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Editorial

Further discussions on choosing the number of animals for an experiment

In the April 2007 issue of Nuclear Medicine and
Biology [1], an editorial discussed justifying the number of
animals for each experiment. In that analysis, an illustration of animals needed for a statistically significant result
at the Pb.05 level was presented using an unpaