Contains Nonbinding Recommendations 
 
 
Establishing the Performance 
Characteristics of In Vitro Diagnostic 
Devices for the Detection or Detection 
and Differentiation of Human 
Papillomaviruses
Guidance for Industry and Food and 
Drug Administration Staff 
GUIDANCE 
Document issued on: September 15, 2017. 
The draft of this document was issued on August 14, 2015. 
This guidance supersedes FDAs guidance entitled Establishing the 
Performance Characteristics of In Vitro Diagnostic Devices for the Detection 
or Detection and Differentiation of Human Papillomaviruses, dated 
November 28, 2011. 
For questions regarding this document contact Natalia Comella, Ph.D., at (301) 796-6226 or by 
email at natalia.comella@fda.hhs.gov or Marina V. Kondratovich, Ph.D., at (301) 796-6036 or 
by email at marina.kondratovich@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 Diagnostics and Radiological Health 
1  Contains Nonbinding Recommendations 
 
 
Preface 
Public Comment 
You may submit electronic comments and suggestions at any time for Agency consideration to 
http://www.regulations.gov . Submit written comments to the Dockets Management Staff, Food 
and Drug Administration, 5630 Fishers Lane, Room 1061, (HFA-305), Rockville, MD 20852. 
Identify all comments with the docket number FDA-2009-D-0386. 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.  You may also send an e-mail request to CDRH-
Guidance@fda.hhs.gov to receive a copy of the guidance. Please use the document number 1740 
to identify the guidance you are requesting. 
2  Contains Nonbinding Recommendations 
 
 
Table of Contents 
I.  INTRODUCTION.................................................................................................5
II.  BACKGROUND...................................................................................................6
III.  SCOPE...................................................................................................................7
IV.  RISKS TO HEALTH ............................................................................................7
V.  DEVICE DESCRIPTION....................................................................................8
VI.  TEST METHODOLOGY....................................................................................8
VII.  ESTABLISHING PERFORMANCE CHARACTERISTICS ..........................9
A. ANALYTICAL STUDIES...................................................................................................9
(1)  Limit of Detection...........................................................................................9
(2)  Precision ........................................................................................................ 10
(3)  Cross-Reactivity ............................................................................................ 13
(4)  Interference ................................................................................................... 15
(5)  Carry-Over and Cross-Contamination Studies (for devices with automated 
liquid handling systems) ......................................................................................... 16 
(6)  Specimen Storage and Shipping Conditions .............................................. 16
(7)  Reagent Storage and Shipping Conditions ................................................. 17
(8)  Evaluation of HPV Detection in the Clinical Dataset ................................ 17
B. CLINICAL PERFORMANCE STUDIES ............................................................................. 19
(1)  Consideration of the Cervical Cancer Screening Guidelines ................... 19
(2)  Intended Use .................................................................................................. 20
(3)  Study Design Considerations Common to ASC-US Triage, Adjunct and Primary 
Screening Intended Uses (and likely any other intended uses): ......................... 21 
(4)  ASC-US Triage Intended Use ...................................................................... 25
(5)  ASC-US Population - HPV Tests for Detection and Differentiation (HPV 
Genotyping Tests) ................................................................................................... 28
(6)  Adjunct Intended Use ................................................................................... 30
(7)  Adjunct Intended Use  HPV Tests for Detection and Differentiation (HPV 
Genotyping Tests) ................................................................................................... 32 
(8)  Primary Screening Intended Use ................................................................ 33
(9)  Study Design to Cover All Three HPV Testing Claims (ASC-US Triage, Adjunct 
and HPV Primary Screening) ................................................................................ 35 
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C. CONTROLS ................................................................................................................... 35
(1)  External Controls ......................................................................................... 36
(2)  Internal Control ............................................................................................ 36
VIII.  REFERENCES .................................................................................................... 37
IX.  APPENDIX  STATISTICAL ANALYSIS ...................................................... 40
4  Contains Nonbinding Recommendations 
 
 
Establishing the Performance 
Characteristics of In Vitro Diagnostic 
Devices for the Detection or Detection 
and Differentiation of Human 
Papillomaviruses 
Guidance for Industry and Food and 
Drug Administration Staff 
This guidance represents the current thinking of the Food and Drug Administration (FDA or 
Agency) on this topic.  It does not establish any rights for any person and is not binding on 
FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff 
or Office responsible for this guidance as listed on the title page.  
I.    Introduction 
FDA is issuing this guidance to facilitate study designs to establish the performance 
characteristics of in vitro diagnostic devices (IVDs) intended for the detection, or detection and 
differentiation, of human papillomaviruses (HPVs).  These devices are used in conjunction with 
cervical cytology to aid in screening for cervical cancer or as first-line primary cervical cancer 
screening devices.  These devices include those that detect a group of HPV genotypes, 
particularly high risk HPVs, as well as devices that detect more than one genotype of HPV and 
further differentiate among them to indicate which genotype of HPV is present.  Approximately 
200 HPV genotypes have been identified, about 40 of which can infect the genital tract [Ref. 1].  
Infection with high-risk types of HPV is considered a necessary cause of virtually all cervical 
cancer [Ref. 2].  Approximately fourteen HPV genotypes are considered carcinogenic or high 
risk [Ref. 3 & Ref. 20].  For the remainder of this document, HPV refers to a high risk 
HPV, except where otherwise noted.  A high risk HPV test refers to an HPV IVD device that 
detects, but does not differentiate between different types of HPV; while a HPV genotyping 
test refers to an HPV IVD device that detects and further differentiates HPV types (some HPV 
tests provide individual HPV genotyping results in addition to the results of pooled probes).   
This guidance provides detailed information on the types of studies the FDA recommends to 
support a premarket application (PMA) for these devices.  It is recommended that you contact 
5  Contains Nonbinding Recommendations 
 
 
FDA prior to beginning your studies to discuss specific study proposals and performance goals 
for your device. 
This guidance is limited to studies intended to establish the performance characteristics of in 
vitro diagnostic HPV devices that are used in conjunction with cervical cytology for cervical 
cancer screening or as first-line primary cervical cancer screening devices.  This guidance 
specifically addresses devices that qualitatively detect HPV nucleic acid from cervical 
specimens, but many of the recommendations will also be applicable to devices that detect HPV 
proteins.  See Section III Scope for more details on what is covered by this guidance document. 
For the current edition of the FDA-recognized standard(s) referenced in this document, see the 
FDA Recognized Consensus Standards Database Web site at 
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfStandards/search.cfm.  
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances 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 guidances means that something is suggested or 
recommended, but not required. 
II.  Background  
This document provides guidance for establishing the performance characteristics of in vitro 
diagnostic devices for the detection, or detection and differentiation, of human papillomaviruses 
in cervical specimens.  These recommendations apply to PMAs for HPV IVDs. 
A manufacturer who intends to market an IVD device for detection, or detection and 
differentiation, of human papillomaviruses must conform to the requirements of the Federal 
Food, Drug, and Cosmetic Act (the FD&C Act) and obtain premarket approval prior to 
marketing the device (sections 513 and 515 of the FD&C Act; 21 U.S.C. 360c and 360e).  
Because HPV diagnostic devices are postamendment devices, they are automatically classified as 
class III under section 513(f)(1) of the FD&C Act.  Devices that have been classified by section 
513(f)(1) into class III require premarket approval in accordance with section 515 of the FD&C 
Act.  See section 515(a)(2) of the FD&C Act (requiring premarket approval for devices classified 
into class III by section 513(f)); see also section 513(a)(1)(C) of the FD&C Act (defining a class 
III device as one that "is to be subject, in accordance with section 515, to premarket approval to 
provide reasonable assurance of its safety and effectiveness").   
Further information on device testing can be found in FDAs guidance entitled In Vitro 
Diagnostic (IVD) Device Studies  Frequently Asked Questions at 
(http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocu
ments/ucm071230.pdf), and FDAs guidance entitled Guidance on Informed Consent for In 
Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually 
Identifiable at 
6  Contains Nonbinding Recommendations 
 
 
(http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocu
ments/ucm071265.pdf). 
III.  Scope 
This document recommends studies for establishing the performance characteristics of in vitro 
diagnostic devices for the qualitative detection, or detection and differentiation, of HPV.  This 
guidance is limited to studies intended to establish the performance characteristics of in vitro 
diagnostic HPV devices that are used in conjunction with cervical cytology for cervical cancer 
screening, or as first-line primary cervical cancer screening devices.  It does not address HPV 
testing from non-cervical specimens, such as pharyngeal, vaginal, penile or anal specimens, or 
testing for susceptibility to HPV infection.  It does not address quantitative or semi-quantitative 
assays for HPV. 
As postamendment devices, HPV diagnostic devices are automatically classified as class III 
devices under section 513(f)(1) of the FD&C Act.  To date, two product codes have been 
established for HPV nucleic acid detection devices: MAQ (HPV DNA detection devices) and 
OYB (HPV RNA detection devices).  Both of these product codes are class III.  The 
recommendations in this guidance apply to HPV diagnostic devices that detect HPV nucleic acid 
(HPV DNA and RNA).  Many of the recommendations will also apply to HPV detection devices 
that utilize targets other than HPV nucleic acid (such as HPV protein).  This guidance therefore 
may encompass future HPV product codes beyond the ones listed.  This guidance does not apply 
to HPV-associated biomarkers (e.g., p16).     
IV.  Risks to Health
 
Failure of devices for the detection, or detection and differentiation, of human papillomaviruses 
to perform as expected, or failure to correctly interpret results may lead to incorrect patient 
management decisions in cervical cancer screening and treatment.  False negative results may 
lead to delays in the timely diagnosis of cervical cancer and treatment, allowing an undetected 
condition to worsen and potentially increasing morbidity and mortality.  False positive results 
could lead many women to unnecessarily undergo more frequent screening and potentially 
invasive procedures such as colposcopy and biopsy.  False positive results for the highest risk 
types of HPV, such as HPV 16 and/or 18, could lead to unnecessarily aggressive treatment of 
cervical lesions that may impair fertility.  Because cervical cancer screening is recommended for 
virtually all sexually active women and a substantial number of these women will be tested for 
HPV, the risk scale for potential harm to public health from false negative and false positive 
HPV results is significant. 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 that are submitted in a PMA to establish the performance of HPV detection devices 
are a key factor for determining the safety and effectiveness of these devices.   
7  Contains Nonbinding Recommendations 
 
 
V.  Device Description 
In your PMA, you are required to provide, amongst other information, a device description (21 
CFR 814.20(b)(3)(ii)).  You should provide in your PMA a device description that includes 
information sufficient to understand what the proposed device is and how it works, such as: 
  A description of the device in text and with pictures, diagrams, and/or engineering 
drawings, as applicable. 
  An explanation of the mechanism of action and principles of operation. 
  Characteristics of the device output (i.e. whether or not genotypes can be differentiated, 
genotypes assessed simultaneously vs. individually in a well or channel, etc.). 
  A detailed technical description of the device including instruments, reagents, 
components, software, and accessories. 
  The proposed indications for use of the device (including sample type(s) and collection 
devices). 
VI.  Test Methodology 
You should describe, in detail, the methodology used by your device in your PMA.  You should 
describe the following elements, as applicable to your device: 
  Test platform.  
  Information and rationale for selection of specific target sequences and the methods used 
to design detection elements. 
  Specimen collection and handling methods. 
  All pre-analytical methods and instrumentation for collection, stabilization, and 
concentration of specimens, as appropriate. 
  Limiting factors of the assay (e.g., saturation level, maximum cycle number, etc.). 
  Reagent components provided or recommended for use, and their function within the 
system (e.g., buffers, enzymes, fluorescent dyes, chemiluminescent reagents, 
oligonucleotides, other signaling/amplification reagents, etc.). 
  The potential for specific and non-specific interference effects from reagents or device 
material. 
  Internal controls and a description of their specific function in the system. 
  External controls that you recommend or provide to users. 
  Instrumentation inherent to using your device, including the components and their 
function within the system. 
  The computational path from raw data to the reported result (e.g., how raw signals are 
processed and converted into a useable result).  This would include adjustment for 
background and normalization, if applicable.  Show how results are reported and 
interpreted. 
  Illustrations, photographs, and a detailed description of non-standard equipment or 
methods, as appropriate. 
8  Contains Nonbinding Recommendations 
 
 
VII. Establishing Performance Characteristics  
A. Analytical Studies 
You should provide in your PMA analytical studies that conform to the following 
recommendations. 
(1) Limit of Detection  
FDA recommends that you determine the limit of detection (LoD) of your device using serial 
dilutions of HPV genomic DNA or RNA transcripts, as appropriate, in sample collection buffer.  
Genomic DNA or RNA transcripts, or both, can be cloned or synthesized material, since HPV 
cannot be cultured.  We recommend that you determine the LoD for each HPV genotype and 
each specimen collection media tested by the device.  
If your assay is indicated for testing with liquid-based cytology (LBC) specimens, and involves 
centrifugation of the cervical cytology sample and removal of the LBC collection media 
(supernatant) prior to processing for HPV testing, you should perform your LoD studies in 
whatever matrix or buffer the cells are re-suspended in after the centrifugation step.  If you use 
LBC mock-samples containing HPV-infected cell lines in any of your analytical studies (as 
recommended under Section VII(A)(2) Precision  below), then you should also perform LoD 
studies with these types of samples.  A human HPV-negative cell line is recommended to serve 
as a surrogate for non-HPV infected cells in LBC samples contrived from HPV-infected cell 
lines (i.e., SiHa and HeLa cell lines).   You should conduct a paired sample LoD study with at 
least one of these HPV-infected cell lines showing that you get the same LoD results in both a 
pooled negative clinical and simulated background matrix (i.e., an HPV negative cell line in 
LBC media).  If these two samples demonstrate equivalence then a negative cell line can be used 
as the background in other analytical studies.  
We recommend that you first define a cutoff for the numerical signal (i.e., the limit of blank 
(LoB)) such that a signal above the LoB in a patient sample indicates that the virus was detected.  
You should also estimate the level of virus that gives a 95% detection rate (the LoD).  There are 
two different types of devices to consider when establishing the cut-off.  One type covers devices 
for which a distribution of numeric signals are obtained when repeatedly testing samples known 
to have zero concentration (true absence of targeted analyte).  For this device type, LoB is a 
threshold for numeric signal with a pre-defined type I error (typically 5%), such that samples 
with a numeric signal above the LoB are considered as HPV detected.  For the second type of 
device, the ultrasensitive devices, samples with zero analyte concentration almost always have 
HPV not detected results (type I error is close to zero). 
We suggest that you refer to the Clinical and Laboratory Standards Institute (CLSI) document 
EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement 
Procedures [Ref. 4] for the basic concepts, design, and statistical analysis of your LoD studies.  
For the first type of device described above, you can use the approach described in CLSI EP17-
A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures, and 
9  Contains Nonbinding Recommendations 
 
 
by Linnet and Kondratovich [Ref. 5] to estimate the LoD using the standard deviation of samples 
with very low concentrations.  For the second type of device, the LoD can be estimated from 
Probit analysis using hit rates (percent of virus detected) of different dilutions [Ref. 6].  Hit rates 
for these dilutions should cover a large part of the range of detection (0% detection to 100% 
detection).  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.  The LoD 
study should include serial dilutions of each targeted HPV genotype, cell line, or specimen type.  
In both approaches to LoD estimation, the appropriate sources of variability should be included 
in the LoD study by testing 3-5 samples over 3-5 days with 2-3 lots of your device.   
Please note that the clinical cutoff, which defines positive and negative results for the HPV test 
on clinical samples, can be higher than the LoB, which analytically defines whether the HPV 
virus is present or absent.  The C  concentration is the concentration of analyte just above the 
95
clinical cutoff such that results of repeated tests of this sample are positive approximately 95% of 
the time.  When the LoB is used as a cutoff, then the concentration C  is the same as the LoD.   
95
For an HPV assay in which the clinical cutoff is higher than the LoB, the concentration C  may 
95
differ from the LoD concentration.    
(2) Precision 
a.  Samples for within-laboratory precision/repeatability studies 
For establishing the precision of HPV tests, you should create 10-20 precision panel members 
with defined analyte levels and HPV genotype(s).  You should establish performance using 
specimens with analyte levels that challenge medical decision points, in addition to specimens 
with moderate analyte levels (as described below). Since HPV cannot be cultured, HPV-infected 
human cell lines (as well as human HPV-negative cell lines) can be used to create panel 
members that mimic clinical specimens and contain a targeted level of HPV analyte.  Utilizing 
cell lines is important for LBC specimens since this helps to account for some of the variability 
that arises due to the sampling and processing of a heterogeneous suspension of cells.  When an 
HPV genotype that you intend to claim to detect is not readily available as an infected cell line, 
you may also use contrived panel members derived from HPV DNA plasmids or RNA 
transcripts, as appropriate.  In addition to these contrived samples with defined levels of HPV 
infected cells or HPV nucleic acid, you should include four or more real clinical samples with 
signal levels that challenge the assay clinical cutoff, plus at least one clinical sample negative for 
HPV in your precision study panels.  Real clinical samples should be used since cell lines and 
plasmids alone cannot address all the variability present in clinical samples.  Clinical samples 
can be pooled to create sufficient volume and to achieve desired levels of virus concentrations.   
In some instances, pooling of clinical samples may significantly increase the variability 
observed; in that event, you should contact FDA to discuss alternative study designs using 
individual clinical samples.  Viral load cannot be defined for clinical specimens, but you should 
challenge medical decision points [i.e., clinical cutoff(s)] by including specimens that test 
positive and/or negative only a fraction of the time (the exact value of this fraction is not critical, 
anything from 5 to 95% positivity is acceptable).  This way, the end user can see what output 
signal levels have a degree of variability associated with their qualitative results.  Panel members 
derived from cell lines and/or real clinical samples should be processed as real LBC specimens, 
10  Contains Nonbinding Recommendations 
 
 
starting from suspension in LBC media before the nucleic acid extraction step.  If you wish to 
utilize a precision panel composed entirely of real clinical specimens (without any simulated 
specimens), please contact FDA to discuss recommendations for additional characterization of 
such specimens.  
We recommend that you conduct within-laboratory precision studies for devices that include 
complex instruments or automated components.  You should include sources of variability (such 
as operators, days, instruments, assay runs, etc.) encompassing a minimum of 12 days (not 
necessarily consecutive), with two runs per day, and two replicates of each sample per run.  You 
should assess precision between three reagent lots; there should be no redundancies in the 
individual reagent lots evaluated within each kit lot, or in any calibrators or controls that are sold 
separately.  Between-instrument imprecision can be evaluated during your in-house precision 
study, but is more commonly assessed during a sponsors reproducibility study (as described 
below in Section VII(A)(2)(b) Reproducibility).   
For simulated precision panel members, the test panel should include at least six samples (two 
HPV genotypes) at three levels of viral load as described below (also see Chart 1):  
  A zero concentration sample with no analyte present. 
  A high negative sample aiming to represent the analyte 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, C  
5
concentration (e.g., for realtime PCR assays, a sample with an analyte concentration not 
more than 10 fold below the clinical cutoff of the assay). 
  A low positive sample (C  concentration) with a concentration of analyte just above 
95
the clinical cut-off such that results of repeated tests of this sample are positive 
approximately 95% of the time.  
  A moderate positive 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). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Chart 1. Three Levels for Precision Studies 
 
 
 
 
 
 
 
 
 
 
 
 
  Clinical Cutoff  
 
 
 
Signal 
 
  High negative C    Low positive C   
5 95
 
 
When the LoB is used as a clinical cutoff, then the concentration C  is the same as the LoD and 
95
the zero concentration (no analyte present in sample) is C  [Ref. 4].  CLSI documents EP05-A3: 
5
Evaluation of Precision of Quantitative Measurement Procedures [Ref. 7] and EP12-A2: User 
Protocol for Evaluation of Qualitative Test Performance [Ref. 8] contain further information 
about designing and performing precision studies.   
For precision studies, it is not necessary to have the high negative and low positive samples at 
exactly C or C .  If the high negative and low positive samples in the precision study are close 
5  95
enough to the cutoff that the standard deviation (or percent coefficient of variation (%CV)) is 
approximately constant over the range around the cutoff, the C  and C  can be evaluated from 
5 95
this within-laboratory precision study.1  The objective of estimating the C  and C
5 95 
concentrations in this manner is to ensure that your precision panel members are adequately 
challenging your medical decision points. 
 
 
 
                                                 
1 If the standard deviations (SD) in the precision studies for concentrations around the cutoff value (C ) 
50
are almost constant, then: C  = C  +1.645 x SD, and C  = C   1.645 x SD. If the coefficient of 
95 50 5 50
variation (CV) in the precision studies for concentrations around the cutoff value are almost constant, then 
C  = C  + 1.645 x CV x C  and C  = C   1.645 x CV x C .  From here, C  = C  / (1  1.645 x CV) 
95 50 95 5 50 5 95 50
and C  = C  / (1 + 1.645 x CV).   
5 50
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b.  Reproducibility 
The protocol for the reproducibility study may vary slightly depending on the assay format.  We 
recommend the following protocol: 
  Evaluate the reproducibility of your test at three testing sites (e.g., two external sites and 
one internal 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.  
  Each contrived cell line sample panel member or clinical sample panel member in the 
reproducibility study should have at least 90 measurements. 
  Use the sample panel as described in the within-laboratory precision study above (include 
C  and C  samples estimated from the within-laboratory precision internal study in your 
5 95
reproducibility study).  For your reproducibility study, the cell line panel members and 
the clinical sample panel members should be processed for each run starting from the 
nucleic acid extraction step with an independent extraction for each run. 
  Between-instrument imprecision is often assessed as part of a sponsors reproducibility 
study (rather than during in-house precision testing) by having each site conduct testing 
using a different instrument.  With this design, instrument precision is confounded with 
site precision and if significant differences in precision are observed between the 
different sites, it is the sponsors responsibility to conduct another study to determine if 
this imprecision is attributable to the sites or the instruments. 
c.  Presentation of precision studies results 
For each sample tested in the precision studies (within-laboratory internal precision study and 
reproducibility study), we recommend you present the mean value of the signal with variance 
components (standard deviation and percent CV).  In addition, you should include the percent of 
values above and below the cutoff for each sample in the precision studies.   For the 
reproducibility study, present the mean value with variance components and percent of values 
above and below the cutoff for each site separately and for the combined data. 
We recommend you consult the CLSI documents EP05-A3: Evaluation of Precision of 
Quantitative Measurement Procedures [Ref. 7] and EP15-A3: User Verification of Precision 
and Estimation of Bias [Ref. 9] for additional information on reproducibility study design and 
statistical analysis. 
(3) Cross-Reactivity  
We recommend that you test your device for potential cross-reactivity with other organisms 
known to colonize the genital tract, including human pathogens that are transmitted by sexual 
contact.  We recommend that you test medically relevant levels of viruses and bacteria (usually 
105 pfu/ml or higher for viruses and 106 cfu/ml or higher for bacteria).  We recommend that you 
confirm the virus and bacteria identities and titers.  Titers in particular are usually estimated by 
13  Contains Nonbinding Recommendations 
 
 
suppliers but are not guaranteed.  The microorganisms recommended for cross-reactivity studies 
are listed below in Table 1.  Specific species are recommended according to prevalence, clinical 
relevance, or both, but additional species may also be tested at the discretion of the sponsor.  Any 
additional species selected should be known to colonize the genital tract.  Additional organisms 
should be tested if there is reason to suspect that cross-reactivity may occur (i.e., clinical 
evidence of cross-reactivity, homology to chosen probe/primer sequences, etc.). 
For devices that target a group of HPV genotypes but do not differentiate among them, you 
should test the most closely related and/or clinically significant non-targeted HPV genotypes for 
cross-reactivity.  For devices that detect more than one genotype of HPV and further differentiate 
among them, you should test for cross-reactivity among targeted genotypes.  Since HPV cannot 
be readily cultured, HPV genotypes may be tested as cloned genomic HPV DNA in plasmids or 
in vitro transcripts, depending upon your targeted analyte. 
Table 1. Microorganisms Recommended for Analytical Specificity (Cross-reactivity) 
Studies. 
Organism 
Bacteria:  Human Papillomaviruses: 
Lactobacillus acidophilus  All non-targeted alpha-HPV genotypes.  Alpha 
Staphylococcus epidermidis  HPV genotypes include the following: HPV 16, 
18, 26, 30, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 
Staphylococcus aureus 
58, 59, 66, 67, 68, 69, 70, 73, 82, 85            
Streptococcus faecalis  HPV 6, 11 
Streptococcus pyogenes  Any non-targeted genital HPV genotypes that 
Streptococcus agalactiae  are likely to cross-react with your assay based 
on probe-homology analysis (such as blast 
Corynebacterium spp. 
search results). 
Chlamydia trachomatis 
Neisseria gonorrhoeae  Other Viruses: 
Escherichia coli  Adenovirus  
Enterococcus spp.  Cytomegalovirus 
Clostridium spp.   Epstein Barr virus 
Peptostreptococcus spp.  Herpes simplex virus 1 
Klebsiella spp.  Herpes simplex virus 2 
Enterobacter spp. 
Proteus spp. 
Pseudomonas spp. 
Bacteroides spp.  Other: 
Bifidobacterium spp.  Candida albicans 
Fusobacterium spp.  Trichomonas vaginalis 
Treponema pallidum 
 
     
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(4) Interference  
We recommend that you conduct a comprehensive interference study using medically relevant 
concentrations of the interferent and at least one of the most clinically relevant HPV genotypes 
(such as HPV 16 or HPV 18) to assess the potentially inhibitory effects of substances 
encountered in cervical specimens. 
Potentially interfering substances include, but are not limited to, the following: whole blood 
(human), leukocytes, contraceptive and feminine hygiene products.  The active ingredients and 
brand names of selected products and tested concentrations should be provided in your labeling.  
Examples of potentially interfering substances are listed in Table 2 below.  We recommend that 
you test for interference using specimens with analyte levels that challenge medical decision 
points around the clinical cutoff (e.g., C ).  We also recommend that you evaluate each 
95
interfering substance at its potentially highest concentration (i.e., the worst case).  One way of 
accomplishing this is to dip a specimen collection device directly into the potentially interfering 
substance and subsequently place the collection device into one aliquot of a split test specimen.  
The other aliquot would be tested without the potential interferent so that the signal between the 
paired samples can be compared.  In this approach, both aliquots (with and without the potential 
interferent) are tested in the same manner as patient specimens with adequate replication (at least 
four to seven replicates) within one analytical run.  An estimate of the observed interference 
effect as the difference between the means of the two aliquots is computed and the 95% two-
sided confidence interval for the interference effect is calculated.  If no significant clinical effect 
is observed, no further testing is indicated.  We recommend that you refer to the CLSI document 
EP07-A2: Interference Testing in Clinical Chemistry; Approved Guideline [Ref. 10] for 
additional information on interference testing.  
Table 2.  Substances Recommended for Interference Studies. 
Substance  
Whole blood (human) 
Leukocytes (1x106 cells/ml) 
Contraceptive jelly 
Douche 
Anti-fungal cream 
Spermicide 
Vaginal lubricant 
Feminine spray 
Intravaginal hormones 
Mucus 
 
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(5) Carry-Over and Cross-Contamination Studies (for devices with 
automated liquid handling systems)  
We recommend that you demonstrate that carry-over and cross-contamination will not occur 
with your device under your recommended instructions for use.  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 five 
runs with alternating high positive and negative samples 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.  The carry-over and 
cross-contamination effect can then be estimated by the percent of negative results for the 
negative samples that are adjacent to high positive samples in the carry-over study compared to 
the percent of negative results in the absence of adjacent high positive samples (i.e., only 
negative samples are run on the plate).  For additional detail, see Haeckel [Ref. 11].  For devices 
that are indicated for HPV testing of residual cytology samples, an analysis of the carryover 
effects of any upstream automated cytology processing system(s) should be provided. 
(6) Specimen Storage and Shipping Conditions 
For your recommended specimen storage conditions, you should demonstrate that your device 
generates equivalent results to time zero for the stored specimens at several time points 
throughout the duration of the recommended storage.  Storage temperatures evaluated should 
represent each extreme of your recommended temperature range.  You should establish your 
specimen storage and shipping conditions utilizing a panel of real clinical samples that represent 
the specimen types claimed in your indications for use and analyte levels that challenge the 
medical decision point(s) of your assay.   The percent change in signal (when compared to time 
zero) should be presented for each panel member at each time point tested, as well as for all 
panel members combined.  Using regression analysis, each sample should be analyzed separately 
such that the absolute and percent difference in signal between the recommended storage time 
and time zero (T ) should be calculated with a 95% confidence interval.  Similar regression 
0
analysis should be performed for all panels combined.   For these studies, detailed information 
about the samples used should be recorded and included in your submission.   
You should record the date [including day and time] the specimens were collected from patients 
as well as the date the samples were tested.  Your records for these dates should include at a 
minimum the day and time for both of these activities   We recommend you submit this 
information for all specimens tested in the clinical study as well as for specimens tested to 
establish T  for your stability studies (among those samples used to establish stability).  
0
Furthermore, if there are concerns about the stability of a specimen type, additional studies may 
be required using contrived samples to more carefully establish T . 
0
   
 
 
16  Contains Nonbinding Recommendations 
 
 
(7) Reagent Storage and Shipping Conditions 
For your recommended reagent storage conditions, you should demonstrate that your device 
generates equivalent results to time zero utilizing the stored reagents at several time points 
throughout the duration of the recommended storage.  Storage temperatures evaluated should 
represent each extreme of your recommended temperature range.  We recommend that you refer 
to the CLSI document EP25-A: Evaluation of Stability in In Vitro Diagnostic Reagents [Ref. 
12] for additional information.  Accelerated stability studies are appropriate for estimating 
reagent stability, but the data provided in your submission should show real-time performance.  
You should establish your reagent storage and shipping conditions utilizing the specimen types 
claimed in your intended use and analyte levels that challenge the medical decision point(s) of 
your assay.  The percent change in signal (when compared to time zero) should be presented for 
each panel member at each time point tested, as well as for all panel members combined.  Using 
regression analysis, each sample should be analyzed separately such that the absolute and percent 
difference in signal between the recommended reagent storage time and T  should be calculated 
0
with a 95% confidence interval.  Similar regression analysis should be performed for all panels 
combined. 
(8) Evaluation of HPV Detection in the Clinical Dataset  
We recommend that you provide an evaluation of your devices ability to detect the targeted 
HPV genotypes in your clinical dataset.  One way to do this is to perform an FDA-approved 
HPV test that detects the same genotypes as your test, or you may perform PCR followed by 
sequencing of the amplicon (PCR/Sequencing) on your clinical specimens and compare these 
results to the results of your device.  Use of an FDA-approved HPV test is recommended 
whenever feasible.  Use of a composite HPV comparator that incorporates multiple FDA-
approved HPV test(s) and/or PCR/Sequencing is also an option.  The nucleic acid amplification 
method used in the composite reference method should be targeted to genomic regions different 
from the one probed by your assay.  You should provide published literature or laboratory data in 
your submission in support of the primers used for amplification. 
For PCR followed by Sanger sequencing, we recommend that you perform the sequencing 
reaction on both strands of the amplicon (bidirectional sequencing) and the generated sequence 
should meet all of the following acceptance criteria: 
  Sequence contains a minimum of 100 contiguous bases,  
  Bases 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), and  
  Sequence matches the reference or consensus sequence, e.g. Expected Value (E-Value) < 
10-30 for the specific target for a BLAST search in GenBank, 
(http://www.ncbi.nlm.nih.gov/Genbank/).   
As Next Generation Sequencing (NGS), also known as High Throughput Sequencing (HTS), 
technologies evolve and mature, they may also be used in the composite reference method.  
Comparator methods based on these technologies should be validated and should meet pre-
17  Contains Nonbinding Recommendations 
 
 
specified quality metrics.  Please contact the FDA Division of Microbiology Devices for 
additional information on the use of the NGS/HTS methods in evaluation of your clinical data. 
A comparison against an FDA-approved HPV genotyping test, or PCR/Sequencing is especially 
important for HPV genotyping assays to establish that the correct HPV genotype has been 
identified by your device.   
Please note that there are two scenarios in which the samples are found negative by the HPV test 
when the clinical cutoff is set above the LoB: 1) the HPV test detected some amount of analyte 
(analyte level is above the LoB) but this amount was below the clinical cutoff that is used to 
define positive and negative results (Detected in Table 3 below = LoB<signal<clinical 
cutoff) or 2) the HPV test did not detect the analyte of interest (Not Detected in Table 3 
below = signalLoB).  For the comparison of the HPV test and an appropriate comparator 
discussed above, please describe whether the analyte was detected or not detected for the 
samples negative by the HPV test as defined above.  You should present the comparison for 
ASC-US (Atypical Squamous Cells of Undetermined Significance) and NILM (Negative for 
Intraepithelial Lesion or Malignancy) 30 populations separately in tables.  For a test with only a 
primary screening indication for population of women 25 years, present the data for NILM and 
ASC-US separately and also for an all-comers population which includes NILM, ASC-US and 
UNSAT (Unsatisfactory cytology results).  An example of a data presentation format is provided 
below in Table 3. 
Table 3.  Example of a Data Presentation Format of HPV Detection in the Clinical 
Dataset. 
Comparator Result  Total 
  High Risk  High Risk  Indeterminate 
Positive  Negative 
HPV Positive 
HPV   Detected 
Negative  Not Detected 
Other  
(Invalid) 
Total 
Evaluation of HPV detection should be presented for each testing site separately and for each 
type of collection media separately.  For the differentiation of HPV genotyping tests, you should 
present the data comparing all outputs of the HPV test versus the same outputs for the 
comparator in a table separately for the ASC-US and NILM 30 populations.  For details, please 
see Section 9 of CLSI MM17-A: Verification and Validation of Multiplex Nucleic Acid 
Assays [Ref. 16].  
An example of a data presentation format for an HPV genotyping test with five possible 
outcomes: HPV16 Positive, HPV18 Positive, HPV16 & HPV18 Positive, Negative, and Invalid 
(Indeterminate), is provided below in Table 4:    
                     
18  Contains Nonbinding Recommendations 
 
 
Table 4.  Example of a Data Presentation Format for HPV Genotyping Test. 
Comparator Result 
No  One High Risk  Two High Risk Types  Multiple 
High  Type  High 
Risk  Risk 
Types  Types 
16  18  Other  16&18  16&Other  18&Other  Other 
Pos:HPV16 
Pos:HPV18 
Pos:HPV16&18 
Negative 
Other 
Invalid/Indeterminate 
Total 
B. Clinical Performance Studies 
You should provide in your PMA clinical performance studies that conform to the following 
recommendations. 
(1) Consideration of the Cervical Cancer Screening Guidelines 
Professional cervical cancer screening guidelines help define the role that an HPV device will 
play in the larger scheme of patient management and are therefore useful in assessing any 
intended use statement for an HPV device and its supporting data.  The guidelines that will be 
considered in this guidance are the 2006 Consensus Guidelines for the Management of Women 
with Abnormal Cervical Cancer Screening Tests (2006 consensus guidelines) [Ref. 13], along 
with the 2012 update to these guidelines (2012 consensus guidelines) [Refs. 23 and 24], which 
are the most current consensus guidelines available on cervical cancer screening to date.   
Consideration should be given to the latest version of the guidelines as the recommendations 
may change. 
Although professional guidelines are considered in FDAs evaluation, intended uses given for an 
HPV test are supported primarily by the data submitted for test approval and are generally 
limited to the populations and sample types evaluated.  Studies should be focused on establishing 
a womans risk for cervical disease in a given population stratified by the HPV test outcomes. 
Intended uses for an HPV test may be written more generally (such as the adjunct intended use 
below) to allow clinicians the flexibility to utilize this risk information as they deem appropriate, 
particularly in the development of future cervical cancer screening guidelines.    
 
 
                                                        
19  Contains Nonbinding Recommendations 
 
 
(2) Intended Use 
The proposed intended use of your device should drive your clinical study design to assess 
performance, as your intended use will ultimately determine how FDA will review your data.  
Below is an example of an intended use statement that could be appropriate for a device for 
detection of HPV: 
The [trade name] HPV Test is a [technology or type of assay] assay for the qualitative 
detection of high-risk types of human papillomavirus (HPV) [indicate target, such as 
DNA, RNA transcript or protein] in cervical specimens.  The HPV types detected by the 
assay are the high-risk HPV types [list types indicate whether test can identify specific 
types].  Cervical specimens that may be tested with the [trade name] HPV Test include 
[insert sample types that may be tested by assay and types of collection devices which 
may be used to collect the samples]. 
The use of this test is indicated: 
1.  To screen patients 21 years and older with atypical squamous cells of undetermined 
significance (ASC-US) cervical cytology results to determine the need for referral to 
colposcopy.   
2.  In women 30 years and older the [trade name] Test can be used with cervical cytology to 
adjunctively screen to assess the presence or absence of high-risk HPV types.  This 
information, together with the physicians assessment of cytology history, other risk 
factors, and professional guidelines, may be used to guide patient management.  
Below is an example of an intended use statement that could be appropriate for a device for 
detection and differentiation of HPV: 
3.  In women 25 years and older, the [trade name] Test can be used as a firstline primary 
cervical cancer screening test to detect high-risk HPV, including genotyping for 16 and 
18.  Women who test negative for high-risk HPV types by the [trade name] Test should 
be followed up in accordance with the physicians assessment of screening and medical 
history, other risk factors, and professional guidelines.  Women who test positive for 
HPV genotypes 16 and/or 18 by the [trade name] Test should be referred to colposcopy.   
Women who test high-risk HPV positive and 16/18 negative by the [trade name] Test 
should be evaluated by cervical cytology to determine the need for referral to colposcopy. 
The first intended use will be referred to as the ASC-US triage intended use, the second 
will be referred to as the adjunct intended use and the third will be referred to as the 
primary screening intended use throughout this guidance.  Study design considerations for 
specific intended uses are described below, following the more general study design 
recommendations. 
 
20  Contains Nonbinding Recommendations 
 
 
(3) Study Design Considerations Common to ASC-US Triage, Adjunct and 
Primary Screening Intended Uses (and likely any other intended uses): 
For general study design guidance, see FDAs guidance entitled Design Considerations for 
Pivotal Clinical Investigations for Medical Devices 
(http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocu
ments/UCM373766.pdf) and FDAs guidance entitled Statistical Guidance on Reporting 
Results from Studies Evaluating Diagnostic Tests 
(http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm0
71148.htm). 
a.  Use of study sites outside the United States (21 CFR 814.15) 
If you rely on clinical data gathered in a study conducted outside the United States to support 
your PMA and not conducted under an investigational device exemption (IDE), you must ensure 
that the data are scientifically valid and that the rights, safety, and welfare of human subjects 
have been protected in accordance with 21 CFR 814.15.  To serve as the sole basis for marketing 
approval, your data must be applicable to the intended use population and the United States 
medical practice (21 CFR 814.15(d)(1)).  Areas of concern for studies conducted outside the US 
include prevalence of specific high-risk HPV types, patient screening intervals, average age of 
onset of screening and sexual activity, cervical cancer risk, cervical sampling methods, 
differences in medical or clinical practice, and ethnicity.  We encourage contacting FDA through 
the pre-submission process if you intend to seek approval based on foreign data, thus reducing 
the risk that the foreign study will not support your intended uses.   
For additional information about the pre-submission process, see FDAs guidance entitled 
Requests for Feedback on Medical Device Submissions: The Pre-Submission Program and 
Meetings with Food and Drug Administration Staff 
(http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocu
ments/UCM311176.pdf) prior to beginning the study.   
b.  Histology review 
FDA considers results of colposcopy and biopsy (if necessary) to be the clinical reference 
standard (i.e., gold standard) for the disease assessment of subjects in the clinical study. You 
may choose to use histology results generated at each of your clinical sites, but we recommend a 
centralized three expert pathologist review (CPR) panel that will likely generate a more 
consistent and accurate disease assessment for your study.  The three pathologists should 
distinguish between Cervical Intraepithelial Neoplasia (CIN) 2 and 3, and should not combine 
these two categories together for reporting purposes (i.e., results of CIN2/3 should not be 
reported).  If you choose to use a centralized panel and/or clinical sites that utilize the new 2-
tiered Lower Anogenital Squamous Terminology (LAST) recommendations [Ref. 25] for 
reporting the results of cervical histology (as low-grade squamous intraepithelial lesion (LSIL) 
and high-grade squamous intraepithelial lesion (HSIL), such that immunohistochemical staining 
for the biomarker p16 is used to clarify any considered intermediate category (CIN2) into either 
LSIL or HSIL, then the 3-tiered result (i.e. CIN1, CIN2, or CIN3) based on the review of 
21  Contains Nonbinding Recommendations 
 
 
hematoxylin and eosin (H&E) stained slides should be denoted along with the 2-tiered diagnosis 
which will be based on the review of the H&E and p16 stained slides  for every patient.   You 
should also provide FDA with the analytical validation data for any non-FDA approved p16 
assay utilized for cervical histology in your study.  Please notify FDA prior to beginning a study 
using the LAST recommendations or any other histology reporting that utilizes the p16 
biomarker. 
We recommend that the CPR panel establish the clinical reference standard (i.e., clinical truth) 
for the subject and that two of the three expert pathologists review the slide independently in a 
masked fashion.  If the two pathologists agree, the diagnosis should be considered the clinical 
reference standard.  If there is no agreement, the third expert pathologist should read the slide 
independently in a masked manner.  If there is agreement among any of the three expert 
pathologist diagnoses, this should be considered the clinical reference standard for the subject.  If 
there is no agreement after the third pathologist review, all three expert pathologists should 
review the slide together at a multi-headed microscope (or equivalent technology) to try and 
reach a consensus diagnosis (with majority rule of 2 of the 3 if a complete consensus cannot be 
reached).  When submitting your data, you should provide information on how discordant 
histology results were resolved. If your CPR panel and/or clinical sites are utilizing the new 2-
tiered LAST recommendations, a consensus should be reached among 2 of the 3 pathologists 
regarding what the preliminary histopathology diagnosis is prior to p16 staining (i.e., CIN1, 
CIN2, CIN3) using the same method for establishing clinical truth among the pathologists as 
when p16 is not used, as described above.  A final consensus should be reached after the p16 
staining is conducted. 
c.  Cytology reporting terminology 
Collection sites should utilize cytology reporting terminology that can be translated to the 2014 
Bethesda System for Reporting Cervical Cytology (2014 Bethesda System), or a more current 
Bethesda system if and when available [Ref. 14].  Cytology results should be converted to the 
2014 (or more current) Bethesda system before reporting the results to FDA. 
d.  Blinding 
Investigators, patients, and clinicians (including those conducting colposcopy and histology) 
should be blinded to a patients HPV status until colposcopy/histology is completed to avoid bias 
in the study.  Additional blinding is recommended for HPV primary screening studies as 
described under Primary Screening Intended Use in Section VII(B)(8) below. 
Also, the protocol should clearly specify what test results will ultimately be released to the 
physician and patient as well as under what circumstances the cytology and HPV results will 
become unblinded as that could inadvertently bias your follow-up study.   
e.  Human papillomavirus genotypes 
For an assay to detect high risk human papillomavirus, the following genotypes categorized as 
carcinogenic by the World Health Organization International Agency for Research on Cancer 
22  Contains Nonbinding Recommendations 
 
 
(IARC) should be targeted: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 [Ref. 15].  If your 
assay does not target any of these recommended HPV genotypes, you should explain why.  
Additional genotypes, such as those deemed probably carcinogenic or possibly carcinogenic 
by IARC (i.e. types 66, 68) may also be included.  We recommend that you discuss with FDA 
the benefits and risks of inclusion of any other human papillomavirus genotypes prior to 
beginning your studies.   
f.  Specimen collection media 
We recommend you perform the described analytical and clinical studies for each type of 
specimen collection media (i.e., specific brand of liquid-based-cytology collection fluid) claimed 
in your intended use.  Clinical performance should be presented for each collection media 
separately.    
g.  Specimen collection devices 
The list of collection devices that may be used to collect specimens for testing by your device 
should be described in your intended use statement and should be approved for use with your 
indicated cytology method(s).  Each claimed collection device (i.e., brush/spatula vs. broom) 
need not be evaluated in your analytical studies.  However, each indicated collection device 
should be evaluated in your clinical studies.  Clinical performance should be presented for each 
collection device separately.    
h.  Specimen collection  general  
For each specimen collected in your clinical study you should capture the date the specimen was 
collected from the patient, the date it was shipped to and received by the testing laboratory, and 
the date the specimen was tested. 
i.  Biopsy methods 
The biopsy methods utilized should be consistent for all patients and all sites within each study.  
If separate studies are conducted for distinct indications (e.g., ASC-US triage vs. adjunct), then 
different biopsy methods may be used for each study.  If the biopsy method is not consistent 
within a dataset for a given indication, it may lead to bias in your study that may prevent proper 
establishment of your performance characteristics for that indication.  A standardized biopsy 
method can have variables associated with it, but these variables should be associated with the 
appearance of the cervix upon visualization during colposcopy, such as the presence or absence 
of visible lesions, or the visibility of the squamocolumnar junction (SCJ).  If additional variables 
are desired, you should discuss them with FDA prior to beginning your studies.  Note that 
biopsies taken from lesioned and non-lesioned areas should be denoted differently on your case 
report forms. 
 
