Journal of Umm Al-Qura University for Applied Sciences

https://doi.org/10.1007/s43994-024-00153-x

REVIEW

Sample size calculation in biomedical, clinical and biological sciences

research
Idris Zubairu Sadiq1 · Abubakar Usman2 · Aliyu Muhammad1 · Kabiru Haliru Ahmad3

Received: 16 September 2023 / Accepted: 5 April 2024

© The Author(s) 2024

Abstract
Sample size determination is a critical aspect of biomedical research, as it dictates the number of samples needed for a suc-
cessful experiment that can yield generalizable results to the broader population. This paper outlines the methodology for
calculating sample sizes in various categories of biomedical and clinical research, encompassing cross-sectional surveys,
comparative studies, case–control studies, cohort studies, and animal studies. Detailed examples are provided for each cat-
egory. The determination of an appropriate sample size holds significant importance from scientific, ethical, and resource
allocation perspectives. It is noteworthy that research outcomes are often directly influenced by the accuracy of sample size
calculations. A robust sample size calculation serves as a cornerstone for researchers, enabling them to draw precise infer-
ences with a high level of precision across a spectrum of biomedical and clinical investigations.

Keywords Sample size · Biomedical research · Clinical research · Animal studies · Surveys

1 Introduction soundness of the supporting research [2]. In statistical terms,

a "population" encompasses the entire group, while a "tar-
The term "sample size" refers to the quantity of subjects, get population" refers to a subset of participants possessing
patients, or units that will be part of a biomedical inves- specific demographic and clinical characteristics, which are
tigation. The process of determining how many observa- the focus of the intervention research. A "sample" denotes
tions or repetitions to incorporate into a statistical sample a portion of the target population selected for inclusion in
is known as sample size determination. In any empirical the study [3].
research endeavor aimed at making inferences about a popu- Crucially, determining the sample size is a pivotal facet
lation based on a sample, careful consideration of sample of the research process as it leads to more accurate findings
size is crucial. The primary objective of calculating sample that can be more broadly applicable. To effectively employ
size is to ascertain the number of samples required to detect sample size calculation formulas, a researcher must have a
unknown clinical parameters, treatment effects, or associa- solid grasp of factors such as significance level, effect size,
tions once data collection is complete [1]. study capacity, and effect size; margin of error; ratio within
Recently, biomedical studies have faced heightened scru- the population; and design effect [4].
tiny within the context of evidence-based medicine. The
strength of evidence is directly contingent on the statistical
2 Sample size calculation: basic statistical
concepts
* Idris Zubairu Sadiq
[email protected]
2.1 Null hypothesis and alternative hypothesis
1
Department of Biochemistry, Faculty of Life Sciences,
Ahmadu Bello University, Zaria, Nigeria The null hypothesis, denoted as ­H0, represents a statement
2
Department of Statistics, Faculty of Physical Sciences, that contradicts the researcher's or experimenter's expecta-
Ahmadu Bello University, Zaria, Nigeria tions or predictions. It essentially posits that the variables
3
Department of Veterinary Microbiology, Faculty lack a precise or genuine relationship. For instance, in a
of Veterinary Medicine, Ahmadu Bello University, Zaria, clinical trial assessing a new drug, the null hypothesis might
Nigeria

Vol.:(0123456789)
 Journal of Umm Al-Qura University for Applied Sciences

state that the new drug, on average, is no more effective strengthens its statistical capability and diminishes the
than the current drug. On the other hand, the alternative chance of making a Type II error, thereby lowering the
hypothesis, denoted as H ­ 1, outlines the purpose of a statisti- likelihood of incorrect negative conclusions. This statisti-
cal hypothesis test. In the same clinical trial scenario, the cal power is denoted as 1 − 1 − β and, in most clinical trials,
alternative hypothesis could propose that the new drug has a power of 0.8 (or 80%) is deemed optimal for effectively
a different average impact compared to the existing drug. An detecting a statistically significant distinction. With an 80%
alternative hypothesis is a statement that suggests or hints at power, there remains a 20% possibility of failing to detect a
a potential finding or result for an investigator or researcher. significant difference, even if it truly exists [4].
These alternative theories are categorized into two groups:
directional and non-directional. 2.4 Effect size

2.2 Significance levels An effect size is a numerical measure, derived from either

actual data or a sample, that quantifies the strength of the
The P value, denoted as p, serves as a crucial metric in relationship between two variables within a population. This
statistical hypothesis testing. It signifies the probability of value might arise from an equation elucidating the contribu-
obtaining a result as extreme as, or more extreme than, the tions of statistics and elements to the effect size, or it could
observed data, assuming that the null hypothesis is true. A represent a parameter value for an imagined population [7].
P value that is less than or equal to the significance level α is Effect sizes can be described in terms of various parameters,
deemed statistically significant. The significance levels com- including the regression coefficient in a regression analysis,
monly used are 0.1, 0.05, and 0.01, representing thresholds the difference in means, or the likelihood of a specific event
that reflect the likelihood of observing such a high value [8] (such as a heart attack) taking place.
by random chance [5]. This metric can be envisioned as a
predictive gauge of how well the data aligns with the model
employed for its analysis. It ranges from 0, indicating com- 2.5 Prevalence rate in the population
plete incompatibility, to 1, denoting full compatibility. This
value offers insight into how effectively the model captures Prevalence, in the context of a specific population, denotes
the essence of the data under consideration [6]. Typically, the proportion of individuals who exhibit a particular dis-
a P value of 0.05 or less is regarded as "statistically sig- ease or characteristic at a specific juncture or over a defined
nificant," implying that the observed result is unlikely to duration [9]. In Epidemiology, prevalence distinguishes
have occurred by chance. Conversely, any P-value greater itself from incidence by encompassing all existing and pre-
than 0.05 is considered "nonsignificant," suggesting that vious cases within the population at the time of assessment,
the observed result may be reasonably explained by random while incidence exclusively accounts for new occurrences
variability [6]. [9]. Point prevalence specifically pertains to the observed
prevalence at a particular moment in time, representing the
2.3 Study power percentage of individuals with a specific disease or attrib-
ute on a specific date. On the other hand, period prevalence
Sample size calculation influences the generalizability of refers to the prevalence of a disease over a specified span,
study findings to the broader population. A study's power signifying the proportion of individuals with a particular
illness or condition at some point within that interval [9].

all new and pre − existing cases in a given period

Prevalence of disease = × 10n (1)
population during the same period

persons having particular attribute in a given period

Prevalence of attribute = × 10n (2)
Population during the same period
Journal of Umm Al-Qura University for Applied Sciences

The value of 1­ 0n is usually 1 or 100 for common attrib- 2.8 Standard deviation (SD) in the population
utes. The value of ­10n might be 1,000, 100,000, or even
1,000,000 for rare attributes and most diseases. The standard deviation gauges the extent of spread within
Example for calculating prevalence in population. An a given dataset. A smaller standard deviation indicates that
evaluation of 2000 pregnant women attending a hospital in the data points are closely clustered around the mean (also
Kano, Nigeria reported a total of 785pregnant women tak- known as the expected value), while a higher standard devia-
ing supplements at least 2 times a week during the second tion signifies a wider dispersion of values [10]. When the
trimester. Calculate the prevalence of frequent supplement standard deviation is smaller, the data consistently centers
use in this group. around the mean, whereas a larger standard deviation indi-
cates greater variability or dispersion in the data. A standard
Prevalance = (785∕2000) × 100 = 0.39 × 100 = 39%
deviation approaching 0 implies that data points are near the
Therefore, the prevalence of using supplement in this mean, whereas a high or low standard deviation suggests that
group is 39% data points deviate from the mean, either above or below it,
respectively [11].

2.6 Incidence rate
3 Sample size calculation for cross‑sectional
An incidence rate is a measure of how easily disease studies/surveys
spreads across a population. The incidence rate is the ratio
of the number of cases to the total time that the popula- A cross-sectional study alternatively called a transver-
tion is exposed to the disease. The incidence, which can sal study, prevalence study or cross-analysis is a type of
be expressed as a risk or an incidence rate, represents the research that focuses on data from a population or a typical
number of new cases of disease over a given period. subgroup at a specific point in time
Number of new cases of disease
Incidence rate = (3)
time each person was observed, the total for all the people

Examples: Calculating Incidence Rates in the population. (Z 1− 𝛼 )2 (p)(q)

Example: In the year 2019, 130,000 new cases of HIV Sample size(n) = 2
(5)
were reported in Nigeria. If the population of people in (d)2
Nigeria is estimated to be 200,000,000 in 2019. Calculate
n = sample size.
the incidence rate of HIV in 2019 in Nigeria.
Z 1−α/2 = Critical value and a standard corresponding
Number of new cases of disease level of confidence.
Incidence rate =
total populationxTimeframe (4) (At 95% CI or 5% level of significance (type-I error) it
is 1.96 and at 99% CI it is 2.58).
Incidence rate = (130,000 ⁄ 200,000,000 × 1) × 100,000. P = prevalence or based on previous research q = 1-p.
= 65 new cases of HIV per 100,000 population. d = Margin of error or precision.
The Incidence rate is therefore 65 new cases of HIV per Example for calculating sample size from the cross-
100,000 population. sectional survey. Calculate the sample size required to
study the prevalence of liver diseases in a capital of a
2.7 Margin of error country if a previous study indicated that liver diseases in
the population was 9%, at 95% CI and 5% margin of error.
The margin of error quantifies the degree of random sam- Given:
pling variability present in the results of a survey. A greater Z1−α/2 = 1.96.
margin of error implies less certainty that a poll's findings P = 9% = 0.09.
accurately represent the broader population. When the mar- q = 1–0.09 = 0.91.
gin of error is elevated, it suggests either an inaccurate sam- d = 5% = 0.05.
pling of the population or a variation in the measure. In Applying
such cases, the variable being measured typically exhibits a
positive variance. (Z 1− 𝛼 )2 (p)(q)
2
Sample size(n) =
(d)2
 Journal of Umm Al-Qura University for Applied Sciences

(1.96)2 (0.09)(0.91) 5 Sample size calculation from the case–

Sample size(n) = 2
(0.05)
= 125.85 control survey

(n) = 126. A case–control study, also known as a case–reference study,

Therefore, the investigator needed a minimum of at is a research method that involves comparing two existing
least 126 samples. groups with different outcomes based on a key causative fac-
tor. Its primary purpose is to investigate whether exposure
is associated with a specific outcome. This type of study is
commonly employed to identify factors contributing to a
4 Calculating sample size for quantitative particular health condition by contrasting individuals with
variable the condition to those without it. Examples of case–control
studies include examining the relationship between tobacco
Quantitative variables are factors that can be counted or smoking and lung cancer, alcohol consumption and hepatic
numerically measured in the population, such as a person's diseases, as well as mortality rates and smoking. There are
height, weight, age, arm length, blood pressure, tempera- two main types of case–control studies:
ture, glucose level, hemoglobin content, and cholesterol Qualitative case–control study: This type aims to estab-
level. These variables can be calculated using the formula lish a non-quantitative relationship, such as the link between
below. tobacco smoking and lung cancer or exposure to carcinogens
Where. versus non-exposure.
Z1−α/2 = Critical value and a standard corresponding level Quantitative case–control study: This type focuses on
of confidence. quantifying the association between two conditions (dis-
SD = Standard Deviation. ease and non-disease).
d = Margin of error or precision
(r + 1)p(1 − p)(Z1−𝛽 + Z 1− 𝛼 )2
2
(Z 1− 𝛼 ) (SD) 2
Sample size(n) = 2
(7)
(6)
2
( )2
Sample size(n) = r p1 + p2
(d)2
where.
Example for calculating sample size with a quantitative
n = number of required sample.
variable. Calculate the sample size require to study fasting
r = ration of case to control.
blood sugar level in the population of a city at a 95% con-
p = population proportion.
fidence interval if the margin of error is 2 mmol/L (mean
p1 = case proportion.
blood sugar) and fasting blood sugar from previous studies
p2 = control proportion.
was recorded as 6.5 mmol.
z1-β = the power of the study.
Given:
z1-α/2 = Critical value.
Z1−α/2 = 1.96.
Calculation of sample size from a qualitative case–con-
SD = 6.5
trol study. Calculate the sample size required to investi-
d = 2.
gate the relationship between coffee consumption and risk
by employing the formula:
of heart diseases at 95% CI and 80% power of the study.
(Z 1− 𝛼 )2 (SD)2 Assumes that the expected proportion in the case is 20%
Sample size(n) = 2
and the control group is 8%.
(d)2 Given;
r = 1.
(1.95)2 (6.5)2 p = 0.14.
Sample size(n) = = 40.164 z1-β = 0.84.
(2)2
z1-α/2 = 1.96.
(n) = 40. p1 = 0.2
In this case, the researcher needed at least 40 samples. p2 = 0.08.
By employing Eq. (6):
Journal of Umm Al-Qura University for Applied Sciences

(r + 1)p(1 − p)(Z1−𝛽 + Z 1− 𝛼 )2 6 Sample size estimation for cohort studies

2
Sample size(n) = )2
A cohort study is a type of medical research aimed at under-
(
r p1 + p2
standing the causes of diseases and how various risk factors
influence health outcomes. It constitutes a specific form of
(1 + 1)(0.14)(1 − 0.14)(0.84 + 1.95)2
Sample size(n) = = 24.08 longitudinal study that observes a group of individuals who
1(0.2 + 0.08)2
share a common characteristic (forming a cross-section of a
(n) = 24. cohort) at different points in time. Cohort studies represent
From the calculations above, the researcher needed at one of the fundamental designs in epidemiology and find
least 24 samples. application in research across diverse fields including social
Calculation of sample size from a quantitative science, nursing, medicine, psychology, and other areas reli-
case–control study. Calculate the sample size required ant on hard-to-obtain evidence-based responses (statistics).
to study the amount of monosodium glutamate associated While clinical trials predominantly serve to evaluate the
with kidney damage at 95% CI and fix the power of the effectiveness of newly developed pharmaceuticals before
study at 80% if a researcher obtained from the previous their market release, epidemiological analysis of how risk
study the mean difference in monosodium consumption factors influence disease occurrence plays a crucial role in
classifying the origins of diseases and furnishing pre-clinical
evidence regarding the viability of protective measures.

�� �2
((Z1− 𝛼2 ) {(1 + 1∕m)p(1 − p)} + (Z1− 𝛽)
√
(p0 (1 − p0 )∕m)p1 (1 − p1 ) )
Sample size(n) = (9)
� �2
p0 − p1

between the case and control groups was 2 mg/day and where.
SD was 10 mg/day. n = number of samples required.
Given that: m = number of control subject per group.
r = 1. p0 = control possibility.
SD = 10. p1 = Experimental possibility.
z1-β = 0.84. p = population proportion.
z1-α/2 = 1.95. z1-β = the power of the study.
d = 2. z1-α/2 = Critical value.
using the following equation: Example of calculating same size estimation from a
cohort studies. Calculate the sample size needed to assess
2
(r + 1)(SD)2 (Z1−𝛽 + Z 1− 𝛼 ) the association between tobacco smoking and risk of mor-
Sample size(n) = 2 (8) tality at 95% CI and 80% power of the study with the equal
r(d)2 number of case and control subjects if a previous study indi-
cated that proportion of risk of mortality in tobacco smokers
(1 + 1)(10)2 (0.84 + 1.95)
2
is 15% and, in the control, group is 24%.
Sample size(n) = = 389.205 Given that;
1(2)2
m = 1.
(n) = 389. p = 0.195.
The researcher, therefore, needed at least 389 samples. z1-β = 0.84.
z1-α/2 = 1.96.
po = 0.24.
p1 = 0.15.
Applying:

�� �2
((Z1− 𝛼2 ) {(1 + 1∕m)p(1 − p)} + (Z1− 𝛽)
√
(p0 (1 − p0 )∕m)p1 (1 − p1 ) )
Sample size(n) = � �2
p0 − p1
 Journal of Umm Al-Qura University for Applied Sciences

√ √ 2
(1.96 {2(0.195)(1 − 0.195)} + 0.84 {(0.24(1 − 0.24))0.15(1 − 0.15)})
Sample size(n) = = 185.6593
(0.24 − 0.15)2

