Considerations for Use of 
Histopathology and Its Associated 
Methodologies to Support 
Biomarker Qualification  
 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
May 2016 
Procedural 
 
   
Considerations for Use of 
Histopathology and Its Associated 
Methodologies to Support 
Biomarker Qualification  
 
Guidance for Industry 
 
 
 
 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
May 2016 
Procedural 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
                       
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.       ESTABLISHING PERFORMANCE CHARACTERISTICS OF BIOMARKERS .. 4 
A.   Methodology for Detection of the Biomarker (Biomarker Assay) ............................................ 4 
B.   Biological Performance of the Biomarker ................................................................................... 5 
1. Performance Characteristics ........................................................................................................... 5 
2.  Biomarker Sensitivity and Temporal Correlation ............................................................................ 5 
3.  Biomarker Specificity ....................................................................................................................... 6 
4.  Reversibility/Resolution ................................................................................................................... 7 
5.  Alternative Testing Models .............................................................................................................. 7 
IV.    SPECIFIC ASPECTS OF HISTOPATHOLOGIC METHODS IN 
CONFIRMATORY BIOMARKER STUDIES .............................................................. 7 
A.  General Planning for Confirmatory Studies ............................................................................... 7 
B.  Slide Examination Procedures to Minimize Bias in Confirmatory Studies .............................. 8 
C.    Timing of Sample Collection ......................................................................................................... 9 
D.     Controls .......................................................................................................................................... 9 
E.  Fixation ........................................................................................................................................... 9 
F.      Number of Sections and Sampling Location (at Necropsy) ....................................................... 9 
G.     Staining ......................................................................................................................................... 10 
H.  Special Methodology .................................................................................................................... 10 
V.        OTHER CONSIDERATIONS FOR HISTOPATHOLOGIC EVALUATION ........ 10 
A.     Digital Pathology and Slide Sharing .......................................................................................... 10 
B.      Lexicons ....................................................................................................................................... 10 
C.      Filtering ........................................................................................................................................ 11 
D.     Other Factors ............................................................................................................................... 11 
1.   Diagnostic Drift ............................................................................................................................ 11 
2.   Chronological Bias....................................................................................................................... 11 
GLOSSARY................................................................................................................................. 12 
REFERENCES ............................................................................................................................ 14 
  Contains Nonbinding Recommendations 
 
Considerations for Use of Histopathology and Its Associated 
Methodologies to Support Biomarker Qualification  
1
Guidance for Industry  
  
 
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 office responsible for this guidance as listed on the 
title page.   
 
 
 
 
I.  INTRODUCTION  
 
This guidance is intended to assist submitters of a biomarker2 for qualification that conduct 
nonclinical studies for which histopathology is used as a reference or truth standard (Zhou et 
al. 2011).  Scientifically rigorous evaluation of biomarker performance in relation to 
histopathologic changes is essential in these studies because they may provide direct evidence to 
support nonclinical biomarker qualification or supportive translational data to aid in the 
development and qualification of clinical biomarkers for a proposed context of use.  This 
guidance discusses the issues that should be considered when generating histopathology data in 
nonclinical biomarker qualification studies and outlines the scientific standards recommended 
for biomarker characterization and qualification. The biomarker qualification process is 
described in a separate guidance, Qualification Process for Drug Development Tools.3 
 
In general, FDAs guidance documents 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.  
 
 
                                                 
1 This guidance has been prepared by the Office of New Drugs and the Office of Translational Sciences in the 
Center for Drug Evaluation and Research (CDER) at the Food and Drug Administration. 
2Terms that appear in bold type upon first use are defined in the Glossary section of this guidance.  
3 See FDA guidance for industry and FDA staff on Qualification Process for Drug Development Tools, available at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm. We update 
guidances periodically.  To make sure you have the most recent version of a guidance, check the FDA Drugs 
guidance Web page at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.  
 
  1  Contains Nonbinding Recommendations 
 
II.  BACKGROUND 
 
The discovery, characterization, qualification, and use of biomarkers have been identified by the 
FDA Critical Path Initiative4 as important means for improving the efficiency and success rate of 
drug5 development.  
 
A biological marker or biomarker is defined as a characteristic that is objectively measured and 
evaluated as an indicator of normal biologic processes, pathogenic processes, or biological 
responses to a therapeutic intervention (Biomarkers Definitions Working Group 2001).   
 
The context of use is a comprehensive and clear statement that describes the manner of use, 
interpretation, and purpose of use of a biomarker in drug development.  Details about the 
elements of context of use and categories of biomarkers can be found at the Biomarker 
Qualification Context of Use Web page 
(http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugDevelopmentToolsQualification
Program/ucm284620.htm).  For nonclinical biomarkers, submitters should specify whether the 
intended use of the biomarker in nonclinical studies will be in normal, healthy animals or in 
animal models of disease.  
 
When a biomarker is qualified, analytically valid measurements of it can be relied upon to have a 
specific and interpretable meaning (e.g., physiologic, toxicologic, pharmacologic, or clinical) in 
drug development and regulatory decision-making.  Industry can then use the biomarker for its 
qualified context of use during premarketing drug development, and FDA reviewers can be 
confident about its qualified context of use without the need to reconfirm its applicability or 
utility.  The qualification process includes a formal review to evaluate data supporting a 
biomarker for a particular context of use.  The types of studies to be conducted in support of a 
biomarker qualification will depend upon the proposed context of use, which dictates the depth 
and extent of data.  Accordingly, data supporting a nonclinical biomarker qualification should be 
reliable and repeatable.  The biomarker also can be used for a purpose outside the qualified 
context of use in investigational new drug application (IND) submissions, subject to review and 
discussion with CDER, on a case-by-case basis.    
 
The terms exploratory study and confirmatory study are used in this guidance similarly to 
their use in describing human efficacy studies  to refer to exploratory nonclinical biomarker 
qualification studies and confirmatory nonclinical biomarker qualification studies, 
respectively.  Exploratory studies should have clear objectives. The results of the studies may 
provide initial information to guide further investigation.  These studies usually occur early in 
the characterization of a biomarker and can use a flexible design that allows for changes in the 
study protocol and analyses in response to the accumulating data.  The studies are often 
informative but would generally not be the sole source of data to support the proposed context of 
use of a biomarker.  In contrast, a confirmatory study is well designed, well controlled, and 
                                                 
4 See FDAs Critical Path Initiative, available at 
http://www.fda.gov/scienceresearch/specialtopics/criticalpathinitiative/default.htm. 
5 The term drug as used in this guidance refers to both human drugs and therapeutic biologics regulated by CDER 
unless otherwise specified. 
  2  Contains Nonbinding Recommendations 
 
hypothesis driven.  Confirmatory studies are expected to provide substantive evidence to support 
the proposed context of use of a biomarker.   
 
A qualification determination is a regulatory conclusion made by the FDA.  Qualification study 
is a general term used in this guidance to refer to nonclinical studies having to do with the 
process of biomarker qualification.  Data quality and integrity are important to support FDAs 
conclusion that the biomarker should be qualified for its specific context of use.  A qualified 
biomarker is intended to support drug development and regulatory decisions.  Thus, the 
qualification data should be reliable and repeatable.   
 
Traditionally, histopathology has been used to identify morphologic changes associated with an 
in vivo diagnosis, evaluation of response to therapy, basic research, and nonclinical safety 
assessment.  There is often a strong correlation between specific histopathologic findings and 
clinical signs, or some clinical chemistry parameters.  Thus, histopathology is currently used in 
some biomarker qualification programs as a reference or truth standard to evaluate the temporal 
correlation of the biomarker with the evolution and reversibility of the morphologic changes. 
This guidance is intended to provide recommendations to investigators who conduct nonclinical 
biomarker qualification studies when histopathology is used as a reference or truth standard.  
 
This guidance is not intended to address the use of histopathology in traditional drug 
development nonclinical safety assessment studies.  Biomarker qualification studies are different 
from nonclinical safety assessment studies and call for a different scientific paradigm.  
Nonclinical safety assessment studies are conducted primarily for hazard characterization of a 
specific drug, and histopathologic assessment assists in identifying effects of the drug.  In safety 
assessment studies, it is common practice for the pathologist to evaluate the histopathology 
knowing the identity of the treatment groups, the clinical signs, hematology, clinical chemistry, 
urinalysis, organ weight, and gross pathology data, and then to integrate this information in a 
unified, physiologically plausible format (Crissman et al. 2004).   
 
In contrast, histopathology is used in nonclinical biomarker qualification studies to establish 
correlative and temporal relationships between the biomarker and morphologic changes.  
Examples include (1) establishing a possible quantitative relationship between biomarker level 
and severity of morphological change; (2) exploring the kinetics of release, clearance of the 
biomarker, and correlative histomorphology; (3) determining the range of normal variability of 
the biomarker and any associated morphologic findings; and (4) identifying potentially 
confounding factors for use of the biomarker.  Particularly in confirmatory nonclinical biomarker 
qualification studies, histopathology can be used as a reference or truth standard.  In these cases, 
the pathologist should function as an impartial interpreter, uninfluenced by other information, 
and unequivocally blinded to novel biomarker value and comparator value (e.g., standard clinical 
chemistry value) and preferably to assigned treatment group.  
 
Although this guidance is not intended to apply to the conduct of nonclinical safety assessment 
studies, biomarker data, either exploratory or confirmatory, can be gathered from safety 
assessment studies to support the qualification of a biomarker.  For example, data collected 
following Best Practices, such as those outlined by Burkhardt et al. 2011, can be used as 
supportive data, regardless of whether the data were derived from safety assessment studies or 
  3  Contains Nonbinding Recommendations 
 
dedicated qualification studies.  We recommend that biomarker developers consult with the FDA 
prior to initiating dual purpose safety assessment-qualification studies. 
 
Qualification of a biomarker is a regulatory conclusion, and thus, the studies used to support 
qualification are expected to be scientifically valid, of high quality, and unbiased.  
 
 
III.       ESTABLISHING PERFORMANCE CHARACTERISTICS OF BIOMARKERS 
 
In this section, we describe biomarker sensitivity, biomarker specificity, analytical 
sensitivity, and analytical specificity (See Sensitivity and Specificity in Glossary).  Diagnostic 
sensitivity and diagnostic specificity of in vitro diagnostic tests are also defined in the Glossary 
(see Sensitivity and Specificity in Glossary) for clarity, but are separate from biomarker 
qualification. 
 
A.   Methodology for Detection of the Biomarker (Biomarker Assay) 
 
Biomarker detection can be based on several different methodologies (e.g., biochemical 
measurements, physiologic organ function tests, or imaging of structural features ranging from 
molecular to anatomic levels).  The method of biomarker detection provides the measurements 
that are the basis for comparison of the biomarker to the reference or truth standard of 
histopathology and, therefore, is critical to biomarker characterization.  Thus, the detection 
system should be well characterized.6  
 
For example, for a biochemical biomarker, the assay used to measure the biomarker should have 
scientifically rigorous analytical sensitivity and analytical specificity.  An assay with poor 
sensitivity or one that is inhibited by other substances in the biological sample or matrix could 
lead to a false negative result by failing to detect initial changes in biomarker levels.  An assay 
with poor specificity would not distinguish injury to a nontarget organ or the assay may cross-
react with nonspecific substances in the specimen.  Thus, the biomarker assay should reliably 
and reproducibly detect changes (increase or decrease) or absence of changes in biomarker 
levels.  Interpretation of any detected change in biomarker levels should ultimately include an 
assessment of whether the change is biologically meaningful through comparison to the 
histopathology. 
 
Considerations for biomarker detection based on biochemical measurements also apply to 
biomarker detection based on other methodologies such as imaging technologies (e.g., positron 
emission tomography, magnetic resonance imaging, radiology). Histopathology used as a 
reference or truth standard provides an independent means of evaluating the same variable that is 
assessed by the biomarker.   
 
                                                 
6 See FDAs draft guidance for industry Bioanalytical Method Validation (Revision 1).  In the Federal Register of 
September 13, 2013 (78 FR 56718), FDA published a notice announcing the availability of the revised draft 
guidance.  When finalized, it will represent the Agencys current thinking on the topic. 
  4  Contains Nonbinding Recommendations 
 
B.   Biological Performance of the Biomarker 
 
1.  Performance Characteristics 
  
It is highly unlikely that any biomarker will be 100 percent specific or 100 percent sensitive.   
The proposed context of use for a biomarker determines the specific performance characteristics 
that can be used to support qualification.  Studies intended to establish the sensitivity, specificity, 
and reproducibility of the biomarker should be relevant to the proposed context of use.  A 
reference or truth standard such as histopathology provides an independent means of evaluating 
the same parameter that the biomarker is intended to assess. 
 
 2.   Biomarker Sensitivity and Temporal Correlation 
 
In contrast to analytical sensitivity, biomarker sensitivity is related to (1) the probability of 
identifying a morphologic effect, (2) the threshold of morphologic change that would cause a 
discernible change in the biomarker level, and (3) the time interval before or after a morphologic 
effect until a change in the biomarker level can be detected. 
 
Biomarker sensitivity is the probability that a biomarker will indicate a specific morphologic 
change when the change is present.  If the biomarker is 100 percent sensitive, it will never be 
negative in the presence of the specific morphologic change that it is designed to detect. 
The temporal relationship between the initial change in biomarker level and the onset of 
histopathologic change defines the biomarker sensitivity.  Accurate characterization of this 
temporal relationship is important to optimizing the biomarker sensitivity.  
 
The timing and frequency of biomarker measurement in qualification studies should allow for a 
clear interpretation of the temporal relationship of the biomarker with morphologic changes.  The 
time frame and mechanism by which an agent (e.g., mechanical, chemical, natural disease) 
produces morphologic changes in tissue should be understood and considered when planning the 
time course of sampling and/or analysis in biomarker qualification studies.   
 
Histopathologic evaluation using light microscopy with hematoxylin and eosin (H&E) staining is 
preferred in qualification studies, when this is the method that will be used in the context of use 
of the biomarker.  For treatment-induced histopathologic changes in animals, the temporal 
relationship between changes in biomarker levels and detectable morphologic changes, as 
observed by light microscopy and H&E staining, usually can be described by one of the 
following statements, as indicated by appropriate supportive data: 
 
  The initial change in biomarker levels precedes the onset of morphologic changes.  
 
  The initial change in biomarker levels is approximately concurrent with the onset of 
morphologic changes.  
 
  The initial change in biomarker levels occurs only after the development of morphologic 
change.  
 
  5  Contains Nonbinding Recommendations 
 
Similarly, when there is a relationship between morphologic and biomarker changes during 
reversal or recovery, morphologic changes may be preceded by, concurrent with, or followed by 
changes in the biomarker levels.  Sometimes recovery from morphologic changes may be 
unrelated to changes in the biomarker levels. 
 
There are several potential causes for false negative biomarker results (when a morphologic 
effect is not accompanied by an expected change in biomarker).  These include (1) low analytical 
sensitivity, (2) interference of other proteins or substances with the assay, (3) inconsistent 
expression of the biomarker under different physiologic or pathologic conditions, (4) a prolonged 
latency in biomarker change relative to the morphologic effect, (5) the morphologic effect being 
caused by a mechanism other than the one measured by the biomarker, (6) mishandling of a 
specimen, or (7) inadequate sampling of a specimen. 
  
Histopathologic sensitivity depends on the histopathologic methods of examination.  The use of 
light microscopy for the initial evaluation of biomarker sensitivity is preferable. Special methods 
(e.g., electron microscopy, immunohistochemistry) may be useful to investigate whether small or 
early changes in biomarker levels are associated with histopathologic changes too subtle to be 
detected by light microscopy with H&E stain.  
 
Biomarker qualification studies should evaluate the quantitative correlation between the 
magnitude of biomarker change and the degree of morphologic change.  For these studies, it is 
critical to obtain a final biomarker measurement as close to the tissue collection as possible (at 
least on the same day) to avoid a temporal disconnect in these two parameters.    
 
3.   Biomarker Specificity  
 
The specificity of a biomarker is the probability of correctly determining that no morphologic 
change has occurred in the tissue of interest.  A biomarker that has 100 percent specificity will 
result in no false positives.  Specificity also can be viewed as a very low or very stable baseline 
value when there is no morphologic change.  A highly specific biomarker will not have a signal 
originating in unaffected or non-target tissues. Appropriate supportive data helps to establish 
specificity of a biomarker. 
 
The specificity of a biomarker candidate can be challenging to evaluate, particularly when it is 
known that multiple isoforms exist with different tissue distributions.  For example, kidney 
injury molecule-1has isoforms specific to liver and kidney (Bailly et al. 2002); troponins have 
isoforms specific to cardiac and skeletal muscle (reviewed in Wei and Jin 2011).   In such cases, 
it is important to examine histologic and biomarker isoform expression in multiple tissue types to 
assure that the biomarker candidate has specificity for the tissue of interest. 
 
There are several causes of a false positive biomarker signal.  Examples include low analytical 
specificity, the release of the biomarker from a nontarget tissue, or expression of the biomarker 
under both physiologic and pathologic conditions.  
 
As noted previously, it is highly unlikely that any biomarker will be 100 percent specific or 100 
percent sensitive.  The proposed context of use of the biomarker will define the appropriate 
  6  Contains Nonbinding Recommendations 
 
balance between biomarker sensitivity and specificity.  Sometimes a single biomarker does not 
fulfill a particular context of use, and therefore the use of multiple biomarkers may be 
considered.  We recommend that the use of multiple biomarkers be discussed with the Agency 
during design of the confirmatory studies for biomarker qualification to ensure that all Agency 
concerns are addressed.  
 
  4.   Reversibility/Resolution 
 
Studies designed to investigate the recovery from or the cessation of a physiologic or pathologic 
process should characterize the temporal relationship between the reversal of biomarker changes 
and the cessation/recovery of the histopathologic changes.  Note, however, that the reversal of 
the biomarker changes may not correlate with the cessation or recovery of the histopathologic 
changes.  For example, in the context of drug-induced injury, reversal of the biomarker levels 
may be caused by the cessation of the biomarker release from the site of injury and/or the 
subsequent degradation or elimination from the body, without cessation/recovery of the 
histopathologic changes.  Thus, an adequate interpretation of the biomarker and histopathologic 
changes is complex and all data should be considered. 
 
  5.   Alternative Testing Models 
 
An alternative to the use of healthy animals for nonclinical biomarker qualification is the use of 
animal models of human disease.  These animal models may be useful for evaluating the change 
in a biomarker candidate in response to a pharmacologic, physiologic, or physical intervention.   
 
 
IV.    SPECIFIC ASPECTS OF HISTOPATHOLOGIC METHODS IN 
CONFIRMATORY BIOMARKER STUDIES 
  
The confirmatory biomarker qualification studies generally will be conducted after many of the 
early questions on the proposed context of use and the assays have been refined.  The focus of 
the histopathology, such as the tissue of interest, the specific lesions, and important features of 
lesions, will have been identified before planning and initiation of confirmatory studies.  The 
topics discussed in this section are critical to confirmatory studies and should be considered and 
addressed to provide convincing study results. 
 
A.  General Planning for Confirmatory Studies 
 
The first step generally entails a systematic review of the literature (Mignini and Khan 2006; 
Piper et al. 1996; Pound et al. 2004; Roberts et al. 2002) conducted in a way that avoids bias in 
the search.  A literature review can be used both to plan the qualification studies and to support a 
qualification effort.   
 
An important factor in planning confirmatory studies is a prospectively written protocol that 
discusses all steps leading to and including the evaluation of the histopathology.  All aspects of 
the necropsy, including timing, handling of the specimen and tissues, fixation, sample storage, 
transportation (if relevant), and slide preparation and evaluation, should be described clearly.  
  7  Contains Nonbinding Recommendations 
 
Some of the important points that should be considered are discussed in the following 
subsections. 
 
B.  Slide Examination Procedures to Minimize Bias in Confirmatory Studies  
 
A pathologist should be free to conduct a blinded assessment of outcomes for any study, 
especially if that is the standard practice for the institution (Holland and Holland, 2011a; Holland 
and Holland, 2011b). Recommendations outlined by Burkhardt et al. provide criteria for when it 
may be appropriate to use blinded evaluations, taking into account the treatment groups and time 
course to characterize changes, devising quantifying and qualifying scoring systems, and 
determining thresholds for background changes (Burkhardt et al. 2011).   
 
For exploratory studies used to support confirmatory studies, a tiered analysis approach may be 
appropriate, using procedures as proposed by the Society for Toxicologic Pathology (Crissman et 
al. 2004).  The first level in this tiered approach can be an unblinded comparison of treated and 
control specimens to identify subtle findings (such as the increase in incidence or severity of 
spontaneous findings) and to develop scoring criteria.  Following this first tier analysis, the 
pathologist might perform a targeted, blinded evaluation from any or all groups as appropriate.  
This allows for identification of subtle, treatment-related findings that can be consistently 
differentiated from those that occur in controls.  A pathology peer review, with targeted 
blinding by a second pathologist who is naive to the study, may also help minimize bias (Morton 
et al. 2010).  Lastly, enlisting a pathology working group may be valuable. 
 
The goal of a confirmatory study is to test whether the biomarker accurately reflects the 
histopathology to support the proposed context of use.  Subjective judgment can influence any 
step of slide evaluation, defeating the purpose of the study.  The risk of bias is greater when the 
histopathologic changes under evaluation are subtle or sparse (the level of tissue change of most 
interest in many biomarker qualification studies).  Slide examination procedures should be 
defined prospectively in the confirmatory study protocol, including steps to minimize bias.  In 
confirmatory studies, we strongly recommend that tissue sections be evaluated with the reader 
blinded to treatment condition, sampling times, novel biomarker results, and any comparator 
biomarker results. 
     
  Slides from concurrent control group animals can be used initially to establish background 
morphology and aid in distinguishing subtle histopathologic changes from normal variation.  
This assessment of normal variations in background histomorphology is important for 
identifying thresholds to distinguish treatment-related lesions and to understand whether certain 
background histomorphology is associated with altered biomarker values.  Extra slides from 
control group animals can be prepared in sufficient number to allow assessment of normal 
variations and background lesions.  When acquiring additional sections from the planned tissue 
samples is not feasible, additional control group animals can be included in the nonclinical 
studies.  For statistical purposes, it is preferable that the data derived from these extra slides not 
be included in the final dataset of the qualification study. The overall peer review assessment 
plan, if any, should be stipulated in the scientific plan and reflected in the protocol, along with a 
plan for resolving disagreements among readers/reviewers. 
 
  8  Contains Nonbinding Recommendations 
 
C.     Timing of Sample Collection  
  
The proposed context of use of a biomarker and the specific goal of a given study will determine 
the timing of tissue sample collection. It is important to determine the variability of biomarker 
measurements and the likely reasons for the variability.  Timing of sample collection may be an 
important factor in variability.  Thus, specific sampling times (study day and hour post-
procedure, if applicable) should be selected carefully, and the rationale should be included in the 
protocol to facilitate interpretation of data and reproducibility.   
 
D.       Controls 
 
The objective of a confirmatory study is to prospectively evaluate the performance of the 
biomarker relative to histomorphologic changes.  The confirmatory study should be designed 
with sufficient rigor to permit a valid comparison of treatment and control groups and permit 
unbiased assessment of histologic material.  To reduce the potential for analytical and biological 
false negative and false positive results, concurrent positive and negative control groups should 
be used to control for environmental factors, enable determination/comparison of biomarker 
sensitivity/specificity, and ensure reagent/method adequacy.  
 
The identification of concurrent control groups and the rationale for their use should be described 
in the protocol.  Adequate historical (external) control data can be used to identify potential 
analytical and biological outlier values in datasets; however, applying the historical data in this 
regard should be determined a priori and prospectively detailed in the protocol.  Historical 
control data should be current and contain only studies for which similar control test agents 
and/or conditions were used (Keenan et al. 2009).  
 
E.  Fixation  
 
Fixation procedures should be described in the study protocol.  Variations in the time to fixation 
and duration of fixation may create variability in the data that obscures a true biomarker-
histomorphologic relationship.  Delays and variations in fixation of tissue should be avoided, and 
deviations from the protocol should be noted.  To avoid experimental bias, animals should be 
randomized for scheduled necropsy. 
 
F.       Number of Sections and Sampling Location (at Necropsy)  
      
The anatomic sites from which tissue is collected and the number of samples collected may 
affect results and their interpretation.  Based on the stated goals, a rationale for the number of 
tissue sections to be collected and the locations from which they will be taken should be 
provided in the protocol. When deciding the number of sections to be collected and analyzed, a 
submitter should consider that there is a greater likelihood of missing subtle or sparse lesions if 
an insufficient number of samples are taken.  A description of each sampling location should be 
sufficient to allow reproducibility across laboratories.  The tissue/organ, lobe, and regional 
position within tissue (e.g., left, right, anterior, posterior, superior, inferior) from which samples 
will be obtained should be described in the protocol.  If serial or step tissue sections will be 
  9  Contains Nonbinding Recommendations 
 
obtained, the procedures for identifying and reporting the precise location of lesions within an 
organ/tissue should be described in the protocol. 
 