 
 
23  Contains Nonbinding Recommendations 
 
 
j.  Cytology sample aliquoting 
Sponsors pursuing intended uses for HPV testing from cytology samples should consider, when 
designing their studies, whether they should be testing from pre-aliquoted cytology samples 
(aliquot taken prior to slide processing) or working from residual cytology samples (aliquot 
taken after slide processing).  Pre-aliquoting of cytology samples should only occur if the 
cytology collection system has been approved for aliquot removal prior to cytology slide 
processing.  This will ensure that patient cytology test results are not compromised by 
inappropriate processing of their cytology specimens.   
Alternatively, sponsors who seek a claim to work from residual cytology specimens should 
analytically assess the effects of carryover during cytology slide processing (see Section 
VII(A)(5) Carry-Over and Cross-Contamination Studies (for devices with automated liquid 
handling systems)).  Sponsors with amplification assays who have concerns about contamination 
may need to work with alternative specimen collection systems or systems approved for pre-
aliquoting to address their contamination issues.  Sponsors who perform their clinical studies on 
pre-aliquoted cytology specimens who subsequently seek a claim to test residual cytology 
specimens should compare the results of a panel of paired pre-aliquoted and residual real clinical 
cytology specimens, in addition to conducting the analytical carryover and cross contamination 
studies discussed above. 
k.  Reporting results for HPV genotyping assays 
Results should be reported in a manner readily interpretable by clinicians.  Groups of HPV 
genotypes with similar risk levels should be reported in groups, instead of individually, where 
appropriate.   
In your PMA, you should describe how each of the reported results or invalid results are 
determined, and how they should be interpreted.  You should indicate the cut-off values for all 
outputs of the assay. 
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). 
l.  HPV vaccination and study populations 
When making sample size estimations, you should consider that as the number of HPV-
vaccinated individuals increases, this will lead to a decrease in the overall prevalence of cervical 
disease in the United States.  Current estimates of vaccine rates and disease prevalence should be 
taken into account when estimating study sample size.  Inclusion of study sites with higher than 
average levels of non-vaccinated individuals may eventually become advisable as the number of 
vaccinated individuals across the US increases.  Please note that, in this scenario, study sites with 
24  Contains Nonbinding Recommendations 
 
 
average levels of vaccinated individuals should also be evaluated.  Sponsors considering this 
type of design should discuss this option with FDA before beginning their studies.  
(4) ASC-US Triage Intended Use  
You should conduct prospective clinical studies using specimens representing the intended use 
population, i.e. patients with ASC-US cervical cytology results, to determine clinical 
performance of your device for all specimen types and specimen collection devices you claim in 
your labeling.  The study should be representative of the entire intended use population, 
including with regard to age.  The clinical performance of a qualitative test (test with two 
outcomes, Positive or Negative) is described by its clinical sensitivity and specificity, positive 
and negative predictive values, and prevalence.  The clinical sensitivity of your device is the 
proportion of individuals who have precancer or cancer [greater than or equal to Cervical 
Intraepithelial Neoplasia 2 (CIN2)2] that are positive by your test.  The clinical specificity of 
your device is the proportion of individuals who do not have precancer or cancer (<CIN2) that 
are negative by your test.  These performance characteristics should be established in prospective 
clinical studies conducted at a minimum of three study sites that are representative of clinical 
sites in the United States.  For a test with more than two outcomes, clinical performance is 
described by likelihood ratios, percentage of subjects in each outcome and prevalence.  
a.  Specimen collection and processing 
Proper specimen collection and processing is critical for establishing the performance 
characteristics of an HPV test.  For an ASC-US triage intended use, the population of women 
studied should be recruited from Ob/Gyn clinics.  Please note that colposcopy clinics are not 
good sources of patients for an ASC-US triage evaluation, as the women who present at 
colposcopy clinics have already been determined to be in need of colposcopy (i.e., have already 
been determined to be HPV positive by other tests, or repeat ASC-US by cytology).  Since 
women who are already known to need colposcopy are not the target population for the ASC-US 
triage intended use, this population should not be used for your study, as the performance 
estimates derived would be inaccurate.  The population of women who present at a colposcopy 
clinic has a higher prevalence of both HPV infection and cervical disease and, due to verification 
bias, device sensitivity would be overstated.   
For tests that are to be performed directly from liquid-based cytology (LBC) specimens, all 
investigative HPV test results should be performed on the same LBC sample that was used to 
generate the cytology result.  This will enable you to avoid any sampling bias in your study (i.e., 
infections that may resolve between the time the original cytology sample and investigative 
sample are taken, removal of a large portion of the HPV infected cells in the first sample, etc.).  
Although one approach to mitigating sampling bias when collecting an extra sample is to 
                                                 
2 Please note that for all places in this document where data for the CIN2 target condition is requested, data for the 
CIN3 target condition should also be presented since CIN3 is more likely to progress to cervical cancer.  If you 
utilize the 2-tiered LAST system, you should also present data for HSIL and above.  HSIL in the LAST system is 
different from HSIL in the 2014 Bethesda System for Reporting Cervical Cytology and this distinction should be 
made clear by indicating for each table containing HSIL results whether cytology or biopsy results are being 
reported. 
25  Contains Nonbinding Recommendations 
 
 
randomize the test procedures performed on the two samples (i.e., cytology and HPV testing), 
this is not an acceptable approach for generating a cytology result in patients.  The first cytology 
sample taken from a patient should always be the sample utilized to generate a cytology result, 
so that this result (and subsequently, the health of the patient) is not compromised.  Therefore, 
randomizing testing on two cytology samples would not mitigate sampling bias for HPV studies.  
One challenge in enrolling patients from Ob/Gyn clinics as opposed to colposcopy clinics is 
fielding the large number of women who are not part of the intended use population.  If you are 
conducting a large study to support multiple HPV testing intended uses, it may be advisable to 
enroll all women, regardless of cytology status, into your study.  Another option, if the ASC-US 
triage intended use is to be pursued in a separate study, is to enroll only patients with ASC-US 
cervical cytology results into your prospective clinical study.  When utilizing the latter approach, 
it is important to establish a procedure for obtaining the original cytology sample that was 
originally used to generate the enrollment ASC-US result in order to avoid sampling bias as 
described above.   
b.  Clinical reference (Gold) standard   
Your study should be designed such that all women with ASC-US cytology from Ob/Gyn clinics 
will proceed to colposcopy, regardless of HPV status or other factors.  Investigators, patients and 
clinicians (including those conducting colposcopy and histology) should be blinded to a patients 
HPV status until colposcopy/histology is completed to avoid bias in the study. 
Time elapsed between collection of a screening cervical cytology specimen and subsequent 
colposcopy procedures should not exceed 12 weeks.  Allowing too much time between these 
procedures could result in higher than normal rates of spontaneous regression of HPV infections 
and their associated cervical lesions, which will adversely affect your estimates of clinical 
sensitivity and specificity.  
You should describe details of the colposcopy procedures used in your clinical study and the 
results of the colposcopy procedures should be categorized as (Negative Colposcopy/No 
Biopsy), Negative Biopsy, CIN 1, CIN 2, CIN 3, Adenocarcinoma In-Situ, and Cancer.  If you 
utilize a 2-tiered LAST system, in addition, you should present the results of the colposcopy 
procedure as (Negative Colposcopy/No Biopsy), Negative Biopsy, LSIL, HSIL, and Cancer.  
c.  Clinical performance evaluation of HPV tests  
The clinical performance of a test for the detection of HPV (a qualitative test) is described by its 
clinical sensitivity and specificity, and by its positive and negative predictive values, along with 
the prevalence of the target condition in the intended use population.   
An example of an acceptable data presentation format for a qualitative test with two outcomes 
(Positive and Negative) is provided below in Table 5: 
 
 
26  Contains Nonbinding Recommendations 
 
 
 
Table 5.Table for Qualitative HPV Test Results 
Central Histology 
Neg  Neg  CIN1  CIN2  CIN3  Cancer 
Colpo 
HPV  A   A   A   A   A   A   A +A +A +A +A +A  
1 2 3 4 5 6 1 2 3 4 5 6
Pos  
HPV  B   B   B   B   B   B   B +B +B +B +B +B  
1 2 3 4 5 6 1 2 3 4 5 6
Neg  
Total  A +B   A +B   A +B   A +B   A +B   A +B   N 
1 1 2 2 3 3 4 4 5 5 6 6
  
We recommend that in addition to the information in Table 5 above, you provide additional 
information for the conditions CIN2 and above (CIN2) with HPV negative results by the 
investigational HPV device.  Please include footnote(s) describing comparator results (i.e., 
sequence positive/negative) for each subjects contributing to B , B , and B  
4 5 6,
The clinical performance of your device for the target condition CIN2 and above (CIN2) 
should be evaluated as follows: 
Sensitivity = (A +A +A )/(A +A +A +B +B +B ); 
4 5 6 4 5 6 4 5 6
Specificity = (B +B +B )/(A +A +A +B +B +B ) 
1 2 3 1 2 3 1 2 3
Positive Predictive Value (PPV)=(A +A +A )/(A +A +A +A +A +A ) 
4 5 6 1 2 3 4 5 6
Negative Predictive Value (NPV)=(B +B +B )/(B +B +B +B +B +B ) 
1 2 3 1 2 3 4 5 6
Prevalence of CIN2=(A +A +A +B +B +B )/N 
4 5 6 4 5 6
Since CIN3 lesions are more likely to progress to cervical cancer than CIN2 lesions [Ref. 17], 
the clinical performance of your device for the target condition CIN3 and above (CIN3) 
should also be presented: 
Sensitivity = (A +A )/(A +A +B +B ); 
5 6 5 6 5 6
Specificity = (B +B +B +B )/(A +A +A +A +B +B +B +B ) 
1 2 3 4 1 2 3 4 1 2 3 4
PPV=(A +A )/(A +A +A +A +A +A ) 
5 6 1 2 3 4 5 6
NPV=(B +B +B +B )/(B +B +B +B +B +B ) 
1 2 3 4 1 2 3 4 5 6
Prevalence of CIN3=(A +A +B +B )/N 
5 6 5 6
The estimates of sensitivity and specificity as well as positive and negative predictive values 
should be provided along with 95% two-sided confidence intervals.  For the 95% confidence 
intervals for sensitivity and specificity, a score method is recommended.  For more details about 
score confidence intervals, see Section IX Appendix  Statistical Analysis and CLSI EP12-A2: 
User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline [Ref. 8].  
The confidence intervals for the predictive values can be calculated (when prevalence is 
constant) based on the confidence intervals of the corresponding likelihood ratios (an estimate of 
the likelihood ratio is a ratio of two independent proportions; therefore, the confidence intervals 
for a ratio of two independent proportions can be used; see Section IX  Appendix  Statistical 
Analysis). 
   
