Guidance for Industry  
and FDA Staff 
 
Establishing the Performance 
Characteristics of In Vitro 
Diagnostic Devices for the Detection 
or Detection and Differentiation of 
Influenza Viruses 
 
    
Document issued on: July 15, 2011 
 
 
The draft of this document was issued on February 15, 2008
 
 
 
 
 
 
For questions regarding this document contact Tamara Feldblyum at 301-796-6195 or by 
email at tamara.feldblyum@fda.hhs.gov. 
 
        U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Devices and Radiological Health 
Office of In Vitro Diagnostic Device Evaluation and Safety 
Division of Microbiology Devices        Contains Nonbinding Recommendations 
 
 
Preface 
 
Public Comment  
You may submit written comments and suggestions at any time for Agency consideration to the 
Division of Dockets Management, Food and Drug Administration, 5630 Fishers Lane, rm. 
1061, (HFA-305), Rockville, MD, 20852.  Submit electronic comments to 
http://www.regulations.gov.  Identify all comments with the docket number FDA-2008-D-
0095.  Comments may not be acted upon by the Agency until the document is next revised or 
updated. 
 
Additional Copies 
Additional copies are available from the Internet at: 
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/
ucm070274.htm.  You may also send an e-mail request to dsmica@fda.hhs.gov to receive 
an electronic copy of the guidance or send a fax request to 301-847-8149 to receive a hard 
 
copy.  Please use the document number 1638 to identify the guidance you are requesting.  
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Table of Contents 
 
 
1.  INTRODUCTION.................................................................................................5 
2.  BACKGROUND...................................................................................................6 
3.  SCOPE...................................................................................................................7 
4.  RISKS TO HEALTH............................................................................................9 
5.  DEVICE DESCRIPTION..................................................................................10 
5.A   INTENDED USE..........................................................................................10 
5.B   TEST METHODOLOGY..............................................................................10 
5.C   INSTRUMENTS - HARDWARE AND SOFTWARE.........................................11 
5.D   ANCILLARY REAGENTS............................................................................12 
6.  LIMITATIONS...................................................................................................13 
7.  CONTROLS........................................................................................................14 
8.  INTERPRETING AND REPORTING TEST RESULTS..............................15 
9.  ESTABLISHING PERFORMANCE CHARACTERISTICS........................15 
9.A   ANALYTICAL PERFORMANCE..................................................................15 
9.A.i  Analytical Sensitivity..................................................................................15 
9.A.ii  Analytical Specificity..................................................................................17 
9.A.iii Cut-off and Equivocal Zone.......................................................................20 
9.A.iv Precision.......................................................................................................21 
9.A.v  Carry-Over and Cross-contamination Studies (for multi-sample assays 
and devices that require instrumentation.)..........................................................24 
9.A.vi Specimen Storage and Shipping Conditions............................................24 
9.B   CLINICAL PERFORMANCE STUDIES.........................................................24 
9.B.i  Study Protocol.............................................................................................25 
9.B.ii  Study Population.........................................................................................26 
9.B.iii Specimens.....................................................................................................26 
9.B.iv  Fresh vs. Frozen Samples...........................................................................28 
9.B.v  Study Sites....................................................................................................28 
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9.B.vi  Reference Methods......................................................................................29 
9.C   POST-MARKET PERFORMANCE VALIDATION..........................................30 
9.D   CLIA WAIVER..........................................................................................31 
9.E   NUCLEIC ACID-BASED INFLUENZA DEVICES...........................................31 
9.E.i  Nucleic Acid Extraction..............................................................................31 
9.E.ii  Controls for Nucleic Acid-based Influenza Assays..................................32 
10.  REFERENCES....................................................................................................33 
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 Guidance for Industry and FDA Staff 
 
Establishing the Performance 
Characteristics of In Vitro Diagnostic 
Devices for the Detection or Detection and 
Differentiation of Influenza Viruses 
 
This guidance represents the Food and Drug Administration's (FDA's) current 
thinking on this topic.  It does not create or confer any rights for or on any person and 
does not operate to bind FDA or the public.  You can use an alternative approach if the 
approach satisfies the requirements of the applicable statutes and regulations.  If you 
want to discuss an alternative approach, 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.  
 
 
1.   Introduction 
 
FDA is issuing this guidance to provide industry and agency staff with recommendations 
for studies to establish the analytical and clinical performance of in vitro diagnostic 
devices (IVDs) intended for the detection, or detection and differentiation, of influenza 
viruses.  These devices are used to aid in the diagnosis of influenza infection.  They 
include devices that detect one specific type or subtype, as well as devices that detect 
more than one type or subtype of influenza virus and further differentiate among them.1  
 
This guidance provides detailed information on the types of data FDA recommends 
submitting in support of Class I and Class II premarket submissions for these devices.  
The guidance includes a list of influenza virus strains recommended for analytical 
sensitivity studies, a list of microorganisms recommended for analytical specificity 
studies, and an example of a suggested format for presenting data from cross-reactivity 
studies.  
 
The scope of this document is limited to types of data intended to establish the 
performance characteristics of devices that detect either influenza viral antigen(s) or 
                                                 
1 There are three types of influenza viruses: A, B, and C. Influenza A viruses are further 
classified by subtype on the basis of the two main surface glycoproteins, hemagglutinin 
(HA) and neuraminidase (NA). Influenza A subtypes and B viruses are further classified 
by strains (http://www.cdc.gov/flu/avian/gen-info/flu-viruses.htm).   
 
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influenza viral gene segment(s).  It includes devices detecting influenza virus protein or 
nucleic acid targets, either single unit test formats or multi-test formats.  It does not 
address performance for assays detecting serological response of the host to the viral 
antigen, nor does it address establishing performance of non-influenza components of 
multi-analyte or multiplex devices.  
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidance documents describe the Agencys current thinking on 
a topic and should be viewed only as recommendations, unless specific regulatory or 
statutory requirements are cited.  The use of the word should in Agency guidance 
documents means that something is suggested or recommended, but not required. 
 
2.  Background  
This document recommends studies for establishing the performance characteristics of in 
vitro diagnostic devices for the detection, or detection and differentiation, of influenza 
viruses directly from human specimens or from culture isolates.  FDA believes that these 
recommended studies will be relevant for premarket submissions (e.g., 510(k)) that may 
be required for a particular device. 
A manufacturer who intends to market an in vitro diagnostic device for detection, or 
detection and differentiation, of influenza viruses must conform to the general controls of 
the Federal Food, Drug, and Cosmetic Act (the Act) and, unless exempt, obtain 
premarket clearance or approval prior to marketing the device (sections 510(k), 513, 515 
of the Act; 21 U.S.C. 360(k), 360c, 360e)).   
 
This document is intended to supplement 21 CFR 807.87 (information required in a 
premarket notification) and other FDA resources such as Premarket Notification: 
510(k), 
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/HowtoMarketYour
Device/PremarketSubmissions/PremarketNotification510k/default.htm; and Guidance 
for Industry and FDA Staff: Format for Traditional and Abbreviated 510(k)s, 
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc
eDocuments/ucm084396.pdf.   
In addition, this document complements two FDA guidance documents that specifically 
address influenza IVDs: In Vitro Diagnostic Devices to Detect Influenza A Viruses: 
Labeling and Regulatory Path, 
(http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc
eDocuments/ucm071270.pdf), and Class II Special Controls Guidance Document: 
Reagents for Detection of Specific Novel Influenza A Viruses, 
(http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc
eDocuments/ucm071274.pdf).  
The guidance document entitled, In Vitro Diagnostic Devices to Detect Influenza A 
Viruses: Labeling and Regulatory Path, addresses recommendations for fulfilling 
labeling requirements applicable to all in vitro diagnostic devices intended to detect 
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influenza A (or A/B) virus directly from human specimens, with a particular emphasis on 
ensuring appropriate labeling for legally marketed influenza A (or A/B) test devices 
whose clearance is not based on data addressing performance with regard to novel 
influenza A viruses infecting humans (including H5N1).  It also discusses the FDA's 
thinking on premarket pathways for new or modified products intended to detect 
influenza A viruses, including a novel influenza A virus, or to detect and differentiate a 
specific influenza A virus.    
 
The guidance document entitled Class II Special Controls Guidance Document: 
Reagents for Detection of Specific Novel Influenza A Viruses, is one of two special 
controls for reagents for detection of specific novel influenza A viruses, classified into 
class II under 21 CFR 866.3332.  This special control guidance document includes 
recommendations for establishing device performance, as well as recommendations for 
labeling and postmarket measures.  Devices classified under 21 CFR 866.3332 are 
subject to an additional special control limiting distribution of these devices to 
laboratories with experienced personnel having training in standardized molecular testing 
procedures and expertise in viral diagnosis and appropriate biosafety equipment and 
containment.   
This guidance is intended to complement the two preceding guidance documents by 
describing the types of studies FDA recommends for establishing the analytical and 
clinical performance of in vitro diagnostic devices (IVDs) intended for the detection, or 
detection and differentiation, of influenza viruses.  FDA recommends that sponsors of 
influenza diagnostic devices use this guidance, in combination with the two existing 
guidances regarding influenza diagnostics, for information on FDAs current thinking 
about the regulation of these devices.    
 
