General Principles of Preclinical

Study Design

Wenlong Huang, Nathalie Percie du Sert, Jan Vollert,

and Andrew S. C. Rice

Contents
1 An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2 General Scientific Methods for Designing In Vivo Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.1 Hypotheses and Effect Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.2 Groups, Experimental Unit and Sample Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.3 Measurements and Outcome Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.4 Independent Variables and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3 Experimental Biases: Definitions and Methods to Reduce Them . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4 Experimental Biases: Major Domains and General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5 Existing Guidelines and How to Use Them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6 Exploratory and Confirmatory Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Abstract
Preclinical studies using animals to study the potential of a therapeutic drug or
strategy are important steps before translation to clinical trials. However, evi-
dence has shown that poor quality in the design and conduct of these studies has
not only impeded clinical translation but also led to significant waste of valuable
research resources. It is clear that experimental biases are related to the poor
quality seen with preclinical studies. In this chapter, we will focus on hypothesis
testing type of preclinical studies and explain general concepts and principles in

W. Huang (*)
Institute of Medical Sciences, School of Medicine, Medical Sciences and Nutrition, University of
Aberdeen, Aberdeen, UK
e-mail: [email protected]
N. Percie du Sert
NC3Rs, London, UK
J. Vollert · A. S. C. Rice
Pain Research Group, Faculty of Medicine, Department of Surgery and Cancer, Imperial College
London, London, UK

# The Author(s) 2019 55

A. Bespalov et al. (eds.), Good Research Practice in Non-Clinical Pharmacology
and Biomedicine, Handbook of Experimental Pharmacology 257,
https://doi.org/10.1007/164_2019_277
56 W. Huang et al.

relation to the design of in vivo experiments, provide definitions of experimental

biases and how to avoid them, and discuss major sources contributing to experi-
mental biases and how to mitigate these sources. We will also explore the
differences between confirmatory and exploratory studies, and discuss available
guidelines on preclinical studies and how to use them. This chapter, together with
relevant information in other chapters in the handbook, provides a powerful tool
to enhance scientific rigour for preclinical studies without restricting creativity.

Keywords
Experimental bias · Hypothesis generating · Hypothesis testing · In vivo studies ·
Preclinical research

This chapter will give an overview of some generic concepts pertinent to the design
of preclinical research. The emphasis is on the requirements of in vivo experiments
which use experimental animals to discover and validate new clinical therapeutic
approaches. However, these general principles are, by and large, generically relevant
to all areas of preclinical research. The overarching requirement should be that
preclinical research should only be conducted to answer an important question for
which a robust scrutiny of the available evidence demonstrates that the answer is
not already known. Furthermore, such experiments must be designed, conducted,
analysed and reported to the highest levels of rigour and transparency. Assessments
of research outputs should focus more on these factors and less on any apparent
“novelty”.

1 An Overview

Broadly, preclinical research can be classified into two distinct categories depending
on the aim and purpose of the experiment, namely, “hypothesis generating” (explor-
atory) and “hypothesis testing” (confirmatory) research (Fig. 1). Hypothesis
generating studies are often scientifically-informed, curiosity and intuition-driven
explorations which may generate testable theories regarding the pathophysiology of
disease and potential drug targets. The freedom of researchers to explore such
innovative ideas is the lifeblood of preclinical science and should not be stifled by
excessive constraints in terms of experimental design and conduct. Nevertheless, in
order to subsequently assess the veracity of hypotheses generated in this way, and
certainly to justify clinical development of a therapeutic target, hypothesis testing
studies which seek to show reproducible intervention effects in relevant animal
models must be designed, conducted, analysed and reported to the highest possible
levels of rigour and transparency. This will also contribute to reducing research
“waste” (Ioannidis et al. 2014; Macleod et al. 2014). Chapter “Resolving the Tension
Between Exploration and Confirmation in Preclinical Biomedical Research” of the
handbook will deal with exploratory and confirmatory studies in details. This chapter
will only focus on general design principles for hypothesis testing studies. We will
General Principles of Preclinical Study Design 57

Fig. 1 Comparison of exploratory (hypothesis generating) and confirmatory (hypothesis testing)

preclinical studies. Descriptive statistics describes data and provides descriptions of the population,
using numerical calculations, graphs, and tables. In contrast, inferential statistics predicts and infers
about a population using a sample of data from the population, therefore one can take data from
samples and make generalisation about a population

address the issue of design principles for hypothesis-generating studies at the end of
this chapter. We advise that when researchers design and conduct hypothesis testing
in vivo studies, they should conform to the general principles for the major domains
that are outlined in Sect. 4 of the chapter and incorporate these principles into a
protocol that can be registered and published. The purpose of using these principles
is to enhance scientific rigour without restricting creativity. It is advisable that
sometimes there can be exploratory elements within the same hypothesis testing
studies; therefore, extra care in terms of applying these principles to reduce experi-
mental biases would be needed before the start of the studies. This chapter will not
cover reporting, which will be detailed in chapters “Minimum Information and
Quality Standards for Conducting, Reporting, and Organizing In Vitro Research”,
“Minimum Information in In Vivo Research”, and “Quality Governance in Biomed-
ical Research” of the handbook.
We would recommend that researchers who conduct hypothesis testing in vivo
studies should prepare clear protocols, which include a statistical analysis plan,
detailing how they are going to set up measures to address the major domains of
experimental biases before the experiments start. Ideally, these protocols should be
preregistered and/or published, so that the methods which will be used to reduce the
impact of bias are documented in an a priori fashion. The process of peer review of a
protocol prior to initiating experiments of course is a valuable opportunity for
refinement and improvement. Registering protocols encourages rigour and transpar-
ency, even if the protocol is not peer-reviewed. Some journals are open to
submissions of these types of protocols, such as BMJ Open Science, and many
journals offer the Registered Reports format. In addition, there are online resources
58 W. Huang et al.

that allow researchers to preregister their experimental protocols, such as preclinical.

eu and osf.io/registries.

2 General Scientific Methods for Designing In Vivo

Experiments

Designing an in vivo experiment involves taking a number of decisions on different

aspects of the experimental plan. Typically, a comparative experiment can be broken
into several component parts.

2.1 Hypotheses and Effect Size

The objective is usually to test a hypothesis. On some occasions, two hypotheses

may be postulated: the null hypothesis and the alternative hypothesis. The alternative
hypothesis refers to the presumption that the experimental manipulation has an effect
on the response measured; the null hypothesis is the hypothesis of no change, or no
effect. In a statistical test, the p-value reports the probability of observing an effect as
large or larger than the one being observed if the null hypothesis was true; the
smaller the p-value, the least likely it is that the null hypothesis is true. The null
hypothesis cannot be accepted or proven true. This also defines the effect of interest,
i.e. the outcome that will be measured to test the hypothesis. The minimum effect
size is the smallest effect the researcher designs the experiment to be able to detect
and should be declared in the protocol; it is set up as the minimum difference which
would be of biological relevance. The effect size is then used in the sample size
calculation to ensure that the experiment is powered to detect only meaningful
effects and does not generate statistically significant results that are not biologically
relevant. In many cases, it will be hard to determine the minimum difference of
biological relevance as for early stage experiments it might be completely unknown,
or translatability between clinical relevance and experimental detection thresholds
will be complex. There is no simple and easy answer to this question, but in general,
a minimum effect size should be set so one can assume to have a beneficial effect for
individuals rather than large cohorts, the difference must be experimentally testable
and reasonable to achieve, and should have a rationale for translation into patients in
the long run.

2.2 Groups, Experimental Unit and Sample Size

In comparative experiments, animals are split into groups, and each group is
subjected to different interventions, such as a drug or vehicle injection, or a surgical
procedure. The sample size is the number of experimental units per group;
identifying the experimental unit underpins the reliability of the experiment, but it
is often incorrectly identified (Lazic et al. 2018). The experimental unit is the entity
General Principles of Preclinical Study Design 59

subjected to an intervention independently of all other units; it must be possible to

assign any two experimental units to different comparison groups. For example, if
the treatment is applied to individual mice by injection, the experimental unit may be
the animal, in which case the number of experimental units per group and the
number of animals per group is the same. However, if there is any contamination
between mice within a cage, the treatment given to one mouse might influence other
mice in that cage, and it would be more appropriate to subject all mice in one cage to
the same treatment and treat the cage as the experimental unit. In another example, if
the treatment is added to the water in a fish tank, two fish in the same tank cannot
receive different treatments; thus the experimental unit is the tank, and the sample
size is the number of tanks per group. Once identified, experimental units are
allocated to the different comparison groups of the desired sample size; this is
done using an appropriate method of randomisation to prevent selection bias (see
Sect. 3). Each comparison group will be subjected to different interventions, at least
one of which will be a control. The purpose of the control group is to allow the
researcher to investigate the effect of a treatment and distinguish it from other
confounding experimental effects. It is therefore crucial that any control group is
treated exactly in the same way as the other comparison groups. Types of control
group to consider include negative control, vehicle control, positive control, sham
control, comparative control and naïve control (Bate and Clark 2014).

2.3 Measurements and Outcome Measures

Measurements are taken to assess the results; these are recorded as outcome
measures (also known as dependent variable). A number of outcome measures can
be recorded in a single experiment, for example, if burrowing behaviour is measured,
the outcome measure might be the weight of gravel displaced, or if neuronal density
is measured from histological brain slides, the outcome measure might be the neuron
count. The primary outcome measure should be identified in the planning stage of
the experiment and stated in the protocol; it is the outcome of greatest importance,
which will answer the main experimental question. The number of animals in the
experiment is determined by the power needed to detect a difference in the primary
outcome measure. A hypothesis testing experiment may also include additional
outcome measures, i.e. secondary outcome measures, which can be used to generate
hypotheses for follow-up experiments. Secondary outcome measures cannot be used
to draw conclusions about the experiment if the experiment was not powered to
detect a minimum difference for these outcome measures.
For the purpose of the statistical analysis, outcome measures fall into two broad
categories: continuous or categorical. Continuous measures are sometimes referred
to as quantitative data and are measured on a numerical scale. Continuous measures
include truly continuous data but also discrete data. Examples of true continuous
data include bodyweight, body temperature, blood/CSF concentration or time to
event, while examples of discrete data include litter size, number of correct response
or clinical score. Categorical responses are measured on a nonnumerical scale; they
60 W. Huang et al.

can be ordinal (e.g. severity score, mild/moderate/severe), nominal (e.g. behavioural

response, left/middle/right arm maze) or binary (e.g. disease state, present/absent).
Continuous responses may take longer to measure, but they contain more informa-
tion. If possible, it is preferable to measure a continuous rather than categorical
response because continuous data can be analysed using the parametric analyses,
which have higher power; this reduces the sample size needed (Bate and Clark
2014).

2.4 Independent Variables and Analysis

There are many ways to analyse data from in vivo experiments; the first step in
devising the analysis plan is to identify the independent variables. There can be two
broad types: independent variables of interest which the researcher specifically
manipulates to test the hypothesis, for example, a drug with different doses, and
nuisance variables, which are other sources of variability that may impact on the
outcome measure, but are not of direct interest to the researcher. Examples of
nuisance variables could be the day of the experiment, if animals used on different
days, or baseline body weight or locomotor activity. Every experiment has nuisance
variables. Identifying them at the protocol stage and accounting for them in the
design and the analysis, for example, as blocking factors, or co-variables, increase
the sensitivity of the experiment to detect changes induced by the independent
variable(s) of interest. The analysis plan should be established before the experiment
starts and any data is collected; it should also be included in the protocol. Additional
analyses can be performed on the data, but if an analysis was not planned before the
data was collected, it should be clearly reported as a post hoc or exploratory analysis.
Exploratory analyses are at greater risk of yielding false positive results.

3 Experimental Biases: Definitions and Methods

to Reduce Them

For any researcher who intends to carry out preclinical in vivo studies, it is important
to understand what experimental biases are. First, we need to know the definition
of bias. It is the inadequacies in the design, conduct, analysis or reporting of an
experiment that cause systematic distortion of the estimated intervention effect
away from the “truth” (Altman et al. 2001; van der Worp et al. 2010), and it will
significantly confound in vivo studies and reduce their internal validity. Sources of
bias are multiple and in many cases context dependant. In this overview chapter, it is
not possible to give an exhaustive list of potential sources of bias, and it behoves the
researcher to systematically identify all potential significant sources of bias for the
particular experiment being in planned and to design appropriate mitigation tactics
into the protocol. Major known types of biases include selection bias, performance
bias, detection bias, and attrition bias. Table 1 gives the definition of each type of
bias and describe the methods to reduce them.
General Principles of Preclinical Study Design 61

Table 1 Bias definition and bias-reducing methods (Lazic et al. 2018)

Name of bias Definition of bias Methods to reduce bias
Selection Refers to the biased allocation of To avoid systematic differences
bias animals to different treatment groups, between animals allocated to different
which could happen at the beginning treatment groups, one shall use a valid
of an animal study or at a stage where randomisation method, e.g. a
reassigning animals to different randomisation software or even a
treatment groups is needed following simple method such as picking a
an initial surgical procedure or number from a hat (Baastrup et al.
treatment. Selection bias results in 2010; Huang et al. 2013; Saghaei
systematic differences in baseline 2004). Detail for randomisation is
characteristics between treatment covered in chapter “Blinding and
groups (Higgins et al. 2011) Randomization”. Note that it is also
necessary to conceal the allocation
sequence from experimenters who
will assign animals to treatment
groups until the time of assignment
Performance Related to the systematic differences One can avoid performance bias by
bias in the care that is provided between improving the study design,
different treatment groups or being e.g. applying the same housing, diet,
exposed to factors other than the location conditions to all the animals
treatment that could influence the and by ensuring proper blinding of the
performance of the animals (Higgins experimenters to treatment groups,
et al. 2011; O’Connor and Sargeant which keeps the experimenters who
2014; van der Worp et al. 2010). perform the experiment, collect data
Performance bias is a result of animals and access outcomes unaware of
being managed differently due to, treatment allocation. Detail for
e.g. housing conditions, diet, group blinding is covered in chapter
sizes per cage, location in the animal “Blinding and Randomization”
house, and experimenters who
provide the care to animals are not
blinded to treatment groups
Detection Defined as the systematic distortion of The only way to avoid detection bias
bias the results of a study that occurs when is a complete blinding of the
the experimenter assessing experimenters, including those who
behavioural outcome measures has the analyse the data, so that they are not
knowledge of treatment assignment to aware which animal(s) belong to
groups (van der Worp et al. 2010). In which treatment group(s). The
this circumstance, experimenters protocol should define at what stage
measuring the outcomes may the blinding codes will be broken
introduce differential measurement of (preferably only after data analysis has
the outcomes rather than the treatment been completed). Detail for blinding is
itself due to inadvertent expectation covered in chapter “Blinding and
Randomization”
Attrition bias Is the unequal occurrence and Experimenters should report attrition
handling of deviations from protocol information for each experimental
and loss to follow-up between group and also include outcomes that
treatment groups (van der Worp et al. will not be affected by attrition. It is
2010). This bias can occur when also advisable to consult a statistician
animals die or are removed from the to minimise the impact of attrition bias
study due to adverse effects of the using some statistical approaches such
treatment or pre-set criteria for as intention-to-treat analysis by
(continued)
62 W. Huang et al.

Table 1 (continued)
Name of bias Definition of bias Methods to reduce bias
removal before observing the imputing the missing data. Excluding
outcomes; therefore, the outcomes are “outliers” from analysis should be
not observed for all animals, causing only undertaken as an extremely
inadvertent bias (O’Connor and measure and should only be done to
Sargeant 2014) pre-stated criteria. Detail for statistics
is covered in chapter “Blinding and
Randomization”

Researchers who conduct hypothesis testing in vivo animal work should under-
stand the importance of limiting the impact of experimental biases in the design,
conduct, analysis and reporting of in vivo experiments. Experimental biases can
cause significant weakness in the design, conduct and analysis of in vivo animal
studies, which can produce misleading results and waste valuable resources. In
biomedical research, many effects of interventions are fairly small, and small effects
therefore are difficult to distinguish from experimental biases (Ioannidis et al. 2014).
Evidence (1960–2012 from PubMed) shows that adequate steps to reduce biases,
e.g. blinded assessment of outcome and randomisation, have not been taken in more
than 20% and 50% of biomedical studies, respectively, leading to inflated estimates
of effectiveness, e.g. in the fields of preclinical stroke, multiple sclerosis,
Parkinson’s disease, bone cancer pain and myocardial infarction research (Currie
et al. 2013; Macleod et al. 2008; Rooke et al. 2011; Sena et al. 2007; van Hout et al.
2016; Vesterinen et al. 2010) and consequently significant research waste (Ioannidis
et al. 2014; Macleod et al. 2014, 2015). Therefore, it is imperative that biomedical
researchers should spend efforts on improvements in the quality of their studies
using the methods described in this chapter to reduce experimental biases which will
lead to increased effect-to-bias ratio.
However, it is worth pointing out that the notion that experimental biases could
significantly impact on in vivo animal studies is often assumed because they are
believed to be important in clinical research. Therefore, such an assumption may be
flawed, as the body of evidence showing the importance of bias-reducing methods
such as randomisation, blinding, etc. for animal studies is still limited and most of the
evidence is indirect. Furthermore, there may also be sources of bias which impact on
preclinical studies which are currently unknown. Thus, systematic review and meta-
analysis of in vivo studies have shown that papers that do not report bias-reducing
methods report larger effect sizes (Vesterinen et al. 2010). However, these studies
are based on reported data alone, and therefore there might be a difference between
what researchers do and what they report in their publications (Reichlin et al. 2016).
Reporting of the precise details of bias reduction methods is often scanty, and
therefore accurate assessment of the precise method and rigour of such procedures
is challenging. Moreover, those papers that do not report one bias-reducing method,
e.g. randomisation, also tend to not report other bias-reducing methods, e.g. blinding
and sample size calculation, suggesting that there could be interactions between
these methods.
General Principles of Preclinical Study Design 63

4 Experimental Biases: Major Domains and General

Principles

In this section, we will describe the major domains, in other words, sources that
could contribute to experimental bias if not carefully considered and if mitigating
tactics are not included in the design of hypothesis testing experiments before data
collection starts. These include sample size estimation, randomisation, allocation
concealment, blinding, primary and secondary outcome measures and inclusion/
exclusion criteria. General descriptions for these domains (Macleod et al. 2009; Rice
et al. 2008; Rice 2010; van der Worp et al. 2010) are shown in the following Table 2.
It is important to note that these domains are key things to be included in a protocol
as mentioned in Sect. 1.

Table 2 General descriptions for the major domains that contribute to experimental biases
Major domains General descriptions
Sample size estimation The sample size refers to the number of experimental units (e.g. a
single animal, a cage of animals) per group. In hypothesis testing
experiments, it should be determined with a power calculation.
Studies that are not appropriately powered are unethical, and both
underpowered and overpowered studies lead to a waste of
animals. The former because they produce unreliable results and
the latter because they use more animals than necessary
Randomisation Refers to the steps to reduce systematic differences between
comparison groups. Failure to conduct randomisation leads to
selection bias
Allocation concealment Refers to the practice of concealment of the group or treatment
assignment (i.e. the allocation) and its sequence of each
experimental unit from the experimenter until the time of
assignment. Failure to conceal allocation will lead to selection
bias. This should not be confused with randomisation
Blinding Refers to the practice of preventing the experimenter who
administer treatments, take care of the animals, assess the
responses and analyse data from knowing the test condition.
Failure of appropriate blinding leads to selection, performance
and detection biases
Primary and secondary Primary outcome measure refers to the outcome measure of most
outcome measures interest, and it is related to the efficacy of an intervention that has
the greatest importance for a given study. Secondary outcome
measure refers to the outcome measure that is related to
intervention efficacy but with less importance than the primary
outcome measure and is used to evaluate additional intervention
effects. It is important to declare what intervention effects are in
the study protocol
Inclusion/exclusion criteria Refers to criteria by which animals will be included or excluded
in a given study, e.g. due to abnormal baselines or not reaching
the required change in thresholds after designed experimental
insult
64 W. Huang et al.

