Document issued on: October 9, 2009
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U.S. Department of Health and Human Services
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Table of Contents
- Background - Premarket Notifications
- Scope of this document and related documents
- Risks to Health
- Device Description
- Intended Use
- Test Methodology
- Ancillary reagents
- Testing Procedures using your device
- Interpreting Test Results/Reporting
- Performance Characteristics
- General Study Recommendations
- Specimen collection and handling
- Nucleic acid extraction
- Assay Cut-off
- Performance Studies
- Clinical Studies
Guidance for Industry and FDA Staff - Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays
This document was developed as a special controls guidance to support the classification into class II (special controls) of a respiratory viral panel multiplex1 nucleic acid assay that includes detection and identification of human metapneumovirus (hMPV) . A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. Nucleic acid assays t esting for hMPV are intended to detect hMPV RNA extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid sequence from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. A respiratory viral panel multiplex nucleic acid assay is intended for detection and identification of a combination of the following viruses:
- Influenza A and Influenza B
- Influenza A subtype H1 and Influenza A subtype H3
- Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B
- Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus
- Human Metapneumovirus
This guidance provides recommendations to manufacturers and FDA staff regarding preparation of premarket notifications for respiratory viral panel multiplex nucleic acid assays that include detection and identification of Human Metapneumovirus (hMPV). The recommendations in this document are applicable to assays that employ technologies such as polymerase chain reaction (PCR), reverse-transcriptase polymerase chain reaction (RT-PCR), bead-based liquid arrays, and microarrays.
This guidance addresses devices that are used in conjunction with clinical presentation and other laboratory tests (e.g., immunofluorescence, bacterial culture, chest x-rays/radiography) to aid in the diagnosis of respiratory hMPV infection. This guidance does not address assays intended for use as the sole basis for diagnosis nor does it address assays meant to differentially diagnose viral from non-viral infections. Additional studies would be needed if using nucleic acid amplification assays to detect hMPV in specimens other than respiratory specimens. For the assays addressed by this guidance, positive results do not rule out bacterial infection, or co-infection with other viruses.
This guidance is issued in conjunction with a Federal Register notice announcing the classification of the respiratory viral panel multiplex nucleic acid assay. Designation of this document as a special control means that any firm submitting a 510(k) for a respiratory viral panel multiplex nucleic acid assay that includes detection and identification of hMPV, will need to address the issues covered in this guidance as well as the issues covered in another special control guidance identified in the classification regulation, "Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay." In addition to these guidance documents, for respiratory viral panels that include detection and differentiation of Influenza A virus subtypes, an additional special control guidance is “Class II Special Controls Guidance Document: Testing for Detection and Differentiations of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays." See 21 CFR 866.3980(b). The firm must show that its device addresses the issues of safety and effectiveness identified in this guidance (and the other special control guidances referenced, as applicable) either by meeting the recommendations of this guidance or by some other means that provides equivalent assurances of safety and effectiveness.
This special controls document identifies the classification regulation and product code for multiplex nucleic acid assays testing for hMPV (refer to Section 3). Other sections of this guidance document identify risks related to this device type and provide recommendations to address these risks.
If you want to discuss an alternative means of satisfying the requirement of special controls for this device, you may contact the FDA staff responsible for implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance.
The Least Burdensome Approach
T he issues identified in this guidance document represent those that we believe need to be addressed before your device can be marketed. In developing the guidance, we carefully considered the relevant statutory criteria for Agency decision-making. We also considered the burden that may be incurred in your attempt to follow the statutory and regulatory criteria in the manner suggested by the guidance and in your attempt to address the issues we have identified. We believe that we have considered the least burdensome approach to resolving the issues presented in the guidance document. If, however, you believe that there is a less burdensome way to address the issues, you should follow the procedures outlined in the document, “A Suggested Approach to Resolving Least Burdensome Issues.” It is available on our Center web page at: https://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm089730.pdf.
A manufacturer who intends to market a device of this generic type must
- conform to the general controls of the Federal Food, Drug, and Cosmetic Act (the act), including the premarket notification requirements described in 21 CFR 807 Subpart E,
- conform to the special control by addressing the specific risks to health associated with hMPV nucleic acid assays identified in this guidance (see Section 4, below)
- satisfy the other special controls designated in 21 CFR 866.3980, the classification regulation for this type of device, and
- obtain a substantial equivalence determination from FDA prior to marketing the device. (See also 21 CFR 807.81 and 807.87).
FDA believes that special controls, when combined with the general controls, will be sufficient to provide reasonable assurance of the safety and effectiveness of nucleic acid assays for testing hMPV.
This document supplements other FDA documents regarding the specific content requirements of a premarket notification submission. You should also refer to 21 CFR 807.87, the guidance, Format for Traditional and Abbreviated 510(k)s2 and the section of CDRH’s Device Advice webpage, Premarket Notification 510(k).3 As described in The New 510(k) Paradigm - Alternate Approaches to Demonstrating Substantial Equivalence in Premarket Notifications; Final Guidance,4 a manufacturer may submit a Traditional 510(k), an Abbreviated 510(k), or a Special 510(k). A manufacturer may choose to submit an abbreviated 510(k) when a guidance document exists, when special controls have been established, or when FDA has recognized a relevant consensus standard. Manufacturers considering certain modifications to their own cleared devices may lessen their regulatory burden by submitting a Special 510(k). For more information on types of Premarket Notification 510(k)s that may be submitted to FDA, see CDRH’s Device Advice webpage, Premarket Notification 510(k).
3. Devices within the Scope of this Document
The scope of this document is limited to the following devices, described in 21 CFR 866.3980:
21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay. A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:
- Influenza A and Influenza B
- Influenza A subtype H1 and Influenza A subtype H3
- Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B
- Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus
- Human Metapneumovirus
In addition to this guidance document, for respiratory viral panels that include detection and identification of hMPV, an additional special control guidance is “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays." For respiratory viral panels that include detection and differentiation of Influenza A subtypes, an additional special control guidance is "Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays."
Product codes applicable for devices described in this guidance and cleared under 21 CFR 866.3980
OCC – Respiratory virus panel nucleic acid assay system
OEM – Human metapneumovirus (hMPV) RNA assay system
As already noted, devices subject to this special control guidance document are also subject to the special control guidance, “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay,” and, if they include testing for detection and differentiation of Influenza A Virus Subtypes, to the special control guidance “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays." See 21 CFR 866.3980(b).