3.  Scope 
 
As previously described, this document recommends studies for establishing the 
performance characteristics of in vitro diagnostic devices for the detection or detection 
and differentiation of influenza viruses, including those for the detection of novel 
influenza viruses in either human specimens or culture isolates.  This document is limited 
to studies intended to establish the performance characteristics of devices that either 
detect influenza viral antigens or influenza viral gene segments (protein or nucleic acid).    
This guidance references serological reagents but does not address detection of 
serological response from the host to the viral antigen, nor does it address establishing 
performance of non-influenza components of multi-analyte or multiplex devices.  
 
The scope of this document includes the devices described in existing classifications, as 
indicated below, and may also be applicable to future influenza diagnostic devices that 
may not fall within these existing classifications.  Those future devices may include 
devices that will be subject to requests for initial classification under section 513(f)(2) of 
the act ("de novo classification"), as well as subsequent devices that seek determinations 
of substantial equivalence to future de novo classified devices. 
 
The following are existing influenza IVD classification regulations: 
 
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21 CFR 866.3330 Influenza virus serological reagents: 
 
(a) Identification.  Influenza virus serological reagents are devices that consist of 
antigens and antisera used in serological tests to identify antibodies to influenza in 
serum.  The identification aids in the diagnosis of influenza (flu) and provides 
epidemiological information on influenza.  Influenza is an acute respiratory tract 
disease, which is often epidemic. 
 
(b) Classification. Class I (general controls).  The device is exempt from the 
premarket notification procedures in subpart E of part 807 of this chapter subject 
to the limitations in  866.9.  
 
Although devices within the classification described in 21 CFR 866.3330 are Class I 
devices, which are generally exempt from premarket notification, (21 U.S.C. 360(l)) 
under FDA regulations a premarket notification may be required for some tests purported 
to fall within this type of device. Specifically, an IVD for detection of influenza is not 
exempt from submission of a 510(k) to the extent that it meets the limitations on 
exemption defined in 21 CFR 866.9: 
 
  Under 21 CFR 866.9(c)(6), an IVD that is intended for use in identifying or 
inferring the identity of a microorganism directly from clinical material is not 
exempt from premarket notification requirements. An IVD that is intended to 
detect an influenza virus directly from a human specimen falls within this 
provision. 
 
  In addition, an IVD to detect influenza may trip the limitations in 21 CFR 
866.9(a) if the new device is intended for a use different from the intended use of 
a legally marketed device classified under 21 CFR 866.3330; or may trip the 
limitations in 21 CFR 866.9(b), if it operates using a different fundamental 
scientific technology from existing influenza tests in that classification. 
 
The following are the product codes for devices cleared under 21 CFR 866.3330: 
 
GNS    Antisera, HAI, Influenza virus A, B, C  
GNT    Antigens, HA (including HA control), Influenza virus A, B, C  
GNX    Antigens, CF, including CF control, Influenza virus A, B, C  
GNW   Antisera, CF, Influenza virus A, B, C 
NIA   Nucleic acid amplification, Influenza virus 
 
21 CFR 866.3332 Reagents for detection of specific novel influenza A viruses  
 
(a) Identification.  Reagents for detection of specific novel influenza A viruses are 
devices that are intended for use in a nucleic acid amplification test to directly 
detect specific virus RNA in human respiratory specimens or viral cultures.  
Detection of specific virus RNA aids in the diagnosis of influenza caused by 
specific novel influenza A viruses in patients with clinical risk of infection with 
these viruses, and also aids in the presumptive laboratory identification of specific 
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novel influenza A viruses to provide epidemiological information on influenza.  
These reagents include primers, probes, and specific influenza A virus controls. 
 
(b) Classification.  Class II (special controls).  The special controls are:  
 
(1) FDAs guidance document entitled Class II Special Controls Guidance 
Document: Reagents for Detection of Specific Novel Influenza A Viruses.  See  
866.1(e) for information on obtaining this document. 
(2) The distribution of these devices is limited to laboratories with experienced 
personnel who have training in standardized molecular testing procedures and 
expertise in viral diagnosis, and appropriate biosafety equipment and 
containment. 
 
The following are the product codes for devices cleared under 21 CFR 866.3332: 
 
NXD  Nucleic Acid Amplification, Novel influenza A virus, A/H5 (Asian 
lineage) RNA  
OMS  Novel influenza A virus, A/H5 NS1 protein 
 
4.  Risks to Health
 
Human influenza is a highly contagious acute respiratory tract disease.  There are three 
genera of human influenza viruses: A, B and C.  Infection with influenza A virus is the 
most severe, with several notable pandemics during the past century.  Influenza A viruses 
are classified into subtypes according to the antigenic composition of their hemagglutinin 
(HA) and neuraminidase (NA) glycoproteins on the viral envelope.  
 
Illness caused by commonly circulating influenza viruses can cause high morbidity and 
mortality, particularly in special populations such as the elderly and the very young.  The 
development of acquired immunity to seasonal influenza viruses is limited because 
influenza viruses mutate in small but important ways from year to year (a process known 
as antigenic drift).  More dramatic changes or major antigenic shifts may result in the 
emergence of a new subtype of influenza A virus, or novel virus that has never circulated 
or has not circulated in humans for several decades.  Novel influenza viruses have the 
potential to cause widespread disease and/or disease of unusually high severity because 
few, if any, people have prior exposure to these viruses.  This lack of immunity, as well 
as additional pathogenic factors that may also increase virulence, results in a greater 
likelihood of morbidity and mortality among those infected. 
In vitro diagnostic devices for the detection, or detection and differentiation, of influenza 
viruses are important for establishing the diagnosis of influenza, for differentiating 
seasonal from novel influenza virus strains, and for obtaining epidemiologic information 
on influenza outbreaks.  Public health officials have emphasized the need for reliable 
influenza diagnostic devices that can differentiate seasonal from emerging viral strains 
and provide rapid test results.   
Failure of devices for detection of influenza viruses to perform as expected, or failure to 
correctly interpret results, may lead to incorrect patient management decisions and 
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inappropriate public health responses.  In the context of individual patient management, a 
false negative report could lead to delays in providing (or failure to provide) definitive 
diagnosis and appropriate treatment and infection control and prevention measures.  A 
false positive report could lead to unnecessary or inappropriate treatment or unnecessary 
control and prevention actions.  Therefore, establishing the performance of these devices 
and understanding the risks that might be associated with the use of these devices is 
critical to their safe and effective use. 
The studies to establish the performance of influenza detection devices as described in 
this guidance document are recommended to support premarket submissions and FDAs 
finding of substantial equivalence for these devices.   
 
5.  Device Description  
 
You must identify a legally marketed predicate device in your 510(k).  21 CFR 
807.92(a)(3).  You should also identify the regulation and the product code for your 
device. We recommend including a table that outlines the similarities and differences 
between the predicate and the new device.  You should include the following descriptive 
information to adequately characterize the new device that is intended to detect or detect 
and differentiate influenza viruses. 
 
5.A  Intended Use 
The intended use statement should specify the influenza virus types and subtypes the 
device detects and identifies, the nature of the analyte (e.g., antigen or RNA), test 
platform, 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 statement should state whether the test is qualitative, whether analyte 
detection is presumptive, and any specific conditions of use.   
 
5.B  Test Methodology 
You should describe in detail the methodology used by your device.  For example, the 
following elements, as applicable to the device, should be included: 
 
  Description of the technology (e.g., immunoassay, RT-PCR, bead array) and 
whether the device is a manual test or run on an instrument.  
  Information and rationale for selection of specific targets and the methods used to 
design antibodies or primers and probes. 
  Specificity of capture and detection reagents for influenza antigens or nucleic acid 
sequences of interest.  
  Specimen types and collection methods (e.g., swabs, aspirates, viral culture 
media, etc.). 
  Assay components provided or recommended for use, and their function within 
the system (e.g., buffers, enzymes, fluorescent dyes, instrumentation and 
software). 
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  Internal controls and a description of their specific function in the system. 
  External controls that are recommend or provided to the users. 
  Types of output generated by the device and system parameters (e.g., 
measurement ranges, units, when applicable). 
  The computational path from raw data to the reported result (please see Section 
5.C below Instruments - Hardware and Software for details). 
  Illustrations or photographs of non-standard equipment or methods.  
 
Your 510(k) should include performance information supporting the conclusion that 
design control requirements for your device have been met as described in 21 CFR 
820.30.  
 
5.C  Instruments - Hardware and Software 
For instruments and systems that measure multiple signals, and for other complex 
laboratory instrumentation that has not been previously cleared, refer to the guidance 
document "Class II Special Controls Guidance Document: Instrumentation for Clinical 
Multiplex Test Systems,"[1] for details on the types of instrument-related data you should 
provide to support clearance. 
 