27  Contains Nonbinding Recommendations 
 
 
The clinical performance for the target condition CIN2 should be stratified by age.  The 
prevalence of CIN2, sensitivity, specificity, PPV and NPV along with 95% CI should be 
presented for each of the following age groups in accordance with screening guidelines: 21-30 
(and stratified further: 21-24 and 25-29), 30-39, and >39. 
d.  Sample size  
When considering sample size for an ASC-US triage intended use, one should consider the 
number of samples from ASC-US patients needed to establish point estimates of clinical 
sensitivity and specificity, along with the lower limits of 95% two-sided confidence intervals.  
Clinical sensitivity for cervical disease (CIN2 and CIN3) is the most critical performance 
parameter for an HPV test, since a false negative HPV test result could lead to delays in cervical 
cancer detection and treatment [Ref. 13].   
If the estimated clinical sensitivity, specificity and subsequent positive and negative predictive 
value(s) of your device do not meet current performance expectations for HPV testing [Ref. 23 
and 24], panel review of your performance data may be necessary to allow assessment of the 
clinical effectiveness of your test. 
e.  Selection of appropriate clinical cutoff for HPV tests  
Selection of the appropriate clinical cutoff can be justified by the relevant levels of sensitivity 
and specificity that are based on Receiver Operating Curve (ROC) analysis of pilot studies with 
clinical samples.  The clinical performance of the HPV test at the selected clinical cutoff is 
ideally estimated using a pivotal clinical study. In some circumstances, the clinical cutoff can be 
determined during the pivotal clinical study using an unbiased procedure and appropriate sample 
size.  If the level of sensitivity that is clinically acceptable is pre-specified (e.g., the level of 
sensitivity of 93%-95% is clinically acceptable in the intended use population), then the pivotal 
study can be used to establish the clinical cutoff corresponding to the pre-specified level of 
sensitivity and to obtain an unbiased estimation of the clinical performance of the HPV test with 
this selected cutoff [Ref. 18 & Ref. 19].  
(5) ASC-US Population - HPV Tests for Detection and Differentiation 
(HPV Genotyping Tests) 
The study principles described in the preceding Section VII(B)(4) ASC-US Triage Intended Use 
to establish clinical sensitivity and specificity for CIN2 in women with ASC-US cytology apply 
to both dual outcome HPV tests (positive or negative for HPV) and multiple outcome HPV 
genotyping tests.   
A test for the detection and differentiation of HPV genotypes usually has multiple outcomes 
(e.g., HPV16+, HPV18+, HPV16/18+, etc.).  The clinical performance of a test for the detection 
and differentiation of HPV genotypes is described by the probabilities of a target condition for 
each outcome of the test, as well as the percent of study subjects with each outcome of the test 
along with disease prevalence for each outcome.  
28  Contains Nonbinding Recommendations 
 
 
In addition to establishing clinical sensitivity and specificity for CIN2 in an ASC-US 
population for an HPV genotyping test, likelihood ratios for each test outcome and the percent of 
study subjects with each test outcome should also be established as described below. 
An example of an acceptable data presentation format for a HPV genotyping clinical study in the 
ASC-US population is provided below in Table 6 (Example shown has outcomes: HPV16+, 
HPV18+, HPV16/18+, etc.): 
Table 6.Data Presentation for HPV Genotyping Test Results 
Central Histology 
Neg  Neg  CIN1  CIN2  CIN3 
Colpo 
Pos:HPV16  A   A   A   A   A   A +A +A +A +A  
11 12 13 14 15 11 12 13 14 15
Pos:HPV18   A   A   A   A   A   A +A +A +A +A  
21 22 23 24 25 21 22 23 24 25
Pos:HPV16&18  A   A   A   A   A   A +A +A +A +A  
31 32 33 34 35 31 32 33 34 35
......  ......  ......  ......  .....  .....  ...... 
Total  N 
The clinical performance of such a test for the target condition CIN2 is evaluated by the 
likelihood ratio for each test outcome X and the percent of study subjects with each test outcome.  
The likelihood ratio (LR) for the test outcome X, LR(T=X), summarizes how many times more 
(or less) likely subjects with the disease (CIN2) are to have that particular result X, 
Pr(T=X|D+),  than subjects without the  disease, Pr(T=X|D-): LR(T=X) = 
Pr(T=X|D+)/Pr(T=X|D-).  
The following calculations should also be described in your PMA for HPV genotyping tests: 
  The likelihood ratios for each of K outcomes (K is a number of different outcomes) 
should be calculated along with 95% confidence intervals.   
  In addition to the likelihood ratios, probabilities that a patient has CIN2 for each of the 
K outcomes of the test should be calculated along with 95% confidence intervals.  As an 
illustrative example, the probability for Outcome HPV16 pos is evaluated as:  Probability 
(CIN2|HPV16 pos)=(A +A )/(A +A +A +A +A ). 
14 15 11 12 13 14 15
  Also present the percent of each of K outcomes in the clinical data set. For example, for 
outcome HPV16 pos: Probability (HPV16 pos)=(A +A +A +A +A )/N (where N is 
11 12 13 14 15
the total number of women with results). 
  In addition, probability of CIN2 for the combined outcomes HPV16/18+ (HPV16/18 is 
defined positive if either HPV16+ or HPV18+ or both) should be calculated as: 
Probability (CIN2| HPV16/18+)=(A +A +A +A +A +A )/(A +A +A +A +A
14 15 24 25 34 35 11 12 13 14 15 
+A +A + A +A +A +A +A +A +A +A ) and the percent of the subjects with 
21 22 23 24 25  31 32 33 34 35
HPV16/18+ results.  
  Prevalence of disease (CIN2) should also be calculated. 
The confidence intervals for the probabilities of CIN2 can be calculated based on the 
confidence intervals of the corresponding likelihood ratios.  
        
29  Contains Nonbinding Recommendations 
 
 
In a similar way, the clinical performance of the HPV test should be estimated for the target 
condition CIN3 (or HSIL and above if 2-tiered LAST system is used). 
(6) Adjunct Intended Use  
a.  General study design options   
Per the 2012 consensus guidelines [Ref. 23], in women 30 years and older, HPV testing is 
recommended as an adjunct to cytology primarily in women with normal cytology.   Establishing 
the clinical sensitivity and specificity of your device in a population of women with normal 
cytology is complicated by the fact that these women are not typically sent for colposcopic 
examination at the time when HPV testing is done due to their low incumbent risk of cervical 
cancer.  However, a subset of women with normal cytology will have cervical abnormalities 
(CIN2) [Ref. 20].  HPV testing may help identify the subset of women 30 years and older with 
normal cytology who are at a higher risk for cervical cancer.  To demonstrate that your device is 
capable of identifying this higher risk subset of women, you should estimate the absolute risks 
and the relative risk for CIN2 in this population for individuals positive vs. negative by your 
assay as described below.  Estimating absolute risks and relative risk for this intended use 
population can be accomplished with at least one of the following prospective clinical study 
designs: 
1.  Characterize a population of women 30 years and older with normal cytology as positive 
or negative by your investigative device and establish agreement with a valid comparator 
HPV detection device, such as an FDA-approved HPV detection device, or 
PCR/sequencing at baseline; then follow the women at yearly time intervals for a 
minimum of three years.  See Section VII(A)(8) Evaluation of HPV Detection in the 
Clinical Dataset above for more details on the baseline analysis of the prospective clinical 
dataset with respect to HPV detection.  All women who develop abnormal cytology 
(ASC-US) during follow-up should be sent to colposcopy regardless of their HPV 
status.  Unlike option two below, women with normal cytology are not sent to colposcopy 
with this study design, particularly if the decision to send to colposcopy is based on 
investigative or approved HPV test results3 at the baseline visit or during follow-up (this 
is important for avoiding bias).  Note that follow-up will end for women who have a 
CIN2 colposcopy result at any time during the study and that these women are 
considered disease positive.  Women who go to colposcopy but are <CIN2 should 
continue to be followed for the remainder of the study duration.  The follow-up data 
should demonstrate a statistically and clinically significant difference in relative risk for 
CIN2 at least at three years for women who are positive as compared to women who are 
negative by your device at enrollment.  In addition, the absolute risk of CIN2 at three 
years for the women who were negative by your test at the baseline should be evaluated 
along with 95% confidence interval.  The data should demonstrate that this absolute risk 
is low enough to ensure your test can be safely used for adjunctive screening in women 
30 and older.  In addition, present the overall risk of CIN2 (regardless of the HPV status 
                                                 
3 An exception would be if a woman was twice cytology negative and HPV positive (at consecutive yearly visits)  
in this scenario she should be sent to colposcopy per the 2012 consensus guidelines [Ref. 23].  The bias created in 
this situation is unavoidable as patient health is paramount.   
30  Contains Nonbinding Recommendations 
 
 
at the baseline). The data analysis should be stratified by age groups (30-39 years and 
40+ years).  The longitudinal follow-up portion of this study may potentially be 
conducted post-approval (see Longitudinal follow-up Section VII(B)(6)(c) below for 
more details).   
2.  Characterize a population of women 30 years and older with normal cytology as positive 
or negative by your device at baseline, then send a subset of those women to colposcopy.  
You should also establish agreement with a valid comparator HPV detection device with 
this second study design option, but a smaller subset of samples may be evaluated since 
you will have more information on risk of CIN2 at baseline.  It is recommended you 
send all HPV positive women (by investigative and/or approved tests) and a random 
subset of HPV negative women to colposcopy.  The data should demonstrate a 
statistically and clinically significant difference in relative risk for CIN2 for women 
who are positive vs. negative by your device at enrollment.  Using multiple imputation, 
absolute risks of CIN2 for the subjects positive and negative by your device should be 
calculated.  For the HPV test for detection and differentiation, the data should also 
demonstrate that the absolute risk of CIN2 at the baseline for some positive outcomes is 
high enough to demonstrate effectiveness of the test in the intended use population.  
Because only a random sample of HPV negative women will have been sent to 
colposcopic examination, the data have a verification bias and therefore, an appropriate 
statistical method such as multiple imputation [Ref. 21] should be used for calculation of 
the absolute and relative risks.   
b.  Enrollment of all comers for adjunct claim: 
Please note that FDA now recommends that you enroll a population of women 30 years and 
older undergoing routine screening (all comers) in your clinical study to evaluate performance 
of an adjunct claim.  Women are HPV tested in parallel with cytology under this claim; 
therefore, women with higher grade cytology are included under the adjunct claim.  FDA has 
historically allowed sponsors to submit only the actionable data on the NILM 30 women, but 
FDA is now considering that 1) the results of an HPV test in women with higher grade cytology 
(>ASC-US) is proving to be an important safety signal in terms of how well the test is working 
overall, 2) a study size that includes all comers 30 years old includes approximately 3% of 
women with >ASC-US cytology and these women usually have colposcopy/biopsy results 
according to the current medical practice, and 3) the 2012 guidelines are including more 
actionable combinations of HPV and cytology results that result from co-testing.  In light of this, 
FDA will be evaluating all cytology categories when reviewing data to support the adjunct claim 
going forward to ensure the safety and effectiveness of HPV IVD devices.  Women with ASC-
US cytology in the all comers adjunct study population should be sent immediately to 
colposcopy.  All patients in the adjunct study population who go to colposcopy but do not have 
histology CIN2 by the CPR panel should be invited to participate in the three year longitudinal 
study.   
 