G.       Staining  
 
Assessment of histologic slides under the conditions of the proposed context of use is generally 
preferable.  For example, most safety assessment studies use light microscopy for H&E-stained 
sections.  The rationale should be provided for each stain used.  If staining methods are 
performed in the laboratory according to previously established procedures, this information can 
be incorporated into the protocol as an appendix or it can be referenced.  For nonstandard stains, 
the methods and scoring systems should be well documented or referenced in the protocol.  We 
encourage using automated or standardized staining techniques for all samples, including 
positive and negative controls. 
 
H.  Special Methodology  
 
In some cases (e.g., to confirm the morphologic process, cell type, or location), there may be a 
scientific need for more specific information or additional supportive data to achieve the study 
objectives.  In these cases, special methodologies may be necessary to assess the morphology of 
particular targets to support a stated hypothesis.  These methods include, but are not limited to, 
the following: 
 
  stereology 
  histochemistry 
  immunohistochemistry 
  in situ hybridization 
  electron microscopy 
 
The rationale for using a special methodology should be provided, and the methodology used 
should be well documented or referenced in the protocol. 
 
 
V.        OTHER CONSIDERATIONS FOR HISTOPATHOLOGIC EVALUATION  
  
Other factors may affect the outcome of the slide evaluation.  This section discusses certain 
factors that should be considered and addressed in the study protocol. 
 
A.       Digital Pathology and Slide Sharing  
 
Digital slides should be shared and examined according to the established guidelines of 
recognized societies of toxicologic pathology (Tuomari et al. 2007) and the Digital Pathology 
Association (DPA) (see https://digitalpathologyassociation.org/).  
 
B.        Lexicons 
 
  10  Contains Nonbinding Recommendations 
 
Lexicons specify the predetermined criteria that classify types and degrees of change.  
Illustrations of the terminology are of great benefit in reducing variability. 
 
For a well-defined morphologic condition, combining associated lesions for data entry is 
appropriate (e.g., chronic progressive nephropathy of rats).  However, combining terms for ease 
of statistical analysis should not obscure individual characteristics appropriate for the unbiased 
analysis of the data and correlation with the biomarker. 
 
C.       Filtering  
 
It is standard practice in diagnostic pathology to interpret certain lesions as background and not 
report them in results (filtering).  Different pathologists can also use different thresholds for 
defining changes as background, thereby affecting the overall interpretation. 
 
We recommend that data be generated to show how much, if at all, the background changes 
contribute to the normal variability of the biomarker (e.g., report all lesions, along with the 
associated biomarker values, independent of the filtering).  An alternative is to comprehensively 
document the type and extent of lesions considered to be incidental background.  The procedures 
for describing and documenting the background lesions should be described in the scientific plan 
or protocol. 
 
D.       Other Factors 
 
Factors such as diagnostic drift and chronological bias are also important to consider, and 
procedures should be included in the protocol to control for these. 
 
     1.     Diagnostic Drift 
 
Diagnostic drift is defined as a gradual change in nomenclature or severity grading of lesions 
within a single study.  It is a source of inconsistency that can negatively affect detection of 
treatment-related lesions/changes or the determination of no-effect levels (Crissman et al. 2004).  
 
     2.       Chronological Bias 
 
Chronological bias is defined as the evolutionary process of a grading system, whereby more 
specific and sensitive criteria for grade assignment are clarified, learned, and publicized. As 
pathologists gain experience, subtleties of the evolving system are applied to interpretation of 
tissue sections (Kondylis et al. 2003).  This is also a source of inconsistency that can negatively 
affect detection of treatment-related lesions/changes or the determination of no-effect levels.  
 
  11  Contains Nonbinding Recommendations 
 
GLOSSARY 
 
Biomarker (or biological marker):  A characteristic that is objectively measured and evaluated 
as an indicator of normal biologic processes, pathogenic processes, or biological responses to a 
therapeutic intervention (Biomarkers Definitions Working Group 2001). 
  
Biomarker qualification study or qualification study:  A general term used in this guidance to 
refer to nonclinical studies specifically pertaining to the qualification of a biomarker. 
 
Chronological bias:  The evolutionary process of a grading system, whereby more specific and 
sensitive criteria for grade assignment are clarified, learned, and publicized (Kondylis et al. 
2003). 
 
Confirmatory nonclinical biomarker qualification study or confirmatory study:  A well-
designed and -controlled nonclinical study in which the hypotheses are stated in advance and 
evaluated.  In such studies, the key hypothesis of interest is predefined and is the hypothesis that 
is tested when the study is complete.  Confirmatory studies are intended to provide firm evidence 
in support of the proposed context of use of the biomarker and, therefore, adherence to the 
protocol is particularly important.  
 
Diagnostic drift:  A gradual change in nomenclature or severity grading of lesions within a 
single study.  Diagnostic drift is a source of inconsistency that can negatively affect detection of 
treatment-related lesions/changes or the determination of no-effect levels (Crissman et al. 2004). 
 
Exploratory nonclinical biomarker qualification study or exploratory study:  A study that 
has clear objectives and provides information that may guide further investigation.  In contrast to 
confirmatory nonclinical studies, an exploratory study may occur early in the characterization of 
a biomarker and can use a flexible design that allows for changes in the study protocol in 
response to the accumulating data.  The analyses may involve exploring data and testing 
different hypotheses.  Such studies cannot be the sole basis to support the proposed context of 
use of a biomarker, but can contribute to the total body of evidence.  Exploratory studies are 
frequently (but not limited to) descriptive science.  
 
Historical (external) control:  A group of subjects, treated or untreated, from an earlier time 
than the study under consideration.  Historical control data may serve to produce a range of 
normal rates of certain findings or changes (Keenan et al. 2009).  
 
Lexicons:  Consist of predetermined criteria used to classify types and degrees of change. A 
lexicon may be illustrated to clarify these predetermined criteria.  
 
Pathology peer review:  A secondary review with the objective of assisting the study 
pathologist to refine, verify, and improve the accuracy and quality of the final pathology 
assessments and interpretations. 
  
Pathology working group:  A group composed of the study pathologist, peer review 
pathologist, and at least one other pathologist.  This group may be convened to resolve 
  12  Contains Nonbinding Recommendations 
 
differences of opinion, review overall pathology interpretations within a study, or resolve and 
document a complicated issue.  
 
Sensitivity:  In the context of this guidance, refers to a biomarker, an assay for detection of a 
biomarker, or a diagnostic test using a biomarker.  Biomarker sensitivity is the probability that 
a biomarker will report a specific morphologic change when the change is present.  A sensitive 
biomarker also demonstrates changes after a relatively low amount of target tissue damage.  
Analytical sensitivity of the detection method for a given biomarker refers to the smallest 
quantity of biomarker that can be measured.  For clarity, the term sensitivity should be qualified 
as being related to the biomarker itself (i.e., biomarker sensitivity) or the methodology used to 
measure the biomarker (i.e., analytical sensitivity).  Diagnostic sensitivity is the probability that 
a person having a disease will be correctly identified by a clinical test (the number of true 
positive results divided by the total number with the disease, which is the sum of the numbers of 
true positive plus false negative results).  
 
Specificity:  Refers to a biomarker, an assay for the detection of a biomarker, or a diagnostic test 
using a biomarker.  Biomarker specificity is the probability that the biomarker does not change 
in the absence of change in the tissue of interest.  A biomarker with high specificity is reliably 
unchanged or stable in the absence of a defined morphologic change.  Analytical specificity for 
the biomarker refers to the ability of the assay to measure one specific substance in the presence 
of other substances expected to be present.  Diagnostic specificity is the probability that a person 
not having a disease will be correctly identified by a clinical test (the number of true negative 
results divided by the total number of those without the disease, which is the sum of the numbers 
of true negative plus false positive results). 
  
Truth Standard:  Any medical procedure or laboratory method or combination of procedures 
and methods that the relevant scientific or clinical community relies upon for diagnosis or other 
specific categorization of the studied tissue, animal, or person and that is accepted by FDA for 
this purpose in biomarker qualification.  Ideal truth standards will have negligible likelihood of 
either a false positive or a false negative result (Zhou, Obuchowski, and McClish, 2011).   
 
  13  Contains Nonbinding Recommendations 
 
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