of accommodating the plate adapter. Spin at 100 to 800 g for 1 to 5 minutes to ensure reagents remain at the bottom of the well and to remove potential bubbles.
3. Immediately following centrifugation, carefully transfer the trays to the Alinity m Assay Tray Carriers. Take care to minimize disturbance to the trays. Load the tray carriers per the Alinity m System Operations Manual, Section 5.
4. If disturbance occurs during the transfer that could potentially introduce bubbles or displace reagents from the bottom of the wells (eg, dropping, bumping, inversion of the trays), re-centrifuge the trays.
5. Proceed with Reagent and sample management per the Alinity m System Operations Manual, Section 5.

For a detailed description of how to run an assay, refer to the Alinity m System Operations Manual, Section 5. Prior to testing specimens, check the control status. If control testing is required, refer to the QUALITY CONTROL PROCEDURES section. Controls may be tested separately or with specimens.
One PCR reaction can detect 1 or more pathogens with the Alinity m Resp-4-Plex assay. Therefore, only 1 patient specimen aliquot is required for detection of the selected assay(s).
To create a test order, select COV2_4PEUA for SARS-CoV-2 and any combination of the other 3 test target names to match the requested tests for each patient specimen.

The testing results will be reported only for those test targets selected for the specimen in the test order.
A test result, for a test target that was not originally selected in the test order, may still be obtained without repeat testing, within a user-configured period of time. Refer to Alinity m System Operations Manual, Section 5 Operating Procedures, Stored Result Retrieval subsection.
The Alinity m System will track the onboard storage time of AMP TRAY 1, ACT TRAY 2, controls, and specimens while on the Alinity m System. The Alinity m System will not allow the use of AMP TRAY 1, ACT TRAY 2, controls, or process specimens that have exceeded the allowable onboard storage time setting by the system.
IMPORTANT: The maximal allowable onboard storage for Alinity m Resp-4-Plex AMP TRAY 1 and ACT TRAY 2 is 96 hours from thaw/ onboarding.

QUALITY CONTROL PROCEDURES

Detection of Inhibition

A defined, consistent quantity of IC is introduced into each specimen and control at the beginning of sample preparation and measured on the Alinity m System to demonstrate proper specimen processing and assay validity.
A Message Code is displayed for the control when the IC Cycle Number (CN) value exceeds the established range.
A Flag or Message Code is displayed for the sample when the IC Cycle Number (CN) value falls outside of the established range:

• For Positive Specimens: If the IC CN is out of range, and any analyte(s) (flu A, flu B, RSV or SARS-CoV-2) in that specimen is detected, the specimen will yield a Positive interpretation for the detected analyte(s). An IC Flag will be reported next to the detected analyte(s).
• For Negative Specimens: If the IC CN is out of range and any of the analyte(s) (flu A, flu B, RSV or SARS-CoV-2) in that specimen is not detected, no result will be reported for that analyte(s) and a Message Code will be generated.
• For Negative and Positive Controls: If the IC CN is out of range, a Message Code will be generated for all the analytes in the Controls.
• Note that for a specimen, each analyte test is treated independently. For example, a specimen may be reported Positive for flu A with an IC flag, but may get a Message Code indicating IC failure for each of the other 3 analytes if they are not detected.

Refer to the Alinity m System Operations Manual, Section 5 for an explanation of the Flags.
Refer to the Alinity m System Operations Manual, Section 10 for an explanation of the corrective actions for Message Codes.

Negative and Positive Controls

A set of Alinity m Resp-4-Plex Negative Control (CTRL-) and Positive Control (CTRL+) are required to be tested at least once every 24 hours, to monitor the performance of the assay and Alinity m System. Valid results for all control levels must be obtained before specimen results are reported. Additional controls may be tested in accordance with local, state, and/or federal regulations or accreditation requirements and your laboratory’s quality control policy.
A Flag is displayed for specimens when a control result is invalid. If the controls for a given target analyte (flu A, flu B, RSV, or SARS-CoV-2) are invalid, all of the specimens for that target analyte processed in parallel with an invalid assay control must be retested. If control results are invalid, refer to the Alinity m System Operations Manual, Section 5 for a description of quality control flags, and Section 10 for troubleshooting information. The presence of flu A, flu B, RSV, or SARS-CoV-2 must not be detected in the negative control. Flu A, flu B, RSV, or SARS-CoV-2 detected in the negative control is indicative of contamination by other samples or by amplified product. If contamination is suspected, clean the Alinity m System and repeat sample processing for controls and specimens following the Procedural Precautions in this package insert. Cleaning and monitoring procedures for the presence of amplification target contamination can be found in the Alinity m System Operations Manual, Section 9.
If negative controls are persistently reactive, contact your Abbott Representative at www.molecular.abbott/portal.