Human metapneumovirus has been associated with acute respiratory tract illnesses in children and adults. The clinical signs associated with hMPV infection appear to be similar to those caused by human respiratory syncytial virus (RSV). The clinical impact of infection by hMPV ranges from mild self-limiting respiratory illness to bronchiolitis and pneumonia that may necessitate mechanical ventilation. Infants and elderly adults may develop severe illness caused by hMPV infection. hMPV may also cause increased morbidity in immunocompromised patients. Therefore, FDA has identified potential risks to health, i.e., issues that may impact safety or effectiveness of a respiratory viral panel multiplex nucleic acid assay that detects and identifies hMPV. These include failure of the device to perform as indicated, leading to inaccurate results or lack of results, and incorrect interpretation of results; both of these potential risks may lead to incorrect patient management decisions.
Failure of the device to perform as indicated:
A false positive result could lead to delayed treatment of respiratory infections caused by other etiologic agents, which may potentially be a more serious infection caused by bacteria or other pathogens. A false negative result could lead to failure to provide a diagnosis and the correct treatment, and may contribute to unnecessary treatment. A lack of result could lead to delayed diagnosis and inadequate treatment.
Failure to interpret results correctly:
A respiratory viral panel multiplex nucleic acid assay that detects and identifies hMPV is intended to aid in the diagnosis of respiratory hMPV infection when used in conjunction with other clinical and laboratory findings. Therefore, failure to interpret assay results in the context of other laboratory results and clinical presentation could lead to inappropriate or delayed treatment. For example, positive assay results do not rule out bacterial co-infection, or co-infection with other viruses, and the hMPV detected by the assay may not necessarily be the cause of the clinical symptoms or disease. In addition, detection of the hMPV nucleic acid may not necessarily indicate active infection. For reasons such as these, additional testing (e.g., bacterial culture, immunofluorescence, and chest x-rays/radiography) is needed in order to obtain the final diagnosis of respiratory hMPV infection.
This special control guidance makes recommendations for mitigating the following specific sources of error that can lead to the risks associated with this type of assay:
Inaccurate results (i.e., false positive or false negative results) or lack of a result may be attributed to the following:
- Failure or improper use of reagents, instrumentation, data management, or software included with the assay.
- Failure or improper use of ancillary reagents or problems with the quality of ancillary reagents.
- Improper testing when performed by laboratory personnel lacking expertise in molecular testing.
False positive results can be caused by the following:
- Persistence of hMPV RNA sequences in vivo, independent of virus viability, as nucleic acid tests do not distinguish non-viable viruses from infective viral particles that are discerned by cell culture.
- The potential of assay primers and probes to cross-react with nucleic acid sequences from other viruses detected by the multiplex assay system, other non-viral pathogens which may be present in patient specimens, or other endogenous nucleic acid sequences.
- In the case of an open assay system, cross-contamination and amplicon contamination if proper control measures are not implemented.
False negative results can be caused by the following:
- Analyte degradation due to improper storage or transport of specimens and extracted nucleic acid, or inadequate extraction of the nucleic acid material.
- The emergence of new genetic subtypes or a genetic mutant of hMPV. Primers and probes detecting hMPV are generally selected for their homology with highly conserved regions within viral RNA segments that are present in all known hMPV genetic subtypes. These primers and probes may fail to react with newly isolated genetic subtypes or a genetic mutant which developed over time, reducing assay performance.
- Competitive inhibition or interference by other substances present in patient specimens or introduced into the analytical system during sample processing / handling.
Failure to properly interpret test results can be due to the following:
- Possibility of bacterial co-infection.
- Inaccurate interpretation and reporting of testing results by laboratory personnel lacking expertise in viral diagnosis.
- Misinterpretation due to performance variability related to seasonal hMPV prevalence and specific patient populations.
In the table below, FDA has identified the risks to health generally associated with the use of nucleic acid amplification assays for detection of hMPV. Measures recommended to mitigate these identified risks are given in this guidance document, as shown in the table below. You should conduct a risk analysis prior to submitting your premarket notification to identify any other risks specific to your device. Risks may vary depending on the type of nucleic acid assays used, the intended use of the test, the sample type, and how the result will be used. The premarket notification should describe the risk analysis method. If you elect to use an alternative approach to address the risks identified in this document, or have identified risks additional to those in this document, you should provide sufficient detail to support the approach you have used to address that risk.
|Identified risk||Recommended mitigation measures|
|Inaccurate results (i.e., false positive or false negative results), or lack of results||Device Description (Section 5)
Performance Characteristics (Section 6)
Labeling (Section 7)
|Failure to interpret results correctly||Labeling (Section 7)|
In your 510(k) submission, you should identify the regulation, the product code(s), and a legally marketed predicate device. We recommend that you include a table that outlines the similarities and differences between the predicate device and your device.
You should include the following descriptive information to adequately characterize your hMPV nucleic acid assay.
5.A Intended Use
The intended use should specify the nature of the analyte and the target (e.g., hMPV F gene RNA), specimen types for which testing will be indicated, the clinical indications for which the test is to be used, and the specific population(s) for which the test is intended. The intended use should state that the test is qualitative and any specific conditions of use.
In your 510(k), you should clearly describe the following information related to the intended use of your product:
- The identity, phylogenetic relationship, or other recognized characterization of the subtypes of hMPV that your device is designed to detect.
- How the device test results will be used to aid in a laboratory identification of hMPV in respiratory specimens from symptomatic patients.
Note: Recognized laboratory methods for definitive identification of hMPV are available, please see e.g., CLSI M41-A5; sequencing of amplicons (bi-directional with quality scores) generated by subtype-specific primers may also be used6 if appropriate validation data can be provided.
You should describe in detail the methodology used by your device. For example, you should describe the following elements, as applicable to your device:
- Test platform (e.g., RT-PCR, bead arrays).
- Specificity of primers and/or probes for the hMPV sequences of interest.
- Information and rationale for selection of specific hMPV target sequences and the methods used to design primers and probes.
- Limiting factors of the assay (e.g., saturation level of hybridization, maximum cycle number).
- Sample type (e.g., swabs, aspirates, and viral culture media), collection and handling methods.
- Reagent components provided or recommended for use, and their function within the system (e.g., buffers, enzymes, fluorescent dyes, chemiluminescent reagents, other signaling/amplification reagents).
- The potential for specific and non-specific probe cross-hybridization.
- Instrumentation required for your device, including the components and their function within the system.
- Types of output generated by the instrumentation and system parameters (e.g., measurement ranges).