If your device includes software, you should submit software information in accordance 
with the level of concern described in the FDA guidance document Guidance for the 
Content of Premarket Submissions for Software Contained in Medical Devices [2].  You 
should determine the level of concern prior to the mitigation of hazards.  In vitro 
diagnostic devices of this type are typically considered a moderate level of concern; 
software flaws could indirectly affect the patient and potentially result in injury because 
inaccurate information may be given to the healthcare provider and the patient.  
 
You should clearly describe how raw signals are converted into a result including 
adjustment to the background signal for normalization, if applicable.  We also 
recommend that you include the following information for software development and 
implementation in the submission: 
 
  System and Software Requirements 
  Hazard Analysis  
  Architecture Design Chart 
  Software Design Specification 
  Software Development Environment Description 
  Verification and Validation 
  Traceability Analysis 
  Unresolved Anomalies  
 
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Before beginning clinical studies, the configuration of the hardware and software 
components should be very similar or identical to the final version of the device.  A risk 
assessment should be performed if any significant changes are made to the hardware or 
software after the completion of the clinical studies and before the clearance and 
distribution of the device. 
 
Below are additional references to help you develop and maintain your device under 
good software life cycle practices consistent with FDA regulations. 
  General Principles of Software Validation; Final Guidance for Industry and  
FDA Staff; 
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDo
cuments/ucm085281.htm 
  Guidance for Off-the-Shelf Software Use in Medical Devices; Final; 
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDo
cuments/ucm073778.htm 
  21 CFR 820.30 Subpart C  Design Controls of the Quality System  
Regulation;http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cf
m?FR=820.30 
  ISO 14971-1; Medical devices - Risk management - Part 1: Application of Risk 
Analysis. 
  AAMI 62304:2006; Medical device software - Software life cycle processes.  
 
 
5.D  Ancillary Reagents 
Ancillary reagents are those reagents that are specified in device labeling as required but 
not provided in order to carry out the assay as indicated in its instructions for use. 
Ancillary agents of concern in this context are those that are specified, for example, by 
manufacturer name, and/or product number.  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 the device from that 
reported in the labeling, then Brand X DNA amplification enzyme is an ancillary reagent 
of concern. In contrast, if your device requires the use of 95% ethanol, and any brand of 
  
95% ethanol will allow the device to achieve the performance characteristics provided in 
the labeling, then 95% ethanol is not an ancillary reagent of concern.
 
 
If the instructions for use of your device specify 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 the instructions, will be consistent with the 
performance established in the premarket submission.  Every effort should be made to 
bring the ancillary reagents under your companys quality system by recommending use 
of only those ancillary reagents that you have determined meet quality standards for your 
test.  The plan may include an application of quality systems approaches, product 
labeling, and other measures.  You should include the elements described below in your 
submission.  
 
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1.  A risk assessment addressing the use of ancillary reagents, including risks 
associated with the management of reagent quality and variability, risks 
associated with any inconsistency between instructions for use provided directly 
with the ancillary reagent and those supplied by the manufacturer with the device, 
and any other issues that could present a risk of obtaining incorrect results with 
your device.  
 
2.  Using your risk assessment as a basis, you should describe how you intend to 
mitigate risks through implementation of any necessary controls over ancillary 
reagents.  These may include, where applicable:  
 
  Plans for assessing user compliance with labeling instructions regarding 
ancillary reagents. 
  Material specifications for ancillary reagents in the labeling. 
  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 your device. 
  Any other issues that must be addressed in order to ensure safe and 
effective use of your test when used in combination with named ancillary 
reagents, in accordance with the devices instructions for use.  
In addition, you should provide testing data to establish that the quality controls supplied 
or recommended are adequate for detecting performance or stability problems with the 
ancillary reagents.  
 
6.  Limitations 
 
You must include a statement of limitations of the procedure in the labeling 
accompanying the product (21 CFR 809.10(b)(10)).  This should include potential issues 
that may affect the performance of your device and were not addressed in your analytical 
or clinical studies.  You should include any potential risks associated with using the 
device in the Warning and Limitation sections of the device labeling.  We recommend 
including statements such as those listed below as they pertain to your device:  
 
  The detection of viral nucleic acid [or viral antigen] 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. 
  Negative results do not preclude influenza virus infection and should not be used as the 
sole basis for treatment or other patient management decisions.  
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  Results from the [device name] should be correlated with the clinical history, 
epidemiological data and other data available to the clinician evaluating the patient.  
  Viral nucleic acids may persist in vivo independent of virus viability.  Detection of 
analyte target(s) does not imply that the corresponding virus(es) are infectious, or are the 
causative agents of clinical symptoms. 
  This device has been evaluated for use with human specimen material only. 
  False negative results may occur if the number of organisms in the clinical specimen is 
below the detection limits of the device. 
  If the virus mutates in the target region, influenza virus may not be detected or may be 
detected less predictably.  
  This device is a qualitative test and does not provide information on the viral load present 
in the specimen.  
  The performance of this device has not been evaluated for patients without signs and 
symptoms of influenza infection.  
  The performance of this device has not been evaluated for monitoring treatment of 
influenza infection.  
  The performance of this device has not been evaluated for the screening of blood or 
blood products for the presence of influenza.  
  This test cannot rule out diseases caused by other bacterial or viral pathogens. 
  The effect of interfering substances has only been evaluated for those listed in the 
labeling.  Interference by substances other than those described below can lead to 
erroneous results. 
  Cross-reactivity with respiratory tract organisms other that those listed below [in the 
labeling] may lead to erroneous results. 
  The performance of this device has not been evaluated for patients receiving intranasally 
administered influenza vaccine. 
  The performance of this device has not been evaluated for immunocompromised 
individuals. 
  The prevalence of infection will affect the tests predictive value. 
 
7.  Controls 
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.  Examples of appropriate external controls include vaccine or prototypic 
vaccine strains, low pathogenic viruses, and inactivated viruses.  Specific information 
about controls for nucleic acid based devices is provided in Section 9.E, Controls for 
Nucleic Acid-based Influenza Assays of this guidance document.  You may contact the 
Division of Microbiology Devices within the Office of In Vitro Diagnostic Device 
Evaluation and Safety (OIVD) at FDA for further information regarding controls.  
  14       Contains Nonbinding Recommendations 
 
 
8.  Interpreting and Reporting Test Results 
 
We recommend that you describe in your submission how positive, negative, equivocal 
(if applicable), or invalid results are determined and how they should be interpreted.  We 
recommend that you indicate the cut-off values for all outputs of the assay including the 
cut-off value for defining a negative (or negative/positive) result of the assay.  If the 
assay has an equivocal zone, the cut-off values (limits) for the equivocal zone should also 
be defined.  If your interpretation of the initial equivocal results requires re-testing, you 
should provide (1) a recommendation whether re-testing should be performed 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 confirms the significance of the assay cut-offs, and the algorithm 
should be followed precisely (e.g., perform re-testing, new extraction, or new patient 
specimen collection) during the clinical study for the performance evaluation of the 
device.  
 
If an assay result can be read or reported as invalid, you should describe how invalid 
results are defined.  If controls are part of the determination of invalid results, you should 
describe each possible combination of control results for defining the invalid result.  You 
should provide a recommendation on how to follow up any invalid result, i.e., whether 
the result should be reported as invalid or the specimen should be re-tested.  If re-testing 
is recommended, provide the information similar to the one for re-testing of equivocal 
results (whether re-testing should be performed from the same nucleic acid preparation, a 
new extraction, or a new patient specimen).  You should include in the submission and 
the labeling the number of initial invalid results and the number of re-tests that were 
needed to determine a final result during your studies.  Additional information on result 
evaluation and reporting can be found in CLSI document ILA 18-A2 [3].  
 
9.  Establishing Performance Characteristics  
 
9.A    Analytical Performance  
We recommend that you establish the following analytical performance characteristics 
for your assay: 
 
9.A.i  Analytical Sensitivity  
Limit of Detection  
The limit of detection (LoD) is defined as the lowest concentration of analyte that can 
be consistently detected (typically in 95% of samples tested under routine clinical 
laboratory conditions) in a defined type of specimen.  You should determine the LoD 
for each specimen type and each analyte that will be tested with your device utilizing 
the entire test system from sample preparation to detection when evaluating assay 
LoD.  This can be accomplished by limiting dilutions of propagated and titered viral 
stocks.  The study should include serial dilutions of at least two strains representative 
of types or subtypes for each claimed influenza virus (please see Table 1 for 
  15       Contains Nonbinding Recommendations 
 
suggested viral strains) and 3-5 replicates for each dilution.  The reference methods 
we recommend for LoD determination are the Tissue Culture Infectious Dose  
50
(TCID ), Egg Infective Dose  (EID ), or plaque assay.  Since the nucleic acid based 
50 50 50
devices detect not only the infective viral particles but also the total viral RNA 
present in the specimen, an additional reference method, quantifying nucleic acids, 
(e.g., genome copy equivalent or g/mL of viral RNA) may also be included.  You 
should report the LoD as the level of virus that gives a 95% detection rate.  The LoD 
should be confirmed by preparing at least 20 additional replicates at the LoD 
concentration and demonstrating that the virus was detected 95% of the time.   
 
We recommend that you determine the LoD for each analyte in the most commonly 
used or most challenging matrix tested by the device.  You may refer to Clinical 
Laboratory Standards Institute (CLSI) document EP17-A [4] for examples of the 
study design. When selecting an appropriate matrix for your analytical studies 
you should choose one of the two alternatives outlined below: 
1. Negative human clinical respiratory specimens can be pooled to create a large 
volume of uniform sample matrix (e.g., negative nasopharyngeal (NP) pools 
prepared from leftover NP swab clinical samples).  The pooled matrix should 
be screened prior to spiking. 
2. Viral transport medium (VTM) or another simulated matrix can be used if you 
can demonstrate in a study that analytical performance of your assay is 
equivalent using the proposed simulated matrix and the 
natural clinical matrix containing viruses.  The study can be conducted in-
house (i.e. within your own company) and include a limited number of samples 
(e.g., 60).     
Analytical Reactivity (Inclusivity) 
We recommend that you demonstrate that the test can detect at least 5 strains for 
influenza B and 10 strains for each influenza A subtype detected by your device.  
Influenza A detection should be tested across all subtypes that have infected humans 
and at viral levels at or near the LoD.  Influenza B strains representing both lineages 
(Victoria and Yamagata) should be included.  Influenza strains selected should reflect 
temporal and geographical diversity with an emphasis on contemporary strains.  For 
each claimed influenza subtype an additional selection of strains representing known 
lineages and clades should be included.  For subtypes for which it is difficult to obtain 
sufficient number of strains to demonstrate reactivity, we recommend that you contact 
the Division of Microbiology Devices to discuss your study.  All virus identities and 
titers should be confirmed.  Additional information on viral culture and identification 
procedures is available in CLSI document M41-A [5] and in the WHO manual [6].  
 
Examples of recommended strains for the LoD and the analytical reactivity studies are 
shown in Table 1.  Vaccine strains (wild type) from recent flu seasons can be included.  
Vaccine strains may vary from one influenza season to another.  The information on 
the current vaccine strains is available from the Centers for Disease Control and 
Prevention (CDC) at http://www.cdc.gov/flu/professionals [7].   
 
  16       Contains Nonbinding Recommendations 
 
Table 1. Examples of influenza strains for analytical sensitivity (LoD) studies. 
Type  Subtype  Influenza Viral Strain 
A  H1N1  A/California/7/2009 (H1N1)  
A  H3N2-like  A/Perth/16/2009 (H3N2)*  
B  B-like   B/Brisbane/60/2008  
A  H1N1  A/PR/8/34   
A  H1N1  A/FM/1/47  
A  H1N1  A/NWS/33  
A  H1N1  A1/Denver/1/57  
A  H1N1  A/New Caledonia/20/1999  
A  H1N1  A/New Jersey/8/76  
A  H1N1  A/Brisbane/59/2007 
A  H1N1  A/Hawaii/15/2001 
A  H3N2  A/Port Chalmers/1/73  
A  H3N2  A/Hong Kong/8/68  
A  H3N2  A2/Aichi2/68  
A  H3N2  A/Victoria/3/75  
A  H3N2  A/New York/55/2004 
A  H3N2  A2/Wisconsin/67/2005 
B    B/Malaysia/2506/2004 
B    B/Lee/40 
B    B/Allen/45 
B    B/GL/1739/54 
B    B/Taiwan/2/62 
B    B/Hong Kong/5/72 
B    B/Maryland/1/59 
A  H5N1  Human and /or Avian 
A  H5N2  Avian 
A  H7N2  Human and /or Avian 
A  H7N7  Human and /or Avian 
A  Other subtypes  Human and/or animal species 
* A/Wisconsin/15/2009 is an A/Perth/16/2009 (H3N2)-like virus and is a 2010 
Southern Hemisphere vaccine virus. 
 
9.A.ii  Analytical Specificity 
Exclusivity  
We recommend that you demonstrate analytical specificity of your assay with 
influenza types and subtypes not detected by your device. An exclusivity panel could 
be comprised of well characterized seasonal or novel influenza strains not detected by 
your device as well as non-human influenza viruses that have been shown to infect 
humans.  Pertaining to nucleic acid-based devices, for large panels or for influenza 
strains that are difficult to culture, purified nucleic acids may be quantified, e.g. 
genome copies/mL instead of determining the viral titers. Nucleic acids can also be 
quantified in cases when highly purified influenza viruses are used in the study, e.g. 
sucrose gradient purification of influenza viruses. 
 
  17       Contains Nonbinding Recommendations 
 
 
Cross-reactivity  
We recommend that you test for potential cross-reactivity with non-influenza 
respiratory pathogens and other microorganisms with which the majority of the 
population may have been infected.  We recommend that you confirm the virus and 
bacteria identities and titers and test the organisms at medically relevant levels 
(usually 106 cfu/ml or higher for bacteria and 105 pfu/ml or higher for viruses).  The 
microorganisms recommended for cross-reactivity studies are listed in Table 2.  
Pertaining to nucleic acid-based devices, for large panels or for organisms that are 
difficult to culture, purified nucleic acids may be quantified, e.g. genome copies/mL 
instead of determining the viral or bacterial titers. Nucleic acids can also be quantified 
in cases when highly purified organisms are used in the study, e.g. sucrose gradient 
purification of viruses. 
 
  Table 2. Microorganisms recommended for analytical specificity (cross-
  reactivity) studies.   
Organism  Type 
Adenovirus   Type 1 
Adenovirus   Type 7 
Human coronavirus*  OC43 and 229E strains 
Cytomegalovirus   
Enterovirus    
Epstein Barr Virus   
Human parainfluenza  Type 1 
Human parainfluenza    Type 2 
Human parainfluenza     Type 3 
Measles   
Human metapneumovirus    
Mumps virus   
Respiratory syncytial virus   Type B   
Rhinovirus   Type 1A 
Bordetella pertussis   
Chlamydia pneumoniae   
Corynebacterium sp.   
Escherichia coli   
Hemophilus influenzae   
Lactobacillus sp.   
Legionella spp   
Moraxella catarrhalis   
Mycobacterium tuberculosis   avirulent 
Mycoplasma pneumoniae   
Neisseria meningitidis   
Neisseria sp.   
Pseudomonas aeruginosa   
Protein A producer, 
Staphylococcus aureus  e.g., Cowan strain 
  18       Contains Nonbinding Recommendations 
 
Staphylococcus epidermidis   
Streptococcus pneumoniae   
Streptococcus pyogenes   
Streptococcus salivarius   
 
For devices detecting multiple analytes, e.g., Flu A and B, and each of the flu A 
subtypes, you should establish that there is no cross-reactivity between types and 
subtypes detected.  We encourage sponsors to present cross-reactivity testing data for 
devices detecting multiple pathogens in the format shown in Table 3).  
 
Table 3. Data presentation example.   
Reference Reagent, Results Positive (+) or 
EXAMPLE 
Negative (-) for Reactivity 
Organism  Strain  Adeno Flu A Flu B Para 1 Para 2 Para 3 RSV
Type 1  +  -  -  -  -  -  - 
Type 3  +  -  -  -  -  -  - 
Type 5  +  -  -  -  -  -  - 
Type 6  +  -  -  -  -  -  - 
Type 7  +  -  -  -  -  -  - 
Type 10  +  -  -  -  -  -  - 
Adenovirus 
Type 13  +  -  -  -  -  -  - 
Type 14  +  -  -  -  -  -  - 
Type 18  +  -  -  -  -  -  - 
Type 31  +  -  -  -  -  -  - 
Type 40  +  -  -  -  -  -  - 
Type 41  +  -  -  -  -  -  - 
Aichi (H3N2)  -  +  -  -  -  -  - 
Mal (H1N1)  -  +  -  -  -  -  - 
Hong Kong (H3N2)  -  +  -  -  -  -  - 
Denver (H1N1)  -  +  -  -  -  -  - 
Influenza A 
Port Chalmers (H3N2)  -  +  -  -  -  -  - 
Victoria (H3N2)  -  +  -  -  -  -  - 
WS (H1N1)  -  +  -  -  -  -  - 
PR (H1N1)  -  +  -  -  -  -  - 
Hong Kong  -  -  +  -  -  -  - 
Maryland  -  -  +  -  -  -  - 
Mass  -  -  +  -  -  -  - 
Influenza B 
Taiwan  -  -  +  -  -  -  - 
GL  -  -  +  -  -  -  - 
Russia  -  -  +  -  -  -  - 
Long  -  -  -  -  -  -  + 
RSV  Wash  -  -  -  -  -  -  + 
9320  -  -  -  -  -  -  + 
 
 
  19       Contains Nonbinding Recommendations 
 
Interference  
We recommend that you conduct a comprehensive interference study using medically 
relevant concentrations of the interferent and at least two strains for each influenza 
type to assess the potentially inhibitory effects of substances 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 4, below.  We recommend 
that you test interference at the assay cut-off determined for each influenza virus 
type/subtype detected by your device 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 effect is observed, no further 
testing is necessary.  Please refer to the CLSI document EP7-A2 [8] for additional 
information.  
 