Table 3 General principles to prevent experimental biases in hypothesis testing in vivo studies
Major domains General principles
Sample size estimation A power calculation (desired power of at least 0.8, and
alpha ¼ 0.05) to estimate the experimental group size should be
carried out before any hypothesis testing study using pilot data or
those relevant data from the literature. This could be done by
using a statistical software. Detail on this can be found in chapter
“A Reckless Guide to P-Values: Local Evidence, Global Errors”
Randomisation There are different methods available to randomly allocate
animals to experimental groups such as computer-generated
randomisation. One should always consider to use the most
robust, appropriate and available method for randomisation.
Detail on this can be found in chapter “Blinding and
Randomization”
Allocation concealment Methods should be used to conceal the implementation of the
random allocation sequence (e.g. numbered cages) until
interventions are assigned, so that the sequence will not be known
or predictable in advance by the experimenters involved in
allocating animals to the treatment groups
Blinding Blinding procedures should be carried out, so that the treatment
identity should not be disclosed until after the outcome
assessments have been finished for all animals and the primary
analysis have been completed. In case that one experimenter
conducts the whole study, any additional steps should be taken to
preserve the blinding. Detail on this can be found in chapter
“Blinding and Randomization”
Primary and secondary Experimenters should decide the outcome of great importance
outcome measures regarding the treatment efficacy before any study starts as the
primary outcome measure. This is also usually used in the sample
size estimation. Primary outcome measure cannot be changed
once the study starts and when the results are known.
Experimenters should also include secondary outcome measures
relating to additional effects of treatments; these may be used for
new hypothesis generating
Inclusion/exclusion criteria Experimenters should set up the exact criteria which will include
and exclude animals from their studies. Every animal should be
accounted for, except under these criteria. They should be
determined appropriately according to the study nature before the
studies commence. Once determined, they cannot be changed
during the course of investigation

General principles to reduce experimental bias in each of the above-mentioned

domains (Andrews et al. 2016; Knopp et al. 2015) are outlined in the following
Table 3.
General Principles of Preclinical Study Design 65

5 Existing Guidelines and How to Use Them

There are resources to assist investigators in designing rigorous protocols and

identify sources of bias. Cross-referencing to experimental reporting guidelines
and checklists (e.g. ARRIVE (NC3Rs 2018a), the NIH guidelines (NIH 2018a)
and the Nature reporting of animal studies checklist (Nature 2013)) can be informa-
tive and helpful when planning an experimental protocol. However, it is important to
bear in mind that these are primarily designed for reporting purposes and are not
specifically designed for use in assisting with experimental design. There are more
comprehensive planning guidelines specifically aiming at early experimental design
stage. Henderson et al. identified 26 guidelines for in vivo experiments in animals in
2012 (Henderson et al. 2013) (and a few more have been published since, like
PREPARE (Smith et al. 2018), developed by the NORECEPA (Norway’s National
Consensus Platform for the advancement of the 3Rs), and PPRECISE for the field of
pain research (Andrews et al. 2016)). Most of them have been developed for a
specific research field but carry ideas and principles that can be transferred to all
forms of in vivo experiments. Notable are, for example, the very detailed Lambeth
Conventions (Curtis et al. 2013) (developed for cardiac arrhythmia research), from
Alzheimer’s research recommendations by Shineman et al. (2011) and generally
applicable call by Landis et al. (2012).
The authors of many of these guidelines state that their list might need adaption to
the specific experiment. This is pointing out the general shortcoming that a fixed-
item list can hardly foresee and account for any possible experimental situation and a
blind ticking of boxes ticking of boxes is unlikely to improve experimental design.
Such guidelines rather serve an educational purpose of making researchers aware of
possible pitfalls and biases before the experimental conduct.
Two examples for a more adaptive and reactive way to serve a similar purpose
should be stated: the NIH pages on rigour and reproducibility (NIH 2018b) provide
in-depth information and collect important publications and workshop updates on
these topics and have a funding scheme specifically for rigour and reproducibility.
Second, using the Experimental Design Assistant (EDA) (NC3Rs 2018b; Percie du
Sert et al. 2017) developed by the UK’s National Centre for the 3Rs (NC3Rs), a free
to use online platform guiding researchers through experimental planning will give
researchers the opportunity to adopt guideline and rigour principles precisely to their
needs. The researcher creates a flow diagram of their experimental set-up grouped in
three domains: the experiment (general questions on hypotheses and aims, animals
used, animal strains, etc.), the practical steps (experimental conduct, assessment,
etc.) and the analysis stage (e.g. outcome measures, statistical methods, data
processing). Unlike a fixed checklist, the EDA checks the specific design as
presented by the experimenter within the tool using logic algorithms. The user is
then faced with the flaws the EDA identified and can adjust their design accordingly.
This process can go through multiple rounds, by that forming a dynamic feedback
loop educating the researcher and providing more nuanced assistance than a static
checklist can.
While this process, however valid, might take time, the following steps of the
EDA actively guide researchers through crucial and complex questions of the
66 W. Huang et al.