- The computational path from raw data to the reported result (e.g., how raw signals are converted into a signal). This would include sufficient software controls for identifying and dealing with obvious problems in the dataset. It would also include adjustment for background and normalization, if applicable.
- Illustrations or photographs of non-standard equipment or methods, if available.
When applicable, you should describe design control specifications for your device that address or mitigate risks associated with an hMPV nucleic acid assay procedure detecting viral RNA segments from hMPV, such as the following examples:
- Minimization of false positives due to contamination or carryover of sample.
- Developing or recommending validated methods for nucleic acid extraction and purification that yield suitable quality and quantity of hMPV nucleic acid from respiratory specimens for use in the test system with your reagents. You should address suitable validated extraction method(s) for different specimen types your assay claims in its intended use.
- Optimizing your reagents and test procedure for recommended instruments.
In your 510(k), you should provide performance information that supports the conclusion that your design requirements have been met. You should also provide information to verify the design of your reagents (e.g., rationale for selection of specific conserved target sequences and the methods used to design primers and probes). (See Section 6 – Performance Characteristics).
5.C Ancillary Reagents
Ancillary reagents are those reagents that an assay manufacturer specifies in device labeling as “required but not provided” in order to carry out the assay as indicated in its instructions for use and to achieve the test performance claimed in labeling for the assay. For the purposes of this document, ancillary reagents of concern are those that must be specified according to manufacturer and catalog or product number, or other specific designation, in order for your device to achieve its labeled performance characteristics. For example, if your device labeling specifies the use of Brand X DNA amplification enzyme, and use of any other DNA amplification enzyme may alter the performance characteristics of your device from that reported in your labeling, then Brand X DNA amplification enzyme is an ancillary reagent of concern for the purposes of this document.7
By contrast, if your device requires the use of 95% ethanol, and any brand of 95% ethanol will allow your device to achieve the performance characteristics provided in your labeling, then 95% ethanol is not an ancillary reagent of concern for the purposes of this document.
If the instructions for use of your device specify one or more ancillary reagents of concern, you should address how you will ensure that the results of testing with your device and these ancillary reagents, in accordance with your instructions, will be consistent with the performance established in your premarket submission. Your plan may include application of quality systems approaches, product labeling, and other measures.
In order to address this aspect of the special control, your 510(k) submission should address the elements described below. FDA will evaluate whether your plan will help to mitigate the risks presented by the device to offer reasonable assurance of the safety and effectiveness of the device and establish its substantial equivalence.
- You should include in your 510(k) a risk assessment addressing the use of ancillary reagents, including risks associated with management of reagent quality and variability, risks associated with inconsistency between instructions for use provided directly with the ancillary reagent and those supplied by you with your assay, and any other issues that could present a risk of obtaining incorrect results with your assay.
- Using your risk assessment as a basis for applicability, you should describe in your 510(k) how you intend to mitigate risks through implementation of any necessary controls over ancillary reagents. These may include, where applicable:
- User labeling to assure appropriate use of ancillary reagents (see “Labeling” for further discussion).
- Plans for assessing user compliance with labeling instructions regarding ancillary reagents.
- Material specifications for ancillary reagents.
- Identification of reagent lots that will allow appropriate performance of your device.
- Stability testing.
- Complaint handling.
- Corrective and preventive actions.
- Plans for alerting users in the event of an issue involving ancillary reagents that would impact the performance of the assay.
- Any other issues that must be addressed in order to assure safe and effective use of your test in combination with named ancillary reagents, in accordance with your device’s instructions for use.
In addition, you should provide testing data to establish that the quality controls you supply or recommend are adequate to detect performance or stability problems with the ancillary reagents.
If you have questions regarding identification, use, or control of ancillary reagents, you should contact FDA for advice.
5.D Testing Procedures Using Your Device
In your 510(k), you should provide a detailed description of the principles of operation for your device. You should specifically describe testing conditions, procedures and controls designed to safeguard against conditions that can cause false positive and false negative results. These include, but are not limited to:
- Description of, or recommendations for, any external controls and internal controls (e.g., a sample negative control that monitors contamination or an internal control that monitors extraction efficiency and amplification inhibition).
- Overall design of the testing procedure, including control elements incorporated into the recommended testing procedures.
- Features and additional controls that monitor procedural errors or factors (e.g., degradation of master mix) that adversely affect amplification and detection.
We recommend that you include a description for all additional procedures, methods, and practices incorporated into your directions for use (See Section 7 - Labeling) that mitigate risks associated with testing for hMPV RNA.
5.E Interpreting Test Results/Reporting
In your 510(k), you should describe how positive, negative, equivocal (if applicable), or invalid results are determined and how they should be interpreted. In your 510(k) submission, you should indicate the cut-off values for all outputs of the assay.
- Specifically, you should provide the cut-off value for defining a negative result of the assay. If the assay has only two output results (negative/positive), this cut-off also defines a positive result of the assay.
- If the assay has an equivocal zone, you should provide cut-off values (limits) for the equivocal zone.
- If your interpretation of the initial equivocal results requires re-testing, you should provide (1) a recommendation whether re-testing should be repeated from the same nucleic acid preparation, a new extraction, or a new patient specimen, and (2) an algorithm for defining a final result by combining the initial equivocal result and the results after re-testing (note that this algorithm should be developed before the pivotal clinical study that evaluates the clinical performance of the assay).
- If one of the reported outputs of your assay can be an equivocal result, you should provide the interpretation and recommendation for how the user should follow up the equivocal results.
- If the assay has an invalid result, you should describe how an invalid result is defined. If internal controls are part of the determination of invalid results, you should provide the interpretation of each possible combination of control results for defining the invalid result. You should provide recommendations for how to follow up any invalid result, i.e., whether the result should be reported as invalid or whether re-testing is recommended. If re-testing is recommended, you should provide information similar to that for re-testing of equivocal results (i.e., whether re-testing should be repeated from the same nucleic acid preparation, a new extraction, or a new patient specimen).
In addition, you should describe how you monitor results over time to identify changes in performance due to biological changes within the virus genetic lineage, or changes in performance when prevalence changes from the existing prevalence at the time your product is evaluated.
6.A General Study Recommendations
In your 510(k) you should provide detailed descriptive information on the studies you conducted to establish each of the performance characteristics outlined below. We recommend that you conduct prospective clinical studies to determine performance of your device. In general, for the clinical studies and the analytical precision studies, we recommend that you conduct testing at 3 sites, representative of where you intend to market the device (e.g., clinical laboratory sites).
You should evaluate your assay performance for each specimen type that you recommend for your assay.