Table 4. Substances recommended for interference studies 
Substance   Active Ingredient  
Mucin:   Purified mucin protein  
bovine submaxillary gland, type I-S 
Blood (human)    
Nasal sprays or drops  Phenylephrine,   
Oxymetazoline, 
Sodium chloride with 
preservatives 
Nasal corticosteroids  Beclomethasone, 
Dexamethasone, Flunisolide, 
Triamcinolone, Budesonide, 
Mometasone, Fluticasone  
Nasal gel   Luffa opperculata, sulfur 
Homeopathic allergy relief medicine  Galphimia glauca,  
Histaminum hydrochloricum 
FluMist  Live intranasal influenza 
virus vaccine  
Throat lozenges, oral anesthetic and  Benzocaine, Menthol  
analgesic 
Anti-viral drugs  Zanamivir 
Antibiotic, nasal ointment  Mupirocin 
Antibacterial, systemic  Tobramycin 
 
9.A.iii  Cut-off and Equivocal Zone 
In your submission, you should explain how the assay cut-off was determined and 
how the cut-off values were validated (see also Section 8, Interpreting and Reporting 
Test Results).  The cut-off should be determined using appropriate statistical 
methods.  For example, provide a result distribution, 95th and 99th percentiles, 
percents of the non-negative (positive or equivocal) results, and other statistics, for 
  20       Contains Nonbinding Recommendations 
 
the clinical samples without any respiratory viruses (zero analyte concentration) 
tested 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 [9]).  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.  
 
9.A.iv  Precision 
We recommend that you provide data demonstrating the precision (i.e., repeatability 
and reproducibility) of your system.  The CLSI documents, EP5-A2 [10] and EP12-
A2 [11], include guidelines that may be helpful for developing an appropriate 
statistical experimental design, computations and a format for establishing 
performance claims.  The precision should be established for each influenza virus 
type and subtype detected by the submitted device.  Any variable that may impact the 
assay precision should be examined.  
 
Site-to-Site Reproducibility 
The protocol for the reproducibility study may vary slightly depending on the assay 
format but it should include an evaluation of all major sources of variability described 
below: 
  Site-to-site and operator-to-operator.  You should include three or more sites 
(for example, two external sites and one in-house site) with multiple operators 
at each site.  Operators should represent end users of the assay in terms of 
education and experience. You should provide training only to the same extent 
that you intend to train users after marketing the device.  We recommend that, 
for rapid testing or point-of-care (POC)2 devices, you include a larger number 
of tests and operators in your evaluation, in order to best represent the settings 
in which the devices will be used. 
 
  Day-to-day variability. The testing should be conducted on five non-
consecutive days, 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 to assess between run and within run imprecision 
components.  Run variability may be combined with operator variability, for 
example, each run can be performed by a different operator. 
 
  Extraction-to-extraction variability.  For devices that require an extraction 
step, such as the nucleic acid amplification assays, samples used in the 
reproducibility testing should be processed at the test site, starting from 
clinical specimens (e.g., nasopharyngeal swabs) using the extraction 
                                                 
2 Tests designed to be used at or near the site where the patient is located, that do not require permanent 
dedicated space, and that are performed outside the physical facilities of the clinical laboratories 
http://www.cap.org/apps/cap.portal 
 
  21       Contains Nonbinding Recommendations 
 
procedure you recommend in the test labeling.  If more than one extraction 
procedure is recommended, each extraction method should be evaluated at 
each site separately (see also Nucleic Acid Extraction in Section 9.E). 
 
  The test sample panel should represent each type and subtype of influenza 
virus detected by your device at three levels of viral load including analyte or 
output concentrations close to the assay cut-off.  The reproducibility panel 
may be prepared by spiking viruses into negative clinical matrix pools 
prepared from leftover clinical specimens. 
 
1.  A high negative sample (C  concentration) with an analyte 
5
concentration below the clinically established 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, (e.g., for 
realtime PCR assays sample with an analyte concentration not more 
than 10 fold below the clinical cut off of the assay). 
2.  A low positive sample (C  concentration) with a concentration of 
95
analyte just above the clinically established cut-off such that results of 
repeated tests of this sample are positive approximately 95% of the 
time. 
3.  A moderate positive sample should reflect a clinically relevant viral 
load3. At this concentration, one can anticipate positive results 
approximately 100% of the time (e.g., approximately two to three 
times the concentration of the clinically established cut-off). 
 
  For an ultrasensitive test for which the clinical cut-off may not be established 
based on the truly negative samples (zero concentration), it may be impossible 
to obtain a C  sample For such a device, the following two concentration 
5 .
levels may be tested in the precision/reproducibility study in place of the C
5 
concentration recommended above: 
 
1.  A negative sample: a sample with an analyte concentration below 
the clinical cut-off such that results of repeated tests of this sample are 
negative 100% of the time, if less than 10% of the clinical samples 
positive by the reference method give results in a real time PCR assay 
below the threshold cycle (Ct) corresponding to LoD,  
  OR 
2.  A near cut-off sample (C20 to C80 ): a sample with a concentration 
of analyte just above or below the assay cut-off such that results of 
repeated tests of this sample are positive approximately 20% to 80% of 
the time, if more than 10% of the clinical samples positive by 
reference method give results below the Ct corresponding to LoD.  
 
                                                 
3 Sample with a typical virus concentration found in the infected subjects in the intended use population. 
  22       Contains Nonbinding Recommendations 
 
When  the limit of blank (LoB)4  is used as a cut-off, then the concentration 
C  is the same as the limit of detection (LoD) and the zero concentration (no 
95
analyte present in sample) is C  if LoB is estimated with Type I error of 5%5 
5
[4].    
 
We recommend that you provide a detailed description of the study design and 
statistical analyses used.  For the factors (e.g., instrument calibration, operators) 
considered in the precision study, we recommend using a balanced factorial design, 
that is, the precision study includes all the possible combinations of these factors.  For 
example, if a precision study is performed over five days using two operators/runs 
and three sites, there are 5 days times 2 operators per runs times 3 sites, which equals 
30 total combinations of the different levels of factors evaluated.  If each combination 
is evaluated using 3 replicates then there are 90 results per panel member from this 
study.  In general, for qualitative tests, variance components should be estimated 
using the appropriate statistical models and methods for each of the factors 
considered in the precision study.  Confounded sources of variability, as well as 
overall variation should be described, with all included factors noted.  For qualitative 
tests that have underlying quantitative output, the component of precision is often 
measured for each source of variation, as well as the total variation, using analysis of 
variance.  For each panel member in the precision study, you should provide both a 
separate analysis by site and a site combined data analysis, including the mean value 
with each variance component estimate (standard deviation and percent CV) as well 
as total variance.  For example, for a combined site data analysis, if a precision study 
is performed at three sites over five days using two operators per run and three 
replicates per run, provide the mean value, standard deviation, and percent CV for 
total variance and variance components for site to site, day to day, operator/run to 
operator/run, and residual (replicate to replicate).  In addition, for each panel member, 
you should provide the percents of the values above and below the cut-off and the 
percent of invalid results for each site separately and for all sites combined. If 
applicable, you should also provide percents of equivocal results for each panel 
member in the precision study for each site and for all sites combined.  The CLSI 
document, EP15-A2 [12], contains additional information on reproducibility study 
design. 
 
Within-Laboratory Precision/Repeatability  
We recommend that you conduct within-laboratory precision studies for devices that 
include instruments or automated components.  These studies may be performed in-
house, i.e., within your own company.  We recommend that you test sources of 
variability (such as operators, days, assay runs, etc.) for a minimum of 12 days (not 
necessarily consecutive), with two operators each performing two runs per day (for a 
total of four runs per day), and two replicates of each sample per run for a total of 96 
results for each test panel member.  These test days should span at least two 
calibration cycles if the calibration cycle is shorter than two months and also should 
                                                 
4 The limit of blank is defined as the highest expected value in a series of results on a sample that contains 
no analyte or the lowest observed test result that can reliably be declared greater than zero.  
 
5 Type I error is the probability of having truly negative samples (with zero analyte concentration) generate 
values that indicate the presence of analyte. Usually, Type I error is set as 5% or less. 
  23       Contains Nonbinding Recommendations 
 
represent different time points within a single calibration cycle.  We recommend you 
use the same sample panel as described in the section, Site-to-Site Reproducibility, 
above.  We recommend using a balanced factorial design; that is, the precision study 
should include all the possible combinations of operator, run, and day. For example, a 
precision study is performed over 12 days, by two operators, over two runs.  In this 
example, there are 12 days times two operators times two runs which equals 48 total 
combinations of the different levels for each factor evaluated. If each combination is 
evaluated in two replicates then there are 96 results per panel member from this 
study. For the two replicates, all factors of potential source of variations should be 
held constant.  We recommend that you report these results in a similar way to that 
described in the section, Site-to-Site Reproducibility. 
 