Flags, Results Codes, and Message Codes

Some results may contain information in the Flags and Codes fields. For a description of the flags and result codes that may appear in these fields, refer to the Alinity m System Operations Manual, Section 5. For a description of message codes refer to the Alinity m System Operations Manual, Section 10.

LIMITATIONS OF THE PROCEDURE

For use under an Emergency Use Authorization only.

• This assay is for in vitro diagnostic use under FDA Emergency Use Authorization only.

• Use of the Alinity m Resp-4-Plex assay is limited to personnel who have been trained in the procedures of a molecular diagnostic assay and the Alinity m System.

• Laboratories are required to report all SARS-CoV-2 results to the appropriate public health authorities.

• The instrument and assay procedures reduce the risk of contamination by amplification product. However, nucleic acid contamination from positive controls or specimens must be controlled by good laboratory practices and careful adherence to the procedures specified in this
  package insert.

• Optimal performance of this test requires appropriate specimen collection, storage, and transport to the test site (refer to the SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST SITE section of this package insert).

• Detection of flu A, flu B, RSV, or SARS-CoV-2 RNA may be affected by sample collection methods, patient factors (eg, presence of symptoms), and/or stage of infection.

• Recent patient exposure to FluMist® or other attenuated influenza vaccines may cause inaccurate positive results for flu A and flu B due to the presence of attenuated viruses.

• Interference was observed for RSV when evaluated with FluMist. Recent patient exposure to FluMist or other attenuated influenza vaccines may cause inaccurate false negative RSV results.

• Interference was observed for flu A, flu B, RSV, and SARS-CoV-2 when evaluated with high concentrations of Pneumocystis jirovecii (PJP). There is a risk of false negative for flu A, B, RSV, and SARS-CoV-2 results under certain co-infection circumstances with Pneumocystis jirovecii (PJP).

• False-negative results may arise from degradation of the viral RNA during storage and transport of the specimens.

• As with any molecular test, mutations within the target regions of Alinity m Resp-4-Plex assay could affect primer and/or probe binding resulting in failure to detect the presence of virus.

• The clinical performance has not been established in all circulating variants but is anticipated to be reflective of the prevalent variants in circulation at the time and location of the clinical evaluation. Performance at the time of testing may vary depending on the variants circulating, including newly emerging strains of SARS-CoV-2 and their prevalence, which change over time.

• Results should be interpreted by a trained professional in conjunction with the patient’s history and clinical signs and symptoms, and epidemiological risk factors.

• The Alinity m Resp-4-Plex assay was validated with nasopharyngeal swabs. Anterior nasal swabs (self-collected under healthcare provider (HCP) supervision or HCP-collected) are also considered acceptable specimen types, but performance has not been established. Specimen types other than nasopharyngeal and anterior nasal swabs should not be tested with this assay.

• Negative results do not preclude infection with the flu A, flu B, RSV, or SARS-CoV-2 and should not be the sole basis of a patient treatment/management or public health decision. Follow up testing should be performed according to the current CDC recommendations.

• Self-collected under supervision of or healthcare provider collected anterior nasal swabs are additional acceptable upper respiratory specimens that can be tested with the Alinity m Resp-4-Plex assay; however, performance with this specimen type has not been validated.

• Influenza was validated in the clinical study by testing archived, selected specimens only. If an influenza result is inconsistent with clinical presentation and/or other clinical and epidemiological information, FDA-cleared Influenza NAATs are available for confirmation if clinically indicated.

• A positive result indicates the detection of nucleic acid from the relevant virus. Nucleic acid may persist even after the virus is no longer viable.

CONDITIONS OF AUTHORIZATION FOR LABORATORIES

The Alinity m Resp-4-Plex assay Letter of Authorization, along with the authorized Fact Sheet for Healthcare Providers, the authorized Fact Sheet for Patients, and authorized labeling are available on the FDA website: https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19 -emergency-use-authorizations-medical-devices/vitro-diagnostics-euas.
However, to assist clinical laboratories using the Alinity m Resp-4-Plex assay (“your product” in the conditions below), the relevant Conditions of Authorization are listed below:

1. Authorized laboratories using your product must include with test result reports of your product, all authorized Fact Sheets. Under exigent circumstances, other appropriate methods for disseminating these Fact Sheets may be used, which may include mass media.
2. Authorized laboratories using your product must use your product as outlined in the authorized labeling. Deviations from the authorized procedures, including the authorized instruments, authorized extraction methods, authorized clinical specimen types, authorized control materials, authorized other ancillary reagents and authorized materials required to use your product are not permitted.
3. Authorized laboratories that receive your product must notify the relevant public health authorities of their intent to run your product prior to initiating testing.
4. Authorized laboratories using your product must have a process in place for reporting test results to healthcare providers and relevant public health authorities, as appropriate.
5. Authorized laboratories must collect information on the performance of your product and report to DMD/OHT7-OIR/OPEQ/CDRH (via email: [email protected]) and Abbott Molecular (email: [email protected]; 1-800-553-7042) any suspected occurrence of false positive or false negative results and significant deviations from the established performance characteristics of your product of which they become aware.
6. All laboratory personnel using your product must be appropriately trained in RT-PCR techniques and use appropriate laboratory and personal protective equipment when handling this kit, and use your product in accordance with the authorized labeling.
7. Abbott, authorized distributor(s), and authorized laboratories using your product must ensure that any records associated with this EUA are maintained until otherwise notified by FDA. Such records will be made available to FDA for inspection upon request.

SPECIFIC PERFORMANCE CHARACTERISTICS

Limit of Detection (Analytical Sensitivity)

Limit of Detection (LoD) studies determine the lowest detectable concentration of SARS-CoV-2, flu A, flu B, and RSV, at which greater than or equal to 95% of replicates test positive.
The LoD was determined by testing dilutions of 7 cultured viruses, including 1 SARS-CoV-2 strain, 2 flu A strains (H1N1 and H3N2), 2 flu B strains
(Victoria and Yamagata lineages), and 2 RSV strains (RSV A and RSV B), spiked in pooled negative clinical nasopharyngeal swab specimens. For each virus, the preliminary LoD was determined by testing a minimum of 3 levels, each in 3 replicates. The final LoD was confirmed by testing 3 to 4 panel members with target concentrations bracketing the preliminary LoD, each panel member in replicates of 21. LoD is defined as the lowest concentration at which greater than or equal to 95% of all replicates tested positive.

Inclusivity

The inclusivity of Alinity m Resp-4-Plex assay for the detection of SARS- CoV-2, flu A, flu B, and RSV was evaluated by testing 6 isolates of SARS- CoV-2, 20 strains of flu A virus (including H1N1, H3N2, H5N1, and H7N2), 9 strains of flu B virus (including Victoria and Yamagata lineages), and 6 strains of RSV (including RSV A and B). Each individual virus isolate or strain (cultured virus or viral RNA) was tested in simulated nasal matrix, in a minimum of 3 replicates. The Alinity m Resp-4-Plex assay detected all replicates of all strains at the concentrations tested (see Table 2). Viral strains present at concentrations below those tested may not be detected.

In addition, in silico analyses were performed in which the sequence of each of the Alinity m Resp-4-Plex analyte primers and probes was analyzed for homology with sequences available in GISAID and/or NCBI Virus Sequences for Discovery (GenBank) databases. A total of 104,885 full-length SARS-CoV-2 sequences available in the GISAID database as of September 28, 2020 and 17,216 full-length SARS-CoV-2 sequences available in the NCBI database as of October 4, 2020 were analyzed for SARS-CoV-2 inclusivity. A total of 67,426 full- length target gene sequences (consisting of 27,550 H1 strains, 39,162 H3 strains, and 714 H7 strains) available in the GISAID database as of May 20, 2020 were analyzed for flu A inclusivity. A total of 20,917 full-length target gene sequences (consisting of 10,934 Victoria strains, 9,967 Yamagata strains, and 16 unclassified strains) available in the GISAID database as of May 22, 2020 were analyzed for flu B inclusivity. A total of 460 full-length sequences (consisting of 316 RSV A strains and 144 RSV B strains) available in the NCBI database as of August 3, 2020 were analyzed for RSV inclusivity.
On February 5, 2021, an in silico analysis of the SARS-CoV-2 primers and probe sets was performed against the variant UK, South Africa, Brazil, and California strains. The in silico analysis predicts that assay performance is unlikely to be impacted by the currently circulating escape variants.

Overall, the results of these in silico analyses and inclusivity testing predict that sequence heterogeneity in Alinity m Resp-4-Plex primer/probe target regions do not impact the detection of SARS-CoV-2, flu A, flu B, and RSV.

Precision

Alinity m Resp-4-Plex assay within-laboratory precision was evaluated using a 9-member panel composed of SARS-CoV-2, flu A, flu B, and RSV each at 2 target concentrations in simulated nasal matrix, as well as a negative panel member in simulated nasal matrix. Each panel member was tested with a target of 6 replicates in a run on each of 5 days.