In order to accurately interpret acceptance criteria and data summaries during the FDA review, we recommend that you provide appropriate specific information concerning protocols. This information is also important to aid users in understanding information in your labeling. For example, when referring to CLSI (Clinical and Laboratory Standards Institute) protocols or guidelines, we recommend that you indicate which specific aspects of the protocols or guidelines were followed.
We recommend that you contact the Division of Microbiology Devices in the Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD) to obtain feedback regarding your planned study and the clinical claims you intend to support. Sponsors may contact OIVD to obtain feedback concerning study plans.
When conducting the performance studies described below, we recommend that you run appropriate external controls every day of testing for the duration of the analytical and clinical studies. You may contact OIVD’s Division of Microbiology Devices at FDA for further information regarding controls. For devices based on nucleic acid technology, we generally recommend that you include the following types of controls:
6.B.i Negative Controls
Blank or no template control
The blank, or no-template control, contains buffer or sample transport media and all of the assay components except nucleic acid. This control is used to rule out contamination with target nucleic acid or increased background in the amplification reaction. It may not be needed for assays performed in single test disposable cartridges or tubes.
Negative sample control
The negative sample control contains non-target nucleic acid or, if used to evaluate extraction procedures, it contains the whole virus (other than hMPV). It reveals non-specific priming or detection and indicates that signals are not obtained in the absence of target sequences. Examples of acceptable negative sample control materials include:
- Patient specimen from a non-hMPV infected individual
- Samples containing a non-target organism (e.g., cell line infected with non-hMPV virus)
- Surrogate negative control, e.g., alien encapsidated RNA8
6.B.ii Positive Controls
Positive control for complete assay
The positive control contains target nucleic acids, and is used to control the entire assay process including RNA extraction, amplification, and detection. It is designed to mimic a patient specimen and is run as a separate assay, concurrently with patient specimens, at a frequency determined by a laboratory’s Quality System (QS). Examples of acceptable positive assay control materials include:
- Patient specimen from an hMPV infected individual
- Cell culture infected with an hMPV strain
- Packaged hMPV target RNA
Positive control for amplification/detection
The positive control for amplification/detection contains purified target nucleic acid at or near the limit of detection for a qualitative assay. It controls the integrity of the sample and the reaction components when negative results are obtained. It indicates that the target is detected if it is present in the sample.
6.B.iii Internal Control
The internal control is a non-target nucleic acid sequence that is co-extracted and co-amplified with the target nucleic acid. It controls for integrity of the reagents (polymerase, primers, etc.), equipment function (thermal cycler), and the presence of amplification inhibitors in the samples. Examples of acceptable internal control materials include human nucleic acid co-extracted with the hMPV RNA and primers amplifying human housekeeping genes (e.g., RNaseP, β-actin). The need for this control is determined on a device case-by-case basis9.
6.C Specimen Collection and Handling
You should specify the specimen type(s) your assay is intended to measure. A specimen has to be collected from the appropriate anatomical site or source at the time in the clinical progression of the disease state during which hMPV will be present at that site. Appropriate specimen types for hMPV nucleic acid tests are respiratory tract specimens (e.g., nasopharyngeal swab (NPS), nasal aspirate (NA), nasal swab, and nasal wash).
The quality and quantity of extracted target can be highly dependent on multiple factors such as specimen source, collection method, handling (e.g., transport and storage times and temperatures). Testing results you provide in your 510(k) should validate that (1) your system provides adequate nucleic acid for hMPV, and (2) the device maintains acceptable performance (e.g., accuracy, reproducibility) under all the various conditions you recommend in your labeling. For example, you should assess the effect of recommended storage times and temperatures on sample stability and recovery using an analysis of specimen aliquots stored and/or transported under your recommended conditions of time and temperature. You should state your acceptance criteria for all specimen stability parameters.
Specimens for pathogen identification should be collected and handled using all applicable state and federal bio-safety guidelines. For standard precautions for handling of specimens, refer to the most current editions of the related Clinical and Laboratory Standards Institute (CLSI) documents.10
Sensitivity for detection of hMPV may differ for frozen specimens when compared to fresh specimens. In developing your test, considerations should include adequate assessment as to whether this is a concern for hMPV RNA and your device performance. You should also assess the effect of repeated freeze/thaw cycles on the yield of the hMPV nucleic acid and its influence on the assay performance.
6.D Nucleic Acid Extraction
Different extraction methods may yield hMPV nucleic acids of varying quantity and quality, and therefore the extraction method can be crucial to a successful result. Purification of hMPV nucleic acids from respiratory specimens can be challenging because biological samples may contain low viral titers in the background of human genomic DNA, as well as high levels of proteins and other contaminants.
For these reasons, you should evaluate the effect of your chosen extraction methods on the performance of the assay with respect to satisfactory hMPV nucleic acid quantity and quality for the intended use of the assay. In addition, you should evaluate your assay’s analytical and clinical performance characteristics using the entire analytical process (including extraction procedures) that you recommend for use with your assay. This should include demonstrating the Limit of Detection (LoD) and reproducibility of your assay with each extraction procedure. Recommendations for conducting the LoD study are provided under “Limit of Detection” (See Section 6.F.i – Analytical Sensitivity). In addition, external site studies (including reproducibility and clinical studies) should include the extraction procedures prescribed in your labeling.
You should perform these evaluations whether you intend to actually provide reagents in your test kit for extraction and preparation of nucleic acid, or whether you simply instruct users concerning appropriate reagents.
If you recommend or include multiple extraction methods, you should demonstrate the LoD and reproducibility for each method. With the assumption that the extraction method introduces minimum variability to the overall assay performance you may be able to combine the extraction method variable with each site performance variable. For example if you recommend three different extraction methods, you can design a reproducibility study by evaluating one of the three extraction methods at each testing site: test extraction method A at site 1, method B at site 2, and method C at site 3. If the results generated from the test panel mentioned above do not show significant differences, no further reproducibility studies are needed. However, if the initial extraction equivalency studies from the three sites indicate statistically significant differences in assay performance, the reproducibility study should be expanded to include testing each extraction method at three study sites (e.g., site 1 extraction method A, B, and C, site 2 extraction method A, B, and C, and site 3 extraction method A, B and C).
In addition to the analytical studies (LoD and Reproducibility), each extraction method should be utilized in at least one clinical site during the clinical trials to generate clinical performance data. If results from the expanded reproducibility testing indicate a significant difference in efficiency among the extraction methods, the data from each clinical testing site (using a different nucleic acid extraction method) are not considered equivalent and should not be pooled, but rather analyzed separately. As a result, additional prospective clinical samples may be called for in order to support the claimed extraction method.