9.A.v  Carry-Over and Cross-contamination Studies (for multi-sample assays 
and devices that require instrumentation.)  
For multi-sample assays and devices that require instrumentation we recommend that 
you demonstrate that carry-over and cross-contamination do not occur with your 
device.  In a carry-over and cross-contamination study, we recommend that high 
positive samples be used in series alternating with negative samples in patterns 
dependent on the operational function of the device.  At least 5 runs with alternating 
high positive (the highest viral load found in clinical specimens or a minimum of 105 
pfu/ml) and high negative samples (as defined in the Site-to-Site Reproducibility 
section, above) should be performed.  We recommend that the high positive samples 
in the study be high enough to exceed 95% or more of the results obtained from 
specimens of diseased patients in the intended use population.  In the case of an 
ultrasensitive device a high negative sample may be replaced with a negative sample 
(please see Section Site-to-Site Reproducibility, above for additional explanations). 
The carry-over and cross-contamination effect can then be estimated by the percent of 
negative results for the negative sample in the carry-over study [13].  
9.A.vi  Specimen Storage and Shipping Conditions 
If you recommend specimen storage and shipping conditions, you should demonstrate 
that your device generates equivalent results for the stored specimens at several time 
points throughout the duration of the recommended storage and transport at both ends 
of your recommended temperature range.  If you recommend viral transport medium 
(VTM) for storage or shipping, you should conduct appropriate studies and provide 
the data, showing that the one or more recommended VTMs are suitable and that the 
device will perform as described when the specimen is preserved in the recommended 
VTM [5].  You should include a statement in the package insert to that effect, also 
indicating the commercial source or chemical composition of the acceptable VTM(s). 
 
9.B  Clinical Performance Studies 
We recommend that you conduct prospective clinical studies to determine the 
performance of your device in comparison to the established reference methods for all 
influenza types and subtypes and each specimen type you claim in your labeling.6  
                                                 
6 Comparing performance of a new assay against an established reference method creates a frame of 
reference for evaluating the device that is useful whether the data is to be considered in an initial 
  24       Contains Nonbinding Recommendations 
 
 
9.B.i  Study Protocol 
We recommend that you develop and include in the premarket submission a detailed 
study protocol that describes:  
 
  Patient inclusion and exclusion criteria.  
  Type and number of specimens needed.  
  Directions for use. 
  A statistical analysis plan that accounts for variances to prevent data bias.  
  Documents supporting compliance with human subject protection regulations.  
  Any other relevant protocol information.  
 
Clinical investigations of unapproved and uncleared in vitro diagnostic devices are 
subject to the investigational device exemption (IDE) provisions of Section 520(g) of 
the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 360j) and the implementing 
regulations. You should consider how 21 CFR part 812 (IDE) applies to your 
particular study and refer to 21 CFR part 50 (Informed Consent), and 21 CFR part 56 
(Institutional Review Board) for other applicable requirements.  Investigational 
devices that differentiate influenza A subtypes and detect novel influenza viruses, 
such as influenza A/H5, are particularly likely to meet the definition of "significant 
risk device" in 21 CFR 812.3(m).  Clinical investigations of significant risk devices 
require the submission of an IDE application to FDA for review and approval, in 
accordance with 21 CFR part 812.7   
For investigational devices that differentiate influenza A subtypes A/H1, A/H3, and 
A/2009 H1N1, but not influenza A/H5, we recommend that the clinical study protocol 
specify procedures for addressing influenza A unsubtypeable results in a timely 
manner, including the following steps:  
  The sample should be tested with the comparator (as per study protocol) in 
order to confirm the result as unsubtypeable.   
  Caution should be used and the laboratory SOP should be followed before 
propagating the unsubtypeable virus in culture.  
  In the event that the sample is confirmed unsubtypeable using the comparator 
method, the appropriate local, state, and/or federal public health authorities 
should be immediately notified and their instructions should be followed.  
We encourage sponsors to contact the Division of Microbiology Devices to request a 
review of their proposed studies and selection of specimen types prior to the initiation 
                                                                                                                                                 
classification action or to facilitate comparison with the performance of a predicate device, in the case of a 
premarket notification and evaluation of substantial equivalence. 
7 You may also refer to the Information Sheet Guidance for IRBs, Clinical Investigators, and Sponsors at 
http://www.fda.gov/oc/ohrt/irbs/devrisk.pdf and Guidance on Informed Consent for In Vitro Diagnostic 
Device Studies Using Leftover Human Specimens that are Not Individually Identifiable at 
http://www.fda.gov/downloads/RegulatoryInformation/Guidances/ucm127033.pdf. 
 
  25       Contains Nonbinding Recommendations 
 
of the studies.  We particularly encourage manufacturers to seek this type of 
discussion when samples are difficult to obtain. 
 
9.B.ii  Study Population 
We recommend that you conduct your studies with specimens obtained from 
individuals presenting with influenza-like illness (e.g., cough, nasal congestion, 
rhinorrhea, sore throat, fever, headache, and myalgia).  Influenza virus concentration 
in nasal and tracheal secretions remains high for 24-48 hours after the onset of the 
symptoms and may last longer in children.  We recommend that the sample be 
collected within three days of the onset of influenza-like symptoms in order to obtain 
optimal assay performance results.  If your device is intended for screening 
individuals for influenza infection, you should include a substantial number of both 
symptomatic and asymptomatic individuals in your study population, and also 
conduct a study during the time period when influenza is not prevalent.  
 
We recommend that you include a representative number of positive samples 
(determined by the reference method) from each age group and present the data 
stratified by age (e.g., pediatric populations aged birth to 5 years, 6 to 21 years, 
[14] adults aged 22-59, and greater than 60 years old) in addition to the overall 
data summary table. 
 
9.B.iii  Specimens 
You should include in the clinical studies samples consisting of all specimen types 
and matrices claimed in the intended use (e.g., nasal swabs, nasopharyngeal swabs, 
nasal aspirates) to demonstrate that correct results can be obtained from each type of 
clinical material.  You should indicate the types of collection devices (swabs, medium 
- VTM) used for collecting the clinical specimens and for establishing the 
performance claims stated in the package insert.  If the swabs are not provided with 
the device, the package insert should contain the information about the commercial 
source and swab specifications (e.g. size, shape, fiber, and shaft type).  Swabs with 
wood shafts or other materials known to inhibit growth of influenza viruses should 
not be used.  Analytical studies demonstrating equivalency of VTMs (e.g., LoD) 
should be included in the submission if the recommended VTM is different from the 
one used for the specimens in the clinical studies or if more than one VTM is 
recommended in the labeling. 
 
The total number of samples needed for substantiating a claim for detection of 
influenza A, influenza B, or H/N subtypes of influenza A, will depend on the 
prevalence of the virus and on the assay performance.  We recommend that all 
influenza detecting devices demonstrate specificity with a lower bound of the 95% 
(two-sided) confidence interval (CI) exceeding 90%. 
 
  For rapid devices detecting influenza A virus antigen, we recommend that you 
include a sufficient number of prospectively collected samples for each 
specimen type claimed to generate a sensitivity result with a lower bound of 
the two-sided 95% CI greater than 60%.  Generally, we recommend testing a 
  26       Contains Nonbinding Recommendations 
 
minimum of 50 samples, determined to be positive using the reference 
method, for each specimen type. 
  For rapid devices detecting influenza B virus antigen, we recommend that you 
include a sufficient number of samples for each claimed specimen type to 
generate a result for sensitivity with a lower bound of the two-sided 95% CI 
greater than 55%.  Generally we recommend a minimum of 30 positive 
samples for each specimen type.   
  Nucleic acid-based tests should demonstrate at least 90% sensitivity for each 
analyte and each specimen type with a lower bound of the two-sided 95% CI 
greater than 80%.  
 
We recommend that you assess the ability of your device to detect influenza viruses 
in fresh specimens collected from patients suspected of having an influenza infection 
who have been sequentially enrolled in the study (all-comers study).    
 
Frozen archived specimens may be useful for analytical performance evaluations, but 
are not recommended for studies to calculate clinical sensitivity or specificity. Freeze-
thawing can change the characteristics of the specimen from those of fresh specimens 
with which the test is intended to be used, possibly affecting assay performance.  
However, for devices intended to detect and/or differentiate influenza viruses for 
which fresh specimens are difficult to obtain, it may be necessary to use, for example, 
frozen archived clinical specimens: 
 
  For novel influenza viruses you may need to use frozen archived clinical 
specimens from patients who are case-confirmed, in accordance with World 
Health Organization (WHO) criteria for laboratory-confirmed cases, to 
demonstrate the performance of your device [6].   
 