(n) = 186. the sample size if he decided to conduct the study at

Based on this calculation, 186 samples are needed to 95% CI with 80% power of the study.
assess the association between tobacco smoking and the
risk of mortality. Given that:
SD = 20 mg/dl.
d = 10 mg/dl.
7 Sample size estimation for comparative z1-β = 0.84.
studies z1-α/2 = 1.96.
Applying
Comparative studies involve the examination, comparison, 2
and contrast of various subjects or concepts. These stud- 2(SD)2 (Z1−𝛽 + Z 1− 𝛼 )
ies employ both quantitative and qualitative methods to Sample size(n) = 2 (11)
(d)2
assess similarities and/or differences between two groups
of subjects and/or patients. For instance, if a researcher
aims to investigate the effects of an antimalarial drug, they 2(20)2 (0.84 + 1.96)2
Sample size(n) = = 62.72
will partition their subjects into two groups: one receiving (10)2
the antimalarial drug and the other receiving a placebo for
comparison. n = 63.
Quantitative comparative studies Therefore, the investigator needed a minimum of at
least 63 samples.
(1) Calculate the sample size required to study the effect Qualitative comparative studies
of new antihypertensive drug if this drug reduces
hypertension by 8 mmHg and the standard deviation (1) If a previous study indicated that 35% of patients suffer-
did on the previous study with other drugs indicated ing from AIDS die within a stipulated time. A clinician
20 mmHg. Assumes that the level of significance is 5% is confident that a new antiretroviral therapy tested and
and study power 80% found to increase survival to 24% would be clinically
significant. Assuming the effect size to be between pro-
Applying portions 0.35 – 0.24 = 0.11. Calculate the sample size
SD = 20 mg/dl. required at a 5% level of significance and study power
d = 8 mg/dl. of 80%.
z1–β = 0.84.
z1–α/2 = 1.96 Given that:
p = 0.295.
2(SD)2 (Z1−𝛽 + Z 1− 𝛼 )
2
p1 = 0.35.
Sample size(n) = 2 (10) p2 = 0.24.
(d)2 zβ = 0.84.
zα/2 = 1.96.
2(20)2 (0.84 + 1.96)2 Applying:
Sample size(n) = = 98
(8)2 2p(1 − p)(Z1−𝛽 + Z 1− 𝛼 )2
(n) = 98. Sample size(n) = ( )2
2
(12)
p0 − p1
Therefore, the investigator needed a minimum of at
least 98 samples.
2(0.295)(1 − 0.295)(0.84 + 1.96)2
Sample size(n) = = 269.5081
(2) A clinician wants to compare a new antidiabetic drug (0.35 − 0.24)2
with a placebo. If the new antidiabetic drug reduces the
blood sugar level by 10 mg/dl as compared to placebo, (n)=270.
which he considered as clinically significant. Assuming Therefore, the investigator needed a minimum of at
a previous study recorded SD as 20 mg/dl. Calculate least 270 samples.
Journal of Umm Al-Qura University for Applied Sciences

8 Sample size formula for animal studies Example. Calculate the minimum and a maximum num-
ber of animals required per group to test a new antidiar-
Taking into account both ethical considerations and finan- rheal drug with control if the research is designed to have 2
cial constraints, it is imperative to conduct experiments groups. Assume ­df to be 10 and 20 respectively.
with a limited number of animals, but with meticulously
n = df∕s + 1 (16)
designed protocols to ensure precise data analysis. It is
strongly advised that researchers engage a statistician in
the early stages of experimental design. Furthermore, df
Minimum = n = +1
embarking on any experiment without a clear understand- s
ing of how the results will be assessed is inadvisable 10
= +1
[12]. The power analysis approach, akin to its applica- 2
tion in human studies, is often the preferred method for =6 animals per group,
determining sample size in animal research. Additionally,
the 'resource equation' method stands as an equally valid Totalsamplesize = 6 × 2 = 12
alternative for ascertaining the appropriate sample size in
studies involving animals [2, 12, 14, 15]. This approach df
aligns with the overarching goal of conducting research Maximum = n = +1
s
that is scientifically sound, cost-effective, and ethically 20
= +1
responsible. 2
The resource equation [13] is given as =11 animals per group,
E =(the total number of experimental units)
(13) Total sample size = 11 × 2 = 22
− (the total number of treatment groups)
Sample size calculation for animal studies having one
where E denotes sample size should be between 10 and 20
control group with two or more experimental groups
[16].
The power analysis. The power, significance level, sided-
(II) for an experiment involving the use of one con-
ness, standard deviation, size effect, and Sample size are six
trol group and two or more experimental groups
variables that are mathematically related to power analysis.
to be treated with the same compound at varying
If the first five are known, the sixth (normally sample size)
concentrations and at the end to be analyzed using
can be calculated [16].
ANOVA, the formula below can be used.
Sample size calculation for animal study having one
experimental group and one control df = (N − 1)(v − 1) (17)