6.E Assay Cut-off
You should explain how the cut-off(s) was determined (see also Section 5.E - Interpreting Test Results/Reporting) as well as how it was validated. The cut-off should be determined using appropriate statistical methods. To support the cutoff you determined you may provide, for example, a result distribution, 95th and 99th percentiles, percents of the non-negative (positive or equivocal) results, and so on, for the clinical samples without any hMPV RNA in your pilot studies. Selection of the appropriate cut-off can be justified by the relevant levels of sensitivity and specificity based on Receiver Operating Curve (ROC) analysis of the pilot studies with clinical samples (for details about ROC analysis, see CLSI document GP10-A Assessment of the Clinical Accuracy of Laboratory Tests Using Receiver Operating Characteristics (ROC) Plots; Approved Guideline: Clinical and Laboratory Standards Institute; Wayne, PA, 1995). If the assay has an equivocal zone, you should explain how you determined the limits of the equivocal zone. The performance of your device using the pre-determined cut-off (and equivocal zone, if applicable) should be validated in an independent population consistent with the defined intended use of your device.
6.F Performance Studies
We recommend that you establish the following performance characteristics for your hMPV RNA assay in your 510(k):
6.F.i Analytical Sensitivity
Limit of Detection
We recommend that you determine the limit of detection (LoD) using limited dilutions of regrown and retitered hMPV viral stocks. The study should include serial dilutions of a representative of each of the four hMPV genetic lineages (A1, A2, B1, B2), and 3-5 replicates for each dilution in pooled hMPV negative human respiratory sample or an equivalent matrix. You should report the LoD as the level of virus that gives a 95% detection rate. The LoD may be confirmed by preparing at least 20 additional replicates at the LoD concentration and demonstrating that the virus was detected 95% of the time. The reference methods we recommend for LoD determination are the tissue culture infectious dose50 (TCID50) assay. We recommend that you determine the LoD for each specimen type tested with the device. We suggest that you refer to Clinical Laboratory Standards Institute (CLSI) document EP17-A11, when designing your studies.
Analytical Reactivity (Inclusivity)
We recommend that you demonstrate that the test can detect at least one additional hMPV virus strain representing each of the four genetic lineages of hMPV at viral levels at or near the LoD. The dilutions should be made in pooled hMPV negative human respiratory sample or an equivalent matrix. All virus identities and titers should be confirmed.
6.F.ii Analytical Specificity
We recommend that you test for potential cross-reactivity with non-hMPV respiratory pathogens and other microorganisms with which the majority of the population may have been infected, e.g., EBV and CMV. We recommend that you test medically relevant levels of viruses and bacteria (usually 106 cfu/ml or higher for bacteria and 105 pfu/ml or higher for viruses). We recommend that you confirm the virus and bacteria identities and titers. The microorganisms recommended for cross-reactivity studies are listed in Table 1.
Table 1. Microorganisms recommended for cross-reactivity studies.
|Epstein Barr Virus|
|Human parainfluenza||Type 1|
|Human parainfluenza||Type 2|
|Human parainfluenza||Type 3|
|Respiratory syncytial virus||A|
|Respiratory syncytial virus||B|
|Mycobacterium tuberculosis avirulent|
|Staphylococcus aureus||Protein A producer|
* We recommend that you include the OC43 and 229E strains of Human coronavirus in your cross-reactivity study.
We recommend that you conduct a comprehensive interference study using medically relevant concentrations of the interferent and at least one strain for each of the hMPV main genetic lineages (A and B) to assess the potentially inhibitory effects of substances normally encountered in respiratory specimens.
Potentially interfering substances include, but are not limited to, the following: blood, nasal secretions or mucus, and nasal and throat medications used to relieve congestion, nasal dryness, irritation, or asthma and allergy symptoms. Examples of potentially interfering substances are presented in Table 2. We recommend that you test interference at the assay cut-off determined for each of the hMPV main genetic lineages (A and B), and for each of the interfering substances. We also recommend that you evaluate each interfering substance at its potentially highest concentration (“the worst case”). If no significant clinical effect is observed, no further testing is necessary. Please refer to the CLSI document EP7-A212 for additional information.
Table 2. Substances Recommended for Interference Studies
bovine submaxillary gland, type I-S
|Purified mucin protein|
|Nasal sprays or drops||Phenylephrine Oxymetazoline Sodium chloride with preservatives|
|Nasal corticosteroids||Beclomethasone dipropionate, flunisolide, triamcinolone acetonide, budesonide, mometasone furoate, f luticasone Fluticasone propionate|
|Nasal gel||Luffa opperculata, Sulfur|
|Homeopathic allergy relief medicine||Galphimia glauca Histaminum hydrochloricum|
|FluMist©||Live, intranasal influenza virus vaccine|
|Throat lozenges, oral anesthetic and analgesic||Benzocaine, Menthol|
|Antibiotic, nasal ointment||Mupirocin|
We recommend that you conduct within-laboratory precision studies for devices that include instruments or automated components. You may perform these studies in-house, i.e., within your own company.
We recommend that you test sources of variability (such as operators, days, assay runs) for a minimum of 12 days (not necessarily consecutive), with 2 runs per day, and 2 replicates of each sample per run. These test days should span at least two calibration cycles, if the calibration cycle is shorter than 2 months. The test panel should consist of 3-6 samples (1-2 viral strains) at three levels of viral load that include:
- A “high negative” sample (C5 concentration): a sample with an analyte concentration below the clinical cut-off such that results of repeated tests of this sample are negative approximately 95% of the time (and results are positive approximately 5% of the time).
- A “low positive” sample (C95 concentration): a sample with a concentration of analyte just above the clinical cut-off such that results of repeated tests of this sample are positive approximately 95% of the time.
- A “moderate positive” sample: a sample with a concentration at which one can anticipate positive results approximately 100% of the time (e.g., approximately two to three times the concentration of the clinical cut-off).
When the limit of blank (LoB) is used as a cutoff, then the concentration C95 is the same as the limit of detection (LoD) and the zero concentration (no analyte present in sample) is C5 if LoB is established with Type I error of 5%.13 CLSI documents EP5-A214 and EP12-A15 contain further information about designing and performing precision studies.
The protocol for the reproducibility study may vary slightly depending on the assay format. As a general guide, we recommend the following protocol:
- Evaluate the reproducibility of your test at 3 testing sites (two external sites and one in-house site).