  During an influenza season when the prevalence of influenza of a particular 
type or subtype is unusually low, prospectively collected archived specimens8 
of known specimen type can be used to supplement the fresh prospectively 
collected specimens.  We also recommend the following: 
 
1.  If the archived specimens were cultured before freezing and the culture 
results are available then the specimens should be tested only with the 
investigational device and the results should be compared to the 
original viral culture results. 
2.  If the culture results are not available, then the archived specimens 
should be thawed and tested with an acceptable NAAT-based 
comparator (e.g. CDC rRT-PCR flu panel) and the investigational 
device.  For details and updates on acceptable NAAT-based 
comparator devices, please contact the Division of Microbiology 
Devices (DMD). 
                                                 
8 For purposes of this guidance, prospectively collected archived specimens are those collected sequentially from all 
patients meeting study inclusion criteria and representing the assays 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 the prevalence is preserved.  These specimens should be 
appropriately stored (e.g., frozen at -70oC). 
  27       Contains Nonbinding Recommendations 
 
3.  A fresh-frozen equivalency study should be performed to demonstrate 
equivalency of the investigational device performance using fresh and 
frozen specimens.  
 
In general, when the number of specimens available for clinical testing is very low 
(e.g., newly emerging strains), the available evidence for FDA's premarket review 
may, of necessity, be obtained from analytical rather than clinical studies. In this 
circumstance, it is particularly critical to have well designed analytical studies. 
Animal studies can be used to supplement analytical studies.   
If a limited number of fresh influenza specimens are available, we recommend that 
you contact DMD to discuss alternative proposals.  
 
9.B.iv  Evaluation of Fresh vs. Frozen samples  
The performance of your device for detection of influenza viruses may change when 
testing frozen specimens as compared to fresh.  If both fresh and frozen specimens 
were included in the clinical study you should assess the effect of repeated 
freeze/thaw cycles on the assay performance and should demonstrate positive 
agreement of at least 95% with a lower bound of a 95% (two-sided) confidence 
interval exceeding 90%.  Either clinical or contrived specimens may be used for this 
study.  The contrived specimens may be prepared by spiking cultured viruses into an 
appropriate negative clinical matrix at different levels of viral load including the titers 
around the assay cut-off.  At a minimum, 60 samples representing swab specimen 
types and 60 samples representing washes and aspirates should be tested.  
 
Propagation of influenza virus in culture is most reliable when fresh specimens are 
used.  Freezing and thawing influenza virus may reduce its viability and generate a 
false negative result especially in specimens with a low viral load.  If these culture 
results are used as a reference method then the result of the investigational device 
may be positively biased.  If frozen specimens are propagated, you should 
demonstrate that there was no substantial loss of viral titer or change in performance 
of the investigational device when compared to propagation of fresh specimens.  The 
study comparing viral titer after propagating fresh and frozen specimens should 
include specimens with a range of viral loads including the titers around the LoD.  
Results should demonstrate a positive agreement of at least 95% with a lower bound 
of the 95% (two-sided) confidence interval exceeding 90%. 
 
9.B.v  Study Sites 
We recommend that you collect specimens and conduct your studies at a minimum of 
three geographically diverse facilities, one of which may be in-house.   
 
We recommend that the performance evaluation for devices intended for POC) use or 
rapid testing include, at minimum, one site at a clinical laboratory as well as sites 
representative of non-laboratory settings where the device is intended to be used (e.g., 
patients bedside, emergency department).  Conducting testing with the device in a 
clinical laboratory with more experienced and trained personnel, in addition to testing 
in non-laboratory sites where the device is intended to be used but operators are likely 
  28       Contains Nonbinding Recommendations 
 
to have less laboratory training, will help to determine whether training of the person 
conducting the test is likely to affect the performance of the device.   
 
9.B.vi  Reference Methods 
We recommend that you compare the results obtained with your device to the results 
obtained by using one or more of the following established reference methods or 
comparators: 
 
1.  Virus culture followed by direct fluorescent antibody (DFA) or other type-
specific antigen detection system (e.g., ELISA). 
2.  A direct specimen fluorescence assay (DSFA) that has been cleared by the 
FDA.  Direct specimen testing using immunofluorescent methods (DSFA) 
provides a specific result that is available faster than culture.  However, in 
order to ensure optimal accuracy it is essential that the operator follow the 
instructions and recommendations in the package insert and follow up all 
DSFA-negative specimens by viral culture. 
 
We recommend that you verify that the virus culture methods used in your study 
follow the CLSI document M41-A, and WHO Manual on Animal Influenza 
Diagnosis and Surveillance [5,6].  It is essential that the specimens be rapidly 
transported to the laboratory for optimal virus recovery or detection.  Culture should 
not be performed on frozen specimens if other options are available as freeze-thawing 
may result in loss of virus infectivity.  If the fluorescent antibody used for virus 
detection in cultured cells is FDA-cleared, no validation information is needed in the 
submission, as long as the laboratory performing the test follows the package insert 
instructions.  If the antibody used in the DFA is a pre-Amendments device,9 then you 
should provide published literature or laboratory data (e.g. LoD and analytical 
reactivity) in your premarket submission in support of the validation for the antibody 
used for typing of influenza virus isolated from culture.   
If your clinical protocol includes the use of frozen specimens, we recommend that 
you contact the Division of Microbiology Devices at FDA to discuss alternative 
proposals.  
 
3.  For devices based on nucleic acid amplification technologies (NAAT), 
alternative comparator methods may be used, including FDA-cleared NAAT-
based assays testing directly from clinical specimens.  An acceptable 
comparator is an assay that (i) demonstrates for each analyte (e.g., type A, B 
or subtype H1N1, H3N2) a sensitivity (compared to viral culture) of at least 
95% and a specificity of at least 92% with a lower bound of the 95% (two-
sided) confidence interval exceeding 90% and (ii) that does not recommend or 
require culture confirmation for negative results.  If the performance of your 
device is established in comparison to an acceptable comparator assay then 
you should calculate results as positive percent agreement and negative 
                                                 
9 Pre-Amendments devices are those devices that were introduced or delivered for introduction into 
interstate commerce for commercial distribution prior to May 28, 1976 (the date of enactment of the 
Medical Device Amendments of 1976). 
  29       Contains Nonbinding Recommendations 
 
percent agreement (rather than sensitivity and specificity).  Performance 
should meet the following criteria: positive and negative percent agreement of 
at least 95% with a lower bound of the 95% (two-sided) CI exceeding 90%.  
As the influenza virus continues to mutate and undergo antigenic drift, the 
performance of comparator devices may also change over time. We 
recommend that you contact DMD for a list of currently acceptable 
comparator assays. 
4.  When FDA-cleared tests or well characterized antibodies are not available, we 
recommend using a validated polymerase chain reaction (PCR) followed by 
bi-directional sequencing of hemagglutinin (HA) or other subtype-specific 
target amplicons, for influenza A subtype identification. This alternative 
method is acceptable for subtyping viruses in cultured cells or in specimens 
determined to be influenza A positive by either culture or an acceptable 
NAAT comparator.  If your test is based on nucleic acid amplification 
technologies, the primer sequences for the non-FDA-cleared comparator PCR 
should be different from the primer sequences included in your device and the 
comparator assay should be validated.   
 
You should provide published literature or laboratory data in support of the 
PCR validation for differentiation of the influenza A subtype.  Validation 
should include LoD and analytical reactivity data. The LoD of this PCR 
should be similar to the analytical sensitivity of the submitted device.  If 
sequencing is used as a component of the comparative method for the 
differentiation of influenza A viruses, we recommend that you perform the 
sequencing reaction on both strands of the amplicon (bidirectional 
sequencing) and that the generated sequence meet all of the following 
acceptance criteria: 
 
  The sequence should contain a minimum of 100 contiguous bases. 
  Bases should have a Quality Value of 20 or higher as measured by PHRED, 
Applied Biosystems KB Basecaller, or similar software packages (this 
represents a probability of an error of 1% or lower) [15]. 
  The sequence should match the reference or consensus sequence with an 
Expected Value (E-Value) < 10-30 for the specific target (for a BLAST search 
in GenBank, http://www.ncbi.nlm.nih.gov/Genbank/).   
 
Additionally, if public health authorities recommend against culturing a specimen 
from a patient who is suspected to be infected with a novel influenza virus, we 
recommend that you use an FDA-cleared NAAT device or validated PCR testing 
followed by sequencing of the amplicons to confirm the identity of the novel virus.   
 
9.C  Post-market Performance Validation 
We recommend that you obtain and analyze post-market data to ensure the continued 
reliability of your device, particularly given the propensity for influenza viruses to mutate 
and the potential for changes in viral strain prevalence over time.  As required by 21 CFR 
820.100(a)(1), Corrective and Preventive Action, you must analyze processes... 
  30       Contains Nonbinding Recommendations 
 
complaints, returned product, and other sources of quality data to identify existing and 
potential causes of nonconforming product or other quality problems.  As updated 
influenza viral sequences become available (from WHO, NIH and other public health 
entities), you should continue to monitor the performance of your assay.  Further, these 
analyses should be evaluated against the device design validation and risk analysis 
required by 21 CFR 820.30(g), Design Validation, to determine if any design changes 
may be necessary.  The aim of your post-market monitoring should be to ensure that your 
device maintains its stated level of performance over time in spite of the antigenic drift 
that is characteristic of influenza viruses.   
 