 
 
31  Contains Nonbinding Recommendations 
 
 
c.  Longitudinal follow-up 
Given that establishing clinical sensitivity and specificity in a population of women with normal 
cytology involves either a very large sample size and/or long term patient follow-up, the FDA 
has considered options that would allow faster access to these important devices while assuring 
their safety and efficacy.  The FDA believes that in cases where an HPV test is receiving, or has 
received, approval for the ASC-US triage intended use and where the test has shown a high 
degree of clinical sensitivity for cervical precancer/cancer (CIN2), there is a high degree of 
confidence that the test performs at a level consistent with current expectations for HPV testing 
[Ref. 2].  In such cases, to receive the adjunctive intended use for the same HPV test, FDA may 
provide for the longitudinal follow-up portion of the adjunctive study described in Option 1 of 
Section VII(B)(6)(a) General study design options above to be completed post-market, as long as 
it has been shown that HPV detection by the investigative test versus the comparator in the 
prospectively collected NILM 30 and older (NILM 30) dataset is comparable to HPV detection 
in the ASC-US population.  In this scenario, the same patients from the prospectively collected 
NILM 30 dataset for whom HPV detection characteristics have been established will be 
followed longitudinally as part of a post-approval study to establish the cumulative three year 
risk of precancer/cancer in patients positive vs. negative by the investigative HPV test in this 
population.  This approach will be considered for tests that detect HPV types that are supported 
for use in the NILM 30 population by current clinical practice guidelines [Ref 13].  Please note 
that post-market studies for devices of this type are only appropriate when the degree of 
uncertainty about certain risks or benefits is acceptable in the context of the overall benefit-risk 
profile of the device at the time of premarket approval.  Also, see FDAs guidance entitled, 
Procedures for Handling Post-Approval Studies Imposed by PMA Order 
(http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm0
70974.htm).  Sponsors should contact FDA to discuss their eligibility to complete their 
longitudinal evaluation post-market.  
(7) Adjunct Intended Use  HPV Tests for Detection and Differentiation 
(HPV Genotyping Tests) 
The study options described in the preceding Section VII(B)(6) Adjunct Intended Use to 
establish relative risk for CIN2 in women 30 and over with normal cytology can be applied to 
both dual outcome HPV tests (positive or negative for HPV) and multiple outcome HPV 
genotyping tests (tests that not only detect, but also differentiate between the different HPV 
types).  The more outcomes an HPV genotyping test has, the more challenging it is to 
demonstrate a statistically significant difference in the relative risk of each outcome.   
In light of recommendations in the 2012 consensus guidelines [Ref. 23], an additional option you 
may wish to pursue for an HPV genotyping assay (aside from the more general adjunct screening 
intended use) is a specific NILM 30 colposcopy triage intended use for the highest risk HPV 
genotypes, such as HPV 16 and 18.  The principles of this type of study design and evaluation 
would be very similar to ASC-US triage, except that you would be dealing with a different study 
population and test outcomes.  If you wish to pursue such an intended use, please contact FDA 
for further assistance. 
32  Contains Nonbinding Recommendations 
 
 
a.  HPV testing in women 30 and over with >ASC-US cytology 
In order to allow for parallel cytology and HPV testing, an adjunct intended use does not need to 
be limited to women with normal cytology (i.e., there is no need to wait for the cytology result to 
order an HPV test).  For all HPV devices with an adjunct intended use, the labeling should 
indicate that a negative HPV result for a woman 30 years and older with >ASC-US cytology 
should not prevent women from going to colposcopy.   
(8) Primary Screening Intended Use 
An advisory meeting of the Microbiology Panel was held on March 12, 2014, which resulted in a 
newly approved indication for an HPV diagnostic device: Primary Cervical Cancer Screening.  If 
you are interested in pursuing a primary screening claim, we recommend you review the 
information on this meeting, which can be found on the CDRH Website at 
https://wayback.archive-
it.org/7993/20170405192832/https:/www.fda.gov/AdvisoryCommittees/CommitteesMeetingMat
erials/MedicalDevices/MedicalDevicesAdvisoryCommittee/MicrobiologyDevicesPanel/ucm388
531.htm. 
To evaluate an HPV primary screening indication, you should characterize a population of 
women 25 years and older undergoing routine screening (an all comers approach) as HPV 
positive or negative by your device at baseline, then send a subset of those women to 
colposcopy.   It is recommended you send all HPV positive (by investigative and/or approved 
tests), cytology positive, and a random subset of HPV negative women with normal cytology to 
colposcopy.  Women with unsatisfactory (UNSAT) cytology results should also be sent to 
colposcopy to assess the risk of disease in these women stratified by their HPV test results.   
The data should demonstrate that primary HPV screening with your device shows acceptable 
clinical performance in detecting CIN2 and CIN3 (sensitivity, specificity, PPV, NPV, absolute 
risk and likelihood ratios) when compared to accepted cervical cancer screening methods [Refs. 
13, 23, 24, and 25].  For tests intended for detection and differentiation, the data should also 
demonstrate that the absolute risk of CIN2 at the baseline for positive outcomes is high enough 
and the absolute risk of CIN2 at the baseline for negative outcomes is low enough to 
demonstrate effectiveness of the test in the intended use population.  Because only a random 
sample of HPV negative women will have been sent to colposcopic examination, the data will 
have a verification bias, and therefore, an appropriate statistical method such as multiple 
imputation should be used for calculation of the absolute and relative risks [Ref. 21].   
a.  Longitudinal follow-up 
All patients in the primary screening study population who underwent colposcopic examination 
and did not have histology CIN2 by CPR should be invited to participate in a three year 
longitudinal study.  Subjects in the follow-up study should undergo annual visits for cervical 
sampling for cytology, and all subjects with ASC-US should be invited to proceed to 
colposcopy.  Colposcopy and biopsies should be performed in a standardized manner as 
described in Section VII(B)(3)(b) Histology review above. All cervical biopsies should be 
33  Contains Nonbinding Recommendations 
 
 
examined by the CPR panel.  All subjects with CIN2 by CPR should exit the study and those 
with <CIN2 by CPR should be invited to proceed to the next years follow-up visit.  In order to 
maximize disease ascertainment, it is recommended that an exit colposcopy and, if needed, an 
endocervical curettage (ECC) be considered for all follow-up subjects in Year 3. 
You should consider enrolling a random subset of women who were cytology and HPV negative 
(by the investigative device and at least one FDA approved device) and were not selected for 
colposcopy at baseline in a parallel longitudinal study where these women are sent to colposcopy 
after a 3 year screening interval.  This will allow a more accurate assessment of CIN3 risk over 
a three year screening interval for these women.  Three year risk estimates for CIN3 obtained in 
this manner will not be affected by detection and treatment of CIN2 lesions at baseline. 
b.  Blinding issues for primary screening study 
Investigators, patients, and clinicians (including those conducting colposcopy and histology) 
should be blinded not only to a patients HPV status, but their cytology status as well, until 
colposcopy/histology is completed to avoid bias in the study.  Patients should be flagged as 
requiring colposcopy/histology without specifying the test results associated with the referral. 
Cytologists are intentionally blinded to all other patient test results for a primary HPV screening 
study to avoid biasing their assessment of the cytology slides based on the knowledge of other 
test results (otherwise performance of cytology alone as a comparator algorithm could be 
potentially biased).  However, cytology performance could be different in a real-life setting in 
the context of using an HPV test as a primary screening device when cytologists know the HPV 
status of the specimens they are screening.  To assess how different the performance of HPV 
primary screening with the investigative device could be in this real-life setting, a subset of 
cytology slides should be re-read at the testing sites with knowledge of the HPV status available 
at the time of the repeat reading.  The effect of this un-blinding on the performance of your 
device for HPV primary screening should be determined. 
c.  Benefit-risk analysis 
You should evaluate the benefit-risk of your HPV primary screening device for detection of 
high-grade cervical disease (CIN2, CIN3) relative to current screening methods.  To permit an 
evaluation of benefit-risk, you should present estimates of the expected number of tests and 
procedures (number of cytology, HPV tests, and colposcopy procedures), and the expected 
number of true positives, false positives, and false negatives per 10,000 screened women for both 
your device and current screening method(s).  This analysis should be performed considering 
both blinded and un-blinded performance estimates for HPV primary screening with regard to 
the cytologists knowledge of HPV results.  For more information, please see FDAs guidance 
entitled Factors to Consider When Making Benefit-Risk Determinations in Medical Device 
Premarket Approval and De Novo Classifications at 
(http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocu
ments/UCM517504.pdf). 
 
34  Contains Nonbinding Recommendations 
 
 
 
d.  Performance in women with cancer 
Sponsors seeking a primary screening claim should ensure that their test is highly sensitive not 
only for cervical pre-cancer, such as CIN2 and CIN3, but for cervical cancer itself.  Therefore, 
additional testing with the investigational device should be conducted on cytology samples 
collected from females who have subsequently been diagnosed with cervical cancer, using 
banked samples from the United States.  We recommend submitting a separate protocol for this 
study to FDA for review using FDAs Pre-Submission program.  For information on how FDAs 
Pre-Submission program, please see FDAs guidance entitled Requests for Feedback on 
Medical Device Submissions: The Pre-Submission Program and Meetings with Food and Drug 
Administration Staff 
(http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocu
ments/UCM311176.pdf)   In your protocol, please provide information on the collection of these 
samples and a detailed description of how you plan to blind these samples.   
e.  General considerations 
Although the basic elements of an HPV primary screening study are provided above, this type of 
study is complex and therefore, it is strongly recommended that any sponsor seeking an HPV 
primary screening indication should submit their detailed clinical protocol to FDA for review via 
the Pre-Submission Program.   
 