Lot-to-Lot Reproducibility

Alinity m Resp-4-Plex assay lot-to-lot reproducibility was evaluated using a positive panel member composed of SARS-CoV-2, flu A, flu B, and RSV in simulated nasal matrix, as well as a negative panel member (simulated nasal matrix). Both panel members were tested using 3 amplification reagent lots with a minimum of 4 replicates per lot.

Analytical Specificity and Microbial Interference

A total of 55 potential cross-reacting microorganisms (viruses, bacteria, and fungi) that are phylogenetically related to the analytes of the assay or that are commonly found in respiratory tract and pooled human nasal wash were tested with Alinity m Resp-4-Plex to assess analytical specificity and microbial interference. The microorganisms were tested at 105 Units/mL for viruses and 106 Units/mL for bacteria and fungi, where these concentrations were available. The unit of measure was specific to each microorganism. Bacteria and fungi were tested as whole microorganisms. Viruses were tested as viral particles or viral lysate unless noted otherwise.
To assess potential cross-reactivity, each microorganism was tested in analyte (flu A, flu B, RSV and SARS-CoV-2) negative samples comprised of simulated nasal matrix. No cross-reactivity was observed in the presence of these potential cross-reactants.
To assess potential microbial interference, microorganisms were spiked into analyte positive samples containing 1 strain each for flu A [A/Switzerland/9715293/13 (H3N2)], flu B [B/Brisbane/33/08 (Victoria lineage)], RSV (RSVB/WestVirginia/14617/85), and SARS-CoV-2 (USA-WA1/2020) targeted to final concentration of 3X LoD. No interference was observed for any of the analyte (flu A, flu B, RSV and SARS-CoV-2) positive samples in the presence of these microorganisms, except when undiluted Pneumocystis jirovecii (PJP) was tested. With a 1:50 dilution of the PJP culture, no interference was observed.

On-Panel Cross-Reactivity

The cross-reactivity of each signal channel of the Alinity m Resp-4-Plex assay for on-panel viruses was evaluated by testing representative strains, at 105 Units/mL. No cross reactivity was observed for on-panel viruses.

Co-Infection (Competitive Interference)

To assess potential competitive interference between SARS-CoV-2, flu A, flu B, and RSV, samples containing low concentrations (≤ 3X LoD) of 3 analyte targets were mixed with a high concentration 105 TCID50/mL of the fourth analyte target and tested in 20 or more replicates. None of the analyte targets present at the high concentration interfered with the detection of low levels of the other 3 analyte targets.

Fresh versus Frozen Specimen Comparison

The performance of Alinity m Resp-4-Plex with fresh and frozen specimens was evaluated for equivalency by testing individual flu A, flu B, RSV and SARS- CoV-2 strains spiked, at two different concentrations representing low positives (3X LoD) and moderate positives (5X LoD), in pooled negative nasopharyngeal clinical swab specimens collected in viral transport medium (VTM). Individual nasopharyngeal swab specimens were prescreened with Alinity m Resp-4-Plex to be negative for flu A, flu B, RSV and SARS-CoV-2 prior to being pooled. In addition to positive samples, negative samples consisted of pooled negative nasopharyngeal clinical swab specimens collected in VTM were also tested.
The influenza, RSV and SARS-CoV-2 viruses selected for this study included one flu A strain (Flu A H3N2/Switzerland/9715293/13), one flu B strain
(Flu B Brisbane/33/08), one RSV strain (RSVB/West Virginia/14617/85) and one SARS-CoV-2 strain (USA-WA1/2020). A minimum of 40 replicates of 3X LoD samples, 10 replicates of 5X LoD samples, and 10 replicates of negative samples were tested for each storage condition. Two sample storage conditions were evaluated, including fresh and after two freeze-thaw cycles. For each freeze-thaw cycle, samples were placed at -70°C or colder for a minimum of 24 hours, after which they were thawed at 5°C ± 3°C for a minimum of 2 hours prior to Alinity m Resp-4-Plex testing. All positive and negative sample replicates were correctly identified using Alinity m Resp-4-Plex for both storage conditions.

Specimen Stability

A stability study was conducted to establish transport and storage claims for nasopharyngeal swab specimens collected in VTM to be analyzed with Alinity m Resp-4-Plex. Samples tested in the study consisted of one flu A strain (Flu A H3N2/Switzerland/9715293/13), one flu B strain (Flu B Brisbane/33/08), and one RSV strain (RSVB/West Virginia/14617/85) that were spiked in pooled negative clinical nasopharyngeal swab specimens to an analyte concentration of approximately 3X LoD.
Positive samples were stored at 5°C ± 3°C and -70°C or colder and five replicates were tested at different time points. All specimens tested at each storage temperature/condition were correctly identified using Alinity m Resp-4-Plex. The study data supports the recommended specimen storage condition (5°C ± 3°C for a maximum of 80 hours and -70°C or colder for a maximum of 7 days).