- Use a five day testing protocol, including a minimum of two runs per day, (unless the assay design precludes multiple runs per day) and three replicates of each panel member per run.
- Each day have at least two operators at each facility perform the test.
- The sample panel should be the same as described in the repeatability study above.
The CLSI document, EP15-A216, contains additional information on reproducibility study design.
6.F.iv Carry-Over/Cross-contamination Studies (for multi-sample assays and devices that require instrumentation.)
We recommend that you demonstrate that carry-over and cross-contamination does not occur with your device (including the nucleic acid extraction method). In a carry-over study, high positive samples should be used in series alternating with high negative samples in patterns dependent on the operational function of the device. We recommend that at least five runs with alternating high positive and high negative samples should be performed during the carry-over study. The high positive sample in the carry-over study should be high enough to exceed 95% or more of the results obtained from specimens of diseased patients from the intended use population. The high negative samples should contain the analyte concentration below the cut-off such that repeat testing of this sample is negative approximately 95% of the time. The carry-over effect can then be estimated by the percent of negative results for the high negative sample in the carry-over study compared with 95%17.
6.G Clinical Studies
You should conduct prospective clinical studies to determine the performance of your device for all the specimen types you claim in your labeling. You should prospectively collect and test the specimens from individuals representing the intended use population, i.e., those with signs and symptoms consistent with acute respiratory tract infections. You should describe the protocol of each clinical study (including the inclusion and exclusion criteria). You should include a sufficient number of samples so that results will be statistically and clinically meaningful. Fresh samples are preferred. However, prospectively collected archived specimens18 may be acceptable if you can demonstrate that freezing or other preservation techniques do not alter the performance of the device in comparison to testing of fresh specimens, if appropriate archives are selected, and appropriate measures are taken to identify and remove or mitigate any biases in the study set. If you evaluate the assay using specimens that were archived after performing viral culture on fresh specimens, you should ensure that the specimens are not utilized selectively (i.e., you should still test all specimens in a prospective manner). Furthermore, samples should be masked to avoid testing bias. If both fresh and archived/frozen samples are tested, we recommend that you analyze the data of these two groups separately. We encourage you to contact the FDA to request a review of your proposed studies.
We recommend that you assess and compare the performance of your device to a predetermined algorithm that uses composite reference methods. The composite reference methods should include laboratory results such as:
- Detection of hMPV RNA from respiratory specimens by well-characterized and validated nucleic acid amplification tests (NAAT) followed by bi-directional sequencing analysis. The NAAT assays should utilize primers generating amplicons of different hMPV genomic regions than your device. Bi-directional sequencing should be performed on both strands of the amplicon and the generated sequence should be of an acceptable quality (quality score of 40 or higher as measured by PHRED or similar software packages) and should match the reference or consensus sequence19,20.
- Isolation of hMPV from respiratory specimens by well-characterized cell culture assay, followed by detection of hMPV from cell culture using well- characterized hMPV specific antibody or well-characterized and validated nucleic acid amplification assay followed by bi-directional sequencing analysis.
The analytical performances of the NAAT followed by sequencing analysis reference assays should be established by performing analytical sensitivity (LoD) and reactivity studies as described in the “Performance Characteristics” section of this special control guidance. (See Section 6.F.i- Analytical Sensitivity).
Literature and/or other validation data for establishing and describing the performances of the NAAT followed by sequencing analysis reference assays should be evaluated prior to clinical testing. You should submit to the FDA validation data from literature to justify your selection of primers for the NAAT followed by sequencing analysis reference assays. This information is important to demonstrate that the reference assays target conserved regions of the hMPV genome and react broadly in detecting hMPV strains. As a part of the pre-market application, primer sequences, target region of the genome, BLAST results, and sequence alignments, should also be submitted to the FDA for review. Appropriate controls, including a positive control that has a low genomic copy number of the virus, a negative control and an independent inhibition control measure, should be incorporated into each NAAT followed by sequencing analysis reference assays.
The analytical performance of an hMPV cell culture assay should also be established by performing analytical sensitivity (LoD) and reactivity studies as described in the “Performance Characteristics” section of this special control guidance. (See Section 6.F.i, above). It is essential that the specimens be rapidly transported to the laboratory for optimal virus recovery and detection. Culture should not be performed on frozen specimens. Literature and/or other validation data for establishing and describing the performance of the hMPV cell culture assay should be provided and evaluated prior to clinical testing. You should submit to the FDA validation data from literature to justify your selection of the cell line used, growth conditions and growth period after inoculation. This information is important to demonstrate that the particular cell culture assay can effectively and efficiently isolate various hMPV strains from clinical specimens. You should also provide to the FDA supporting information from scientific publications and/or laboratory data demonstrating how the antibody was validated for detecting hMPV in culture. The cell culture assay protocol, including controls utilized in the culture assay, should also be a part of the pre-market application. We recommend that you include a positive control that has a low titer of the virus, and a negative control in the cell culture assay.
You may contact the FDA for further information regarding establishing a predetermined algorithm that uses composite reference methods.
6.G.i Study Protocol
We recommend that you develop a detailed study protocol that includes patient inclusion and exclusion criteria, type and number of specimens needed, directions for use, and a statistical analysis plan that accounts for variances to prevent data bias. We recommend that you include this, and any other relevant protocol information, in your premarket submission.
We encourage sponsors to contact the FDA to request a review of their proposed studies and selection of specimen types. This is referred to as the pre-IDE process. We particularly encourage manufacturers to seek this type of discussion when samples are difficult to obtain or sponsors are planning to submit a premarket notification for the first time.
6.G.ii Specimen Type(s)
The total number of samples you should include in your study for substantiating a claim for detection of hMPV from respiratory specimens will depend on the prevalence of the virus and on assay performance.
For devices detecting hMPV from respiratory specimens, we recommend that you include a sufficient number of prospectively collected fresh samples for each specimen type you claim to generate a result with at least 90% sensitivity with a lower bound of the two-sided 95% CI greater than 80%. All hMPV detecting devices should demonstrate specificity with a lower bound of the two-sided 95% CI greater than 90%.
If you have questions regarding the choice of appropriate specimen type(s) and numbers, please contact the FDA.
6.G.iii Study Sites
We recommend that you conduct your studies at a minimum of three different geographical sites representing the testing environment where the device will ultimately be used (e.g., clinical laboratory), by individuals who will use the test in clinical practice. At least one of the study sites should be a US site. One of the study sites may be in-house.
6.G.iv Study Population
We recommend that you conduct your studies on individuals presenting with signs and symptoms of acute respiratory tract infections. We recommend that you include samples from each age group in your clinical studies and present the data stratified by age (e.g., less than 5, 6- 21, 22-59, and greater than 60 years old) in addition to the overall data summary table.
6.G.v Presentation of Clinical Study Results
In your 510(k) you should describe how the samples were selected, and any reasons that samples were excluded.
We recommend that you initially analyze and present data from each study site separately to evaluate any inter-site variation and include results of the analysis in the 510(k). It may be possible to pool clinical study results from the individual sites in the package insert if you can demonstrate that there are no significant statistical or clinical differences in the results or populations among sites.
We also recommend that you analyze and present data separately for fresh prospective clinical samples evaluated against “Laboratory Diagnosis Truth” (determined by a predetermined algorithm that uses composite reference methods) and prospectively collected archived specimens (frozen) evaluated against “Laboratory Diagnosis Truth.”
We recommend that you provide line data, including appropriate daily external control testing data during all clinical studies. You may supply this information electronically using Microsoft EXCEL, delimited text files, or SAS files
6.G.vi Prevalence (Expected Values)
You should establish the prevalence of hMPV in relevant patient populations from your clinical study sites, showing signs and symptoms consistent with acute respiratory tract infection. You should assay a statistically determined number of specimens that are representative of the intended use, including the specified sample matrix(ces). You should provide these results based on your new device performance, and summarize the distribution of the population according to age groups (infants <1, children="" 1-5,="" 6-10,="" 11-15,="" 16-21,="" and="" adults=""> 18 years) and gender.
Human metapneumovirus (hMPV) RNA assay system, like other devices, are subject to statutory requirements for labeling (sections 502(a), 201(n) of the Act; 21 USC § 352(a), 321(n)). These IVD devices must provide adequate directions for use and adequate warnings and precautions. (Section 502(f); 21 USC § 352(f).) Specific labeling requirements for all IVD devices are set forth in 21 CFR 809.10. See also 21 CFR § 801.119 (IVDs labeled in accordance with 21 CFR 809.10 are deemed to satisfy section 502(f)(1).)
Although final labeling is not required for 510(k) clearance, final labeling for in vitro diagnostic devices must comply with the requirements of 21 CFR 809.10 before an in vitro diagnostic device is introduced into interstate commerce.
To ensure compliance with section 502 of the Act and 21 CFR 809.10, FDA recommends that labeling for these devices ( a respiratory viral panel multiplex nucleic acid assay that detects and identifies hMPV nucleic acid) address the items identified below. These labeling recommendations also help to mitigate the risks identified previously in this guidance to ensure safe and effective use of these devices.
Your labeling should clearly describe the identity, phylogenetic relationship, or other recognized characterization of hMPV that your device is designed to detect, and the associated clinical aspects of human infection.
In addition to specific elements that describe the analyte detected, your intended use i should specify indications for testing respiratory specimens from patients with signs and symptoms of acute respiratory tract infections. The intended use should specify that the device should be used in conjunction with other laboratory testing and clinical observations.
FDA also recommends that your statement of intended use be clarified by statements such as:
The detection and identification of hMPV RNA from individuals exhibiting signs and symptoms of acute respiratory infection aids in the diagnosis of respiratory hMPV infection if used in conjunction with other clinical and laboratory findings.
Negative results do not preclude hMPV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.
Positive results do not rule out bacterial infection, or co-infection with other viruses.
The hMPV RNA detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial culture, immunofluorescence, x-ray findings, etc) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of hMPV infection.”
In the device description, you should briefly describe the test methodology used in this type of device.
This section should include a general description of the entire analysis procedure, from physician sampling to result reporting.
Directions for use
You should provide clear and concise instructions that delineate the procedures for using the device, and types of controls that will minimize risks of inaccurate results. Instructions should encourage use of additional control measures and testing of control materials to ensure use in a safe and effective manner.
If your reagents will not include reagents for extraction and preparation, you should recommend and list the nucleic acid extraction method that demonstrated safety and effectiveness with your device. You should provide the name and catalogue number of each method you recommend.
For test systems that call for ancillary reagents of concern (see Section 5.C) you should:
- Emphasize through conspicuous labeling that proper product performance requires use of specific ancillary reagents as directed. This labeling may include warnings against use of the test system if specified ancillary reagents are not available.
- Assure that users can clearly identify which ancillary reagents are suitable for use with your test. For example, if only certain lots of a named ancillary reagent are appropriate for use, the labeling for your test system should identify those lots by number. (See 21 CFR 809.10(b)(8)(ii)).
When your labeling calls for ancillary reagents that are supplied with instructions for use or other warnings or limitations by the ancillary reagent manufacturer, your should ensure that users of your test system will understand which instructions they should follow when using those ancillary reagents in your test system. If there is a conflict between the directions and warnings provided by the manufacturer of the ancillary reagent and the instructions for use that you supply with your test system, you should assess and address the risk that users may mistakenly follow the labeling provided directly with the ancillary reagent and consequently obtain invalid results with your test system. We note that in some circumstances, statements in the labeling of the test system may not be sufficient to address the risks created by this conflict.
Quality control recommendations should include types of procedures and materials that can be used as additional quality control measures, and the expected results for acceptability of control testing.
If controls are included with your device, the specifications for control materials, including level of virus, source of that virus, method of inactivation and method for determining non-infectiousness should be provided, if applicable.
Precautions, Warnings, and Limitations
In addition to any other limitations and warnings that are relevant to your specific assay, we recommend providing statements such as the following under Limitations, as applicable:
- A trained health care professional should interpret assay results in conjunction with the patient’s medical history, clinical signs and symptoms, and the results of other diagnostic tests.
- Analyte targets (hMPV sequences) may be present persistently in vivo, independent of virus viability. Detection of hMPV RNA does not imply that the hMPV is infectious, nor is the causative agent for clinical symptoms.
- The detection of hMPV RNA sequences is dependent upon proper specimen collection, handling, transportation, storage and preparation (including extraction). Failure to observe proper procedures in any one of these steps can lead to incorrect results. There is a risk of false negative values resulting from improperly collected, transported, or handled specimens.
- There is a risk of false positive values resulting from cross-contamination by target organism, its nucleic acids or amplified product, or from non-specific signals in the assay.
- There is a risk of false negative values due to the presence of hMPV sequence variants in the assay’s viral target.
- Assay performance was not established in immunocompromised patients.
- Positive and negative predictive values are highly dependent on prevalence. The assay performance was established during the e.g., 2006/2007 season. The performance may vary depending on the prevalence and population tested.
If positive or negative interference has been reported for any commonly used collection materials or substances that may be endogenously or exogenously introduced into a specimen prior to testing, you should advise users of the possibility of false negative or false positive results due to such interference.
We recommend that you state that inadequate or inappropriate specimen collection, storage, and transport are likely to yield false negative test results. We also recommend that you state that training in specimen collection is highly recommended because of the importance of specimen quality.
We recommend that you incorporate into the Results sectiondirections for reporting results that include statements such as the following, as applicable:
Report negative test results as “hMPV RNA not detected. This result does not exclude hMPV infection.”
Report positive test results as “hMPV RNA detected. This result does not rule out co-infections with other pathogens.”
Interpretation of Results
Your interpretation of the results section in the package insert should list all possible assay outputs and determinations of the presence or absence of hMPV RNA and assay control.
If internal controls are part of the determination of valid positive and negative results, you should provide the interpretation of each possible control result and a recommendation for how to follow up with any invalid or indeterminate result.
If your assay has an equivocal zone, you should provide the interpretation and the recommendation for how to follow up with the equivocal result. (e.g., whether the equivocal result should be reported as such, or whether testing should be repeated).
If your interpretation of the results requires repeat testing of an invalid or equivocal result, you should provide the recommendation whether testing should be repeated from the same nucleic acid preparation, a new extraction, or a new patient specimen.
If your assay performance (i.e., sensitivity) demonstrated a lower bound of the two-sided 95% CI less than 90%, negative results may need to be interpreted as presumptive prompting a recommendation for confirmation by an alternate method (e.g., cell culture).
This section should include the expected values using your test and the explanation of the result. It should also include the number of samples, age, gender, and demographics of the population used to determine the expected values.
You should include in the package insert a summary of the study designs and the results that would aid users in interpreting test results. This includes clinical and analytical performance characteristics. Clinical performance characteristics typically comprise prospective clinical study results summarizing performance (sensitivity, specificity, 95% confidence intervals) by your assay. In cases where some retrospective clinical samples were also used, these results should be presented separately from the prospective clinical study results, as positive and negative agreement. Analytical performance characteristics contain descriptions of the results and methodology used for the studies outlined in Section 6.
We recommend that the Performance Characteristics section describe the population(s) (i.e., geographical location, specimen types, and age groups) used to establish the performance characteristics of the device and provide the season.
1 In this guidance, multiplex assays are defined as those assays in which two or more targets are assayed through a common process of sample preparation, amplification and/or detection, and interpretation.
5 Clinical and Laboratory Standards Institute. 2006. Viral Culture; Approved Guidelines. CLSI document M41-A [ISBN 1-56238-623-9] Clinical and Laboratory Standards Institute, Wayne PA.
6 Clinical and Laboratory Standards Institute. 2004. Nucleic Acid Sequencing Methods in diagnostic Laboratory Medicine; Approved Guideline. CLSI document MM9-A [ISBN 1-56238-558-5] Clinical and Laboratory Standards Institute, Wayne PA.
7 Even if you establish that one or more alternative ancillary reagents may be used in your assay, each of those named alternatives may still be an ancillary reagent of concern. If you are unsure whether this aspect of the special controls applies to your device, we recommend you consult with the FDA.
8 Pasloske BL, Walkerpeach CR, Obermoeller RD, Winkler M, and DuBois DB. Armored RNA Technology for Production of Ribonuclease-Resistant Viral RNA Controls and Standards. J. Clin. Microbiol. 1998; 36:3590-3594.
9 Clinical and Laboratory Standards Institute. 2006. Molecular Diagnostic Methods for Infectious Disease; Approved Guideline. CLSI document MM3-A2 [ISBN 1-56238-596-8] Clinical and Laboratory Standards Institute, Wayne PA.
10 Clinical and Laboratory Standards Institute. 1997. Protection of Laboratory Workers from Infectious Disease Transmitted by Blood, Body Fluids, and Tissue. CLSI document M29-A. Clinical and Laboratory Standards Institute, Wayne PA.
11 Clinical and Laboratory Standards Institute. 2004. Protocol for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline. CLSI document EP17-A [ISBN 1-56238-551-8] Clinical and Laboratory Standards Institute, Wayne PA
12 Clinical and Laboratory Standards Institute. 2005. Interference Testing in Clinical Chemistry; Approved Guideline. CLSI document EP7-A2 [ISBN 1-56238-584-4]
13 Type I error is the probability of having truly negative samples (those with zero concentration) give values that indicate presence of analyte. Usually, Type I error is set at 5% or less.
14 Clinical and Laboratory Standards Institute. 2004. Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline. EP5-A2 [ISBN 1-56238-542-9] Clinical and Laboratory Standards Institute, Wayne PA.
15 Clinical and Laboratory Standards Institute. 2002. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline. EP12-A. [ISBN 1-56238-468-6] Clinical and Laboratory Standards Institute, Wayne PA.
16 Clinical and Laboratory Standards Institute. 2008. User Verification of Performance for Precision and Trueness; Approved Guideline-Second Edition. EP15-A2. [ISBN 1-56238-574-7] Clinical and Laboratory Standards Institute, Wayne PA.
17 Haeckel R. Proposals for the Description and Measurement of Carry-over Effects in Clinical Chemistry. Pure Appl. Chem. 1991; 63:302-306.
18 In this guidance, we define prospectively collected archived specimens as specimens collected sequentially from all patients meeting study inclusion criteria and representing assay intended use population (i.e., not pre-selected specimens with known results) coming in to a clinical testing facility between two pre-determined dates (e.g., from the beginning to the end of one flu season), so there is no bias and prevalence is preserved. These specimens should be appropriately stored (e.g., frozen at -70oC) and, as noted in the text, the sponsor should show that there is no change in device performance due to banking/freezing/storage of the specimens.
19 Clinical and Laboratory Standards Institute. 2004. Nucleic Acid Sequencing Methods in diagnostic Laboratory Medicine; Approved Guideline. CLSI document MM9-A [ISBN 1-56238-558-5] Clinical and Laboratory Standards Institute, Wayne PA.
20 Clinical and Laboratory Standards Institute. 2006. Molecular Diagnostic Methods for Infectious Disease; Approved Guideline. CLSI document MM3-A2 [ISBN 1-56238-596-8] Clinical and Laboratory Standards Institute, Wayne PA.