experiment, by suggesting fitting methods of statistical analyses of the experiment

and subsequently carrying out sample size calculations. The EDA can then also
generate a randomization sequence or compile a report of the planned experiment
that can, e.g. be part of a preregistration of the experimental protocol.

6 Exploratory and Confirmatory Research

It is necessary to understand that there are in general two types of preclinical

research, namely, exploratory and confirmatory research, respectively. Figure 1
shows that exploratory studies mainly aim to produce theories regarding the patho-
physiology of disease (hypothesis generating), while confirmatory studies seek to
reproduce exploratory findings as clearly defined intervention effects in relevant
animal models (hypothesis testing). The next chapter will deal with exploratory and
confirmatory studies in details. Similar standards of rigour are advisable for both
forms of studies; this may be achieved by conforming to the general principles for
the major domains that are outlined in Table 2 and incorporating these principles into
a protocol that can be registered and published. It is important to note that both
exploratory and confirmatory research can be closely linked: sometimes there can be
exploratory and confirmatory components within the same studies. For example, a
newly generated knockout mouse model is used to examine the effect of knockout on
one specific phenotype (hypothesis testing – confirmatory) but may also describe a
variety of other phenotypic characteristics as well (hypothesis generating – explor-
atory). Therefore, extra care in terms of applying these principles to reduce experi-
mental bias would be needed before the commence of the studies. It also worth
noting that sometimes it might not be compulsory or necessary to use some of the
principles during exploratory studies such as sample size estimation and blinding
which are albeit of highest importance in confirmatory research.
However, it is necessary to recognise how hypothesis confirming and hypothesis
generating research relate to each other: while confirmatory research can turn into
exploratory (e.g. if the findings are contrary to the hypothesis, this can lead to a new
hypothesis that can be tested in a separate experiment), under no circumstances
exploratory findings should be disseminated as the result of hypothesis confirming
research by fitting a hypothesis to your results, i.e. to your p-values (often called
HARKing ¼ hypothesising after results are known or p-hacking ¼ sifting through a
multitude of p-values to find one below 0.05).
In conclusion, this chapter provides general concepts and principles that are
important for the design and conduct of preclinical in vivo experiments, including
experimental biases and how to reduce these biases in order to achieve the highest
levels of rigour for hypothesis generating research using animals. The chapter should
be used in conjunction with other relevant chapters in the handbook such as chapters
“Blinding and Randomization”, “Minimum Information and Quality Standards for
Conducting, Reporting, and Organizing In Vitro Research”, “Minimum Information
in In Vivo Research”, “A Reckless Guide to P-Values: Local Evidence, Global
Errors”, and “Quality Governance in Biomedical Research”.
General Principles of Preclinical Study Design 67

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