9.D  CLIA Waiver  
If you are seeking waiver categorization for your device under the Clinical Laboratory 
Improvement Amendments of 1988 (CLIA),10  we recommend that you consult with 
Division of Microbiology Devices staff regarding the design of specific studies to support 
the CLIA waiver application for your device.  The guidance, Recommendations for 
Clinical Laboratory Improvement Amendments of 1988 (CLIA) Waiver Applications, is 
also available [16]. 
 
9.E  Nucleic Acid-based Influenza Devices 
The information described here is relevant to studies intended to determine the 
performance of nucleic acid-based influenza assays [17,18].  This section complements 
the recommendations for performance studies described earlier in this document.  Where 
applicable, you should describe design control specifications that address or mitigate 
risks associated with primers, probes, and controls used to detect viral RNA segments, 
such as the following examples:  
 
  Prevention of probe cross-contamination for multiplexed tests in which many of 
the probes are handled during the manufacturing process.  
  Minimization of false positives due to contamination or carryover of sample.  
  Use of multiple probes for a single analyte to enable detection of virus variants 
appearing due to mutations within the target RNA segment(s), or variants within a 
designated Influenza virus strain (or lineage). 
  Developing or recommending validated methods for nucleic acid extraction and 
purification that yield suitable quality and quantity of viral nucleic acid for use in 
the test system with your reagents.  
 
9.E.i  Nucleic Acid Extraction  
Different extraction methods may yield nucleic acids of varying quantity and quality 
and, therefore, the extraction method can be crucial to a successful result.  Purification 
of viral nucleic acids can be challenging as biological samples may contain low viral 
titers masked by a background of human genomic DNA, as well as high levels of 
proteins and other contaminants.   
 
                                                 
10 See 42 U.S.C.  263a(d)(3). 
 
  31       Contains Nonbinding Recommendations 
 
For these reasons, you should evaluate the effect of your chosen extraction method on 
the performance of the assay.  If you include or recommend multiple extraction 
methods for use with your assay, you should demonstrate analytical and clinical 
performance of your assay with each extraction method and each claimed influenza 
virus type and subtype.  Specifically, you should demonstrate the LoD and 
reproducibility for each recommended extraction procedure.  You may be able to 
combine the extraction method variable with each site performance variable in the 
reproducibility study.  For example, if you recommend three different extraction 
methods, you can design a reproducibility study to evaluate one of the three 
extraction methods at each of three testing sites: test extraction method A at site 1, 
method B at site 2, and method C at site 3.  However, if the 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 methods A, B and C, site 2 extraction methods 
A,B and C, and site 3 extraction methods A, B and C). 
 
In addition to the analytical studies (LoD and Reproducibility at external sites), each 
extraction method should be utilized in at least one clinical site during the clinical 
studies 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 the individual clinical testing sites (using different 
nucleic acid extraction methods) are not considered equivalent and should not be 
pooled, but rather should be analyzed separately.  As a consequence, further testing of 
prospective clinical samples may be needed in order to support the claimed extraction 
method. 
 
9.E.ii  Controls for Nucleic Acid-based Influenza Assays  
We recommend that you use quality control material for verification of assay 
performance in analytical and clinical studies.  If your device is based on nucleic acid 
technology, we generally recommend that you include the following types of 
controls: 
 
Negative Controls 
Blanks 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.  These controls are 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 non-target organism.  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: 
  32 Contains Nonbinding Recommendations 
 Patient specimen from a non-influenza infected individual.
 Samples containing a non-target organism (e.g., cell line infected with non-
influenza virus).
 Surrogate negative control, e.g., alien encapsidated RNA [19].
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 laboratorys Quality System (QS).  
Examples of acceptable positive assay control materials include: 
 Cell lines infected with an inactivated or a non-virulent strain of influenza
virus.
 Packaged influenza 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 
patient sample and the reaction components when negative results are obtained. It 
indicates that the target is detected if it is present in the sample.    
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 
inhibitors in the samples.  This type of control can also assure specimen adequacy or 
that human cellular material was sampled (host target).  Examples of acceptable 
internal control materials include MS2 bacteriophage or human nucleic acid co-
extracted with the influenza virus and primers amplifying human housekeeping genes 
(e.g., RNaseP, -actin).   
10. References
1. Class II Special Controls Guidance Document: Instrumentation for Clinical
Multiplex Test Systems.
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDo
cuments/ucm077819.htm
2. Guidance for the Content of Premarket Submissions for Software Contained in 
Medical Devices.
https://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/
GuidanceDocuments/ucm089593.pdf
3. Clinical and Laboratory Standards Institute. 2001. Specifications for Immunological
Testing for Infectious Diseases; Approved Guideline, Second Edition. ILA18-A2.
33       Contains Nonbinding Recommendations 
 
4.  Clinical and Laboratory Standards Institute. 2004. Protocol for Determination of 
Limits of Detection and Limits of Quantitation; Approved Guideline. EP17-A. 
5.  Clinical and Laboratory Standards Institute. 2006. Viral Culture; Approved 
Guideline. M41-A. Clinical and Laboratory Standards Institute, Wayne PA. 
6.  WHO Manual on Animal Influenza Diagnosis and Surveillance. 2002, Geneva, 
(World Health Organization). (Complete document 
WHO/CDS/CSR/NCS/2002.5, available at: 
http://www.wpro.who.int/NR/rdonlyres/EFD2B9A7-2265-4AD0-BC98-
97937B4FA83C/0/manualonanimalaidiagnosisandsurveillance.pdf 
7.  Centers for Disease Control and Prevention. 2006-07 Influenza vaccine 
composition in, MMWR Recommendations and Reports: Prevention and Control 
of Influenza: Recommendations of the Advisory Committee on Immunization 
Practices (ACIP). 2006; July 28; 55(RR10):1-42. 
www.cdc.gov/flu/professionals/vaccination/composition0607.htm 
8.  Clinical and Laboratory Standards Institute. 2005. Interference Testing in Clinical 
Chemistry; Approved Guideline. EP7-A2. Clinical and Laboratory Standards 
Institute, Wayne PA. 
9.  Clinical and Laboratory Standards Institute. 1995. Assessment of the Clinical 
Accuracy of Laboratory Tests Using Receiver Operating Characteristics (ROC) 
Plots; Approved Guideline. GP10-A. Clinical and Laboratory Standards Institute, 
Wayne PA. 
10. Clinical and Laboratory Standards Institute. 2004. Evaluation of Precision 
Performance of Quantitative Measurement Methods; Approved Guideline. EP5-
A2. Clinical and Laboratory Standards Institute, Wayne PA. 
11. Clinical and Laboratory Standards Institute. 2002. User Protocol for Evaluation of 
Qualitative Test Performance; Approved Guideline. EP12-A2. Clinical and 
Laboratory Standards Institute, Wayne PA. 
12. Clinical and Laboratory Standards Institute. 2006. User Verification of 
Performance for Precision and Trueness; Approved Guideline. EP15-A2. Clinical 
and Laboratory Standards Institute, Wayne PA. 
13. Haeckel R. (1991) Proposals for the description and measurement of carry-over 
effects in clinical chemistry.  Pure Appl. Chem. 63:302-306.  
14. Guidance for Industry and FDA Staff Premarket Assessment of Pediatric Medical 
Devices. 2004. 
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/G
uidanceDocuments/ucm089742.pdf 
15. Patton, S.J.,Wallace, A.J., Elles, R. (2006) Benchmark for Evaluating the Quality 
 
of DNA Sequencing: Proposal from an International External Quality Assessment 
Scheme. Clinical Chemistry, 52:728-736.  
16. Guidance for Industry and FDA Staff: Recommendations for Clinical Laboratory 
Improvement Amendments of 1988 (CLIA) Waiver Applications for 
Manufacturers of In Vitro Diagnostic Devices. 2008. 
  34       Contains Nonbinding Recommendations 
 
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/G
uidanceDocuments/ucm070890.pdf 
17. Clinical and Laboratory Standards Institute. 2004. Nucleic Acid Sequencing 
Methods in Diagnostic Laboratory Medicine; Approved Guideline. MM9-A. 
Clinical and Laboratory Standards Institute, Wayne PA. 
18. Clinical and Laboratory Standards Institute. 2006 Molecular Diagnostic Methods 
for Infectious Disease; Proposed Guideline. MM3-A2. Clinical and Laboratory 
Standards Institute, Wayne PA. 
19. Pasloske BL, Walkerpeach CR, Obermoeller RD, Winkler M, and DuBois DB. 
(1998) Armored RNA Technology for Production of Ribonuclease-Resistant Viral 
RNA Controls and Standards. J. Clin. Microbiol, 36:3590-3594. 
 
 
 
  35