(9) Study Design to Cover All Three HPV Testing Claims (ASC-US Triage, 
Adjunct and HPV Primary Screening) 
Please note that the study design described above to evaluate the HPV Primary Screening 
intended use can also be used to evaluate the ASC-US Triage and Adjunct intended uses, noting 
that some additional women will need to be enrolled who are 21-24 years old with ASC-US 
cytology.  These young women with ASC-US can be invited into the study after their cytology 
result is known, and therefore, the number of additional women needed to evaluate all three HPV 
testing claims in a single study will be minimal (i.e. beyond the number needed for a primary 
screening claim).  Please see recommendations under Section VII(B)(4) ASC-US Triage 
Intended Use above for considerations when evaluating ASC-US Triage performance from a 
population of women enrolled based on their cytology result (in particular recommendations on 
the source of these specimens).  
C. Controls 
When conducting the performance studies described above, we recommend that you run 
appropriate external controls every day of testing for the duration of the analytical and clinical 
studies.  Since HPV cannot be readily cultured, appropriate external controls include HPV 
genomic DNA contained within plasmids or synthetic HPV RNA transcripts (depending on 
35  Contains Nonbinding Recommendations 
 
 
whether your test targets HPV DNA or RNA) in a matrix that mimics clinical samples as closely 
as possible.  The HPV genotype(s) selected for use in your controls should be among the most 
clinically relevant HPV genotypes (e.g., HPV 16).  As the clinical significance of HPV strains 
shift due to vaccination programs, appropriate control sequences may need to be re-assessed.   
We recommend that you consult with FDA when designing specific controls for your device.  If 
your device is based on nucleic acid technology, we recommend that you include the following 
types of controls: 
(1) External Controls 
a.  Negative control  
The negative external control contains an appropriate buffer or sample transport media and is run 
through the entire assay process in the same manner as a clinical specimen.  This control is used 
to rule out contamination with target nucleic acid or increased background in the amplification 
and/or detection reaction.   
b.  Positive control  
The positive external control contains target nucleic acids at levels approximately two-fold above 
the C  concentration of the assay in an appropriate buffer or sample transport media, and is run 
95
through the entire assay process in the same manner as a clinical specimen.  For a test that targets 
HPV DNA, the cloned HPV 16 genome in carrier plasmid DNA suspended in sample transport 
media would be an appropriate control.  The complete targeted conserved region of the HPV 16 
genome, such as the L1 region, can also be utilized in lieu of a full-length genomic clone.  For a 
test that targets HPV RNA transcripts, synthetic full-length transcripts of the targeted genes 
suspended in sample transport media would be an appropriate control.  For controls with analyte 
levels that do not adequately challenge medical decision points, as part of ensuring compliance 
with 21 CFR 809.10(b)(8)(vi), the following warning should be included in the labeling: 
The Positive and Negative Controls are intended to monitor for substantial reagent failure.  The 
Positive Control should not be used as an indicator for cut-off precision and only ensures reagent 
functionality.  Quality control requirements must be performed in conformance with local, state 
and/or federal regulations or accreditation requirements and your laboratorys standard Quality 
Control procedures.  
For manufacturers that do not wish to provide external controls, instructions should be included 
in the package insert to instruct end-users how to make their own external controls.  This option 
is only appropriate for devices that include an internal positive control for all samples and that do 
not have a primary screening claim. 
(2) Internal Control 
The internal control is a non-target nucleic acid sequence that is co-processed (i.e., extracted and 
amplified) with the target nucleic acid.  It controls for integrity of the reagents (polymerase, 
36  Contains Nonbinding Recommendations 
 
 
primers, etc.), equipment function (thermal cycler), and the presence of inhibitors in the samples.   
Examples of acceptable internal control materials include human nucleic acid co-processed with 
the HPV and primers amplifying human housekeeping genes (e.g., RNaseP, -actin).  An internal 
control for a human "housekeeping gene may also help ensure adequate cellular sampling of the 
aliquot material.  This type of control is needed for all HPV devices with a primary screening 
claim to help reduce the likelihood of a false negative result; otherwise, the need for this control 
should be determined on a device case-by-case basis [Ref. 22].   
VIII.  References  
1.  Bernard HU, Burk RD, Chen Z, van Doorslaer K, zur Hausen H, de Villiers EM. 
Classification of Papillomaviruses (PVs) Based on 189 PV types and Proposal of Taxonomic 
Amendments. Virology. 2010; 401(1): 70-79. 
2.  Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, 
Peto J, Meijer CJ, Munoz.  Human papillomavirus is a necessary cause of invasive cervical 
cancer worldwide.  Journal of Pathology. 1999; 189(1):12-19. 
3.  Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer 
CJ, International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. 
Epidemiologic Classification of Human Papillomavirus Types Associated with Cervical 
Cancer. New England Journal of Medicine. 2003; 348(6):518527. 
4.  Clinical and Laboratory Standards Institute. 2012. Evaluation of Detection Capability for 
Clinical Laboratory Measurement Procedures; Approved Guideline  Second Edition. EP17-
A2. 
5.  Linnet, K. and Kondratovich, M. Partly nonparametric approach for determining the limit of 
detection. Clinical Chemistry. 2004; 50(4), 732-740. 
6.  Weusten J.J. A statistical approach to assess analytical sensitivity of diagnostic assays in the 
presence of false-positive results. Journal of Virological Methods; 151(2):301-10.  
7.  Clinical and Laboratory Standards Institute. 2014. Evaluation of Precision of Quantitative 
Measurement Procedures; Approved Guideline. EP05-A3. Clinical and Laboratory Standards 
Institute, Wayne PA. 
8.  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. 
9.  Clinical and Laboratory Standards Institute. 2014. User Verification of Precision and 
Estimation of Bias; Approved Guideline. EP15-A3. Clinical and Laboratory Standards 
Institute, Wayne PA. 
37  Contains Nonbinding Recommendations 
 
 
10. Clinical and Laboratory Standards Institute. 2005. Interference Testing in Clinical Chemistry; 
Approved Guideline. EP07-A2. Clinical and Laboratory Standards Institute, Wayne PA. 
11. Haeckel R. Proposals for the description and measurement of carry-over effects in clinical 
chemistry.  Pure Applied Chemistry 1991; 63:301-306. 
12. Clinical and Laboratory Standards Institute. 2009.  Evaluation of Stability of In Vitro 
Diagnostic Reagents; Approved Guideline. EP25-A. Clinical and Laboratory Standards 
Institute, Wayne PA. 
13. Wright T, Massad L, Dunton C, Dunton J, Spitzer M, Wilkinson E, et al.  2006 consensus 
guidelines for the management of women with abnormal cervical cancer screening tests. 
American Journal of Obstetrics and Gynecology. 2007; 197(4):346-55.    
14. Nayar R, Wilbur DC, eds. The Bethesda system for Reporting Cervical Cytology. 
Definitions, Criteria, and Explanatory Notes. 3rd ed. Springer; 2015.  
15. Schiffman M, Clifford G, Buonaguro F.  Classification of weakly carcinogenic human 
papillomavirus types; addressing the limits of epidemiology at the borderline.  Infectious 
Agents and Cancer. 2009; 4:1-8. 
16. Clinical and Laboratory Standards Institute. 2008. Verification and Validation of Multiplex 
Nucleic Acid Assays: Approved Guideline. MM17-A. Clinical and Laboratory Standards 
Institute, Wayne PA. 
17. Schiffman M, Rodriguez AC. Heterogeneity in CIN3 diagnosis. Lancet Oncology. 2008; 
9(5):404-6. 
18. Linnet K., Brandt E.  Assessing diagnostic tests once an optimal cutoff point has been 
selected. Clinical Chemistry. 1986; 32(7): 1341-6. 
19. Kondratovich M, Yousef WA. Evaluation of accuracy and optimal cutoff of diagnostic 
devices in the same study.  Joint Statistical Meeting. 2005. ASA Section on Statistics in 
Epidemiology, p.2547-2551. 
20. Khan MJ, Castle PE, Lorincz AT, Wacholder S, Sherman M, Scott DR, Rush BB, Glass, AG, 
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39  Contains Nonbinding Recommendations 
 
 
IX.   Appendix  Statistical Analysis 
Calculating Score Confidence Intervals for Percentages and Proportions 
The following are additional recommendations for performing statistical analyses of percentages 
or proportions.  There are several different methods available.  We suggest that either a score 
method described by Altman, et al. (Altman D.A., Machin D., Bryant T.N., Gardner M.J. eds. 
Statistics with Confidence. 2nd ed. British Medical Journal; 2000) or a Clopper-Pearson Method 
(Clopper CJ, Pearson E.  The use of confidence or fiducial limits illustrated in the case of 
binomial. Biometrika 1934; 26:404-413) be used.  The advantages with the score method are that 
it has better statistical properties and it can be calculated directly.  Score confidence limits tend 
to yield narrower confidence intervals than Clopper-Pearson confidence intervals, resulting in a 
larger lower confidence limit.  Thus when n=70 samples and 65/70=92.9%, the score lower limit 
of two-sided 95% confidence interval is 84.3%.  In contrast, the Clopper-Pearson lower 
confidence limit is 84.1%.  In this document, we have illustrated the reporting of confidence 
intervals using the score approach.  For convenience, we have provided the formulas for the 
score confidence interval for a percentage.   
A two-sided 95% score confidence interval for the proportion of A/B is calculated as: 
[ ( ) ( ) ]
100% Q -Q /Q ,100% Q +Q /Q , where values Q , Q , and Q are computed from the 
1 2 3
1 2 3 1 2 3
data using the formulas below.  For the proportion of A/B: 
Q =2A+1.962 = 2A+3.84
1
Q =1.96 1.962 +4A(B- A)/B =1.96 3.84+4A(B- A)/B
2
Q =2(B+1.962)=2B+7.68
3
In the formulas above, 1.96 is the quantile from the standard normal distribution that corresponds 
to 95% confidence.   
For an example of proportion if (65/70), Q =133.84, Q =9.28, and Q =147.68, then the  two-
1 2 3
sided 95% score confidence interval is 84.3% to 96.9% 
Calculation of Confidence Intervals for Positive Predictive Value (PPV) and Negative 
Predictive Value (NPV) based on Confidence Intervals for Likelihood Ratios (Prevalence is 
Constant) 
PPV is (1+PLR-1*(1-)/)-1, where PLR is positive likelihood ratio (PLR=se/(1-sp)); NPV is 
(1+NLR*/(1-))-1 , where NLR is negative likelihood ratio (NLR=(1-se)/sp)) and  is 
prevalence.   For the calculation of 95% confidence intervals for the likelihood ratios, use 
calculation of confidence intervals for the ratio of two independent proportions (the estimate of 
Se and the estimate of (1-Sp) for PLR and the estimate of (1-Se) and the estimate of Sp for 
NLR).  There are several different methods available for calculation of the confidence intervals 
for the likelihood ratios (see Altman D.A., Machin D., Bryant T.N., Gardner M.J. eds. Statistics 
with Confidence. 2nd ed. British Medical Journal; 2000, pages 18-110).  We suggest that a score 
method described in paper by Nam (Nam J. Confidence limits for the ratio of two binomial 
40  Contains Nonbinding Recommendations 
 
 
proportions based on likelihood scores: non-iterative method.  Biom J 1995; 37:375-9) be used.   
Using the 95% confidence interval for the corresponding likelihood ratio, it is easy to calculate 
the 95% CI for the corresponding predictive value where  (prevalence) is a constant.    
Note: 
Suppose that [L, U] is a 1-r level confidence interval for b and suppose that G is a function 
defined on the parameter space.   
If G is increasing, then [G(L), G(U)] is 1-r level confidence interval for G(b).   
If G is decreasing, then [G(U), G(L)] is 1-r level confidence interval for G(b).                  
(Functions (1+x-1*(1-)/)-1 and(1+x*/(1-))-1 are monotonic functions when  is a constant.) 
41