Use of Simulated Nasal Matrix

In comparing simulated nasal matrix (SNM) to natural clinical matrix, it was found that all replicates, prepared near the LoD for each on-panel analyte, in either natural clinical matrix or SNM, tested positive for all on-panel analytes.

Interfering Substances

Potentially interfering substances that may be encountered in respiratory specimens were evaluated by testing 1 strain of SARS-CoV-2 (USA-WA1/2020), and 2 strains each of flu A [A/Switzerland/9715293/13 (H3N2) and A/Brisbane/59/2007 (H1N1)], flu B [B/Brisbane/33/08 (Victoria lineage) and B/Massachusetts/02/2012 (Yamagata lineage)], and RSV [RSVB/WestVirginia/14617/85 and RSVA/Long/MD/56 (RSV A)] at a target concentration of 3X LoD. No interference resulting in a negative result (except for RSVA/Long/MD/56, where one of the six replicates was not detected in the presence of FluMist) was observed in the presence of any of the substances at the concentrations.

Clinical Performance Evaluation

The performance of Alinity m Resp-4-Plex was evaluated by testing individual banked clinical nasopharyngeal (NP) swab specimens in viral transport medium.
A total of 114 specimens were analyzed for SARS-CoV-2 detection by both Alinity m Resp-4-Plex and a comparator EUA SARS-CoV-2 assay. The positive percent agreement (PPA) between the 2 assays was 100% (55/55) and the negative percent agreement (NPA) was 96.6% (57/59).
An additional 22 specimens were analyzed for SARS-CoV-2 detection by both Alinity m Resp-4-Plex and a different comparator EUA SARS-CoV-2 assay. These specimens all had Ct values within 3 Cts of the mean Ct at the LoD for the comparator. The positive percent agreement (PPA) between the two assays was 100% (22/22).
A total of 239 specimens were analyzed for flu A detection by both Alinity m Resp-4-Plex and a comparator FDA-cleared assay for flu A, flu B, and RSV. The positive percent agreement (PPA) between the 2 assays was 100% (63/63) and the negative percent agreement (NPA) was 100% (176/176). A total of 239 specimens were analyzed for flu B detection by both Alinity m Resp-4-Plex and a comparator FDA-cleared assay for flu A, flu B, and RSV. The positive percent agreement (PPA) between the 2 assays was 100% (63/63) and the negative percent agreement (NPA) was 99.4% (175/176). A total of 240 specimens were analyzed for RSV detection by both Alinity m Resp-4-Plex and a comparator FDA-cleared assay for flu A, flu B, and RSV. The positive percent agreement (PPA) between the 2 assays was 100% (64/64) and the negative percent agreement (NPA) was 100% (176/176).

BIBLIOGRAPHY

1. US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories. 5th ed. Washington, DC: US Government Printing Office; December 2009. [Also available online. Type> www.cdc.gov, search> BMBL> look up sections III and IV.]
2. US Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910.1030. Bloodborne Pathogens.
3. Clinical and Laboratory Standards Institute. Protection of Laboratory Workers from Occupationally Acquired Infections: Approved Guideline—Fourth Edition. CLSI Document M29-A4. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
4. World Health Organization. Laboratory Biosafety Manual. 3rd ed. Geneva, Switzerland: World Health Organization; 2004.
5. Clinical and Laboratory Standards Institute. Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods; Approved Guideline. CLSI Document MM13-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.
6. Centers for Disease Control and Prevention (CDC). Interim Guidelines for Collecting, Handling, and Testing for Patients with Suspected Novel Influenza A (H1N1) Virus Infection. Available online at: https://www.cdc.gov/h1n1flu/specimencollection.htm
7. Centers for Disease Control and Prevention (CDC). Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons Under Investigation (PUIs) for Coronavirus Disease 2019 (COVID-19). Available online at: https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelines-clinical-specimens.html

TECHNICAL ASSISTANCE

For technical assistance, call Abbott Molecular Technical Services at 1-800-553-7042 in the US and from outside the US at +49-6122-580, email [email protected], or visit the Abbott Molecular website at www.molecular.abbott/portal.
Abbott Molecular Inc. is the legal manufacturer of the Alinity m Resp-4-Plex AMP Kit (List No. 09N79-096)

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