(I) For an experiment involving the use of one experimen- where N is the total number of subjects and v is the number
tal group and one control to be compared using an inde- of measurements repeated
pendent T-test, the number of animals can be calculated N = df∕(v − 1) + 1 (18)
using the formula below.
To obtain the minimum and maximum numbers of ani-
df = N − s = sn − s = s(n − 1) (14) mals available, substitute the df in the formulas below with
where N denotes the total number of subjects, s denotes the 10 and 20, respectively:
number of groups, and n denotes the number of subjects per df 10
group, ­df denotes the degree of freedom. n is obtained by Minimum = N = +1= +1 (19)
v−1 v−1
rearranging the formula:
n = df∕s + 1 (15) df 20
Maximum = N = +1= +1 (20)
v−1 v−1
The ­df in the formulas is substituted with the minimum
(10) and maximum (20) based on the appropriate range of Example. Calculate the sample size required to test the
the ­df to achieve the optimal and maximum numbers of ani- toxicity of different doses of cisplatin 4.5 mg/kg,5.5 mg/
mals per group: kg, 6.5 mg/kg, and 7.5 mg/kg against control.
For minimum, n = 10/s + 1 and for maximum, n = 20/s + 1.
 Journal of Umm Al-Qura University for Applied Sciences

Table 1  Software and online calculators for computing sample size

Software/Calculator Description Website

Epi Info A suite of free, user-friendly tools for public health CDC Epi Info Website
professionals, including a sample size calculator
Sample Size Calculator by Creative Research An online tool for determining sample size needed Creative Research Systems Website
Systems for surveys and experiments
Power and Sample Size Calculation (PS) A software package for calculating power and sam- PS Website
ple size for a wide range of study designs
ClinStat A statistical calculator with a suite of tools, includ- ClinStat Website
ing sample size calculations for clinical research
Stata A comprehensive statistical software that includes Stata Website
tools for sample size determination among its
features
Sample Size Calculator by Creative Research An online tool for determining sample size needed Creative Research Systems Website
Systems for surveys and experiments

10 treated with two different compounds at varying

Minimum = N = +1
v−1 concentrations and at the end to be analyzed using
10 ANOVA, the formula below can be used.
= +1
(5 − 1)
df
10
= +1
n=
gv
+1 (21)
4
=2.5 + 1 = 3.5 where ­df denotes the degree of freedom, N denotes the total
3.5 should be rounded to 4 number of subjects, g denotes the number of groups, n denotes
the number of subjects per group, and v denotes the number of
Maximum = N =
20
+1 measurements repeated.
v−1 Example. If a single dose of freshly made streptozotocin of
=
20
+1 45 mg/kg body weight was given to animals to induce diabe-
5−1 tes. Calculate the sample size needed to test the hypoglycemic
20 effect of moringa olifera extract in diabetic and non-diabetic
= +1
4 mice with the following groups(G): G1: non-diabetic (nor-
=5 + 1 = 6 mal saline), G2(diabetic untreated), G3:diatetic (100 mg/kg),
Since this experiment may require sacrificing animals, G4:(diabetic 400 mg/kg), and G5:(Diabetic 800 mg/kg) and
the total sample size required can be calculated as follows G6: positive control(metformin 42 mg/kg).
Note that the number of treatment group can be consid-
Minimum = N × v = 4 × 5 = 20 animals ered as (negative control, positive control, treated groups,
and diabetic untreated which is 4) and number of repeated
Maximum = N × v = 6 × 5 = 30 animals measurement=6
As this experiment involve cisplatin which is toxic, it is n = df∕gv + 1 (22)
advisable to use the maximum number of animals as some
may die during the experiment. In this case, 30animals
df
may be divided into 5 groups with each group containing minimum = n = +1
gv
6 animals per group.
10
The animal sample size for one control group and two = +1
or more experimental groups to treated with different 4×6
10
compounds = +1
24
=0.4 + 1 = 1.4 ∼ 2
(III) for an experiment involving the use of one control
group and two or more experimental groups to be
Journal of Umm Al-Qura University for Applied Sciences

df Open Access This article is licensed under a Creative Commons Attri-

Maximum = +1 bution 4.0 International License, which permits use, sharing, adaptation,
gv distribution and reproduction in any medium or format, as long as you give
=
20
+1 appropriate credit to the original author(s) and the source, provide a link
4×6 to the Creative Commons licence, and indicate if changes were made. The
20 images or other third party material in this article are included in the article's
= +1 Creative Commons licence, unless indicated otherwise in a credit line to
24
the material. If material is not included in the article's Creative Commons
=0.8 + 1 = 1.8 ∼ 2 licence and your intended use is not permitted by statutory regulation or
exceeds the permitted use, you will need to obtain permission directly from
In this case Minimum = Maximum the copyright holder. To view a copy of this licence, visit http://​creat​iveco​
mmons.​org/​licen​ses/​by/4.​0/.
Total sample size = Minimum = Maximum = N × g = 2 × 4 = 8

As the experiment will involve sacrificing an animal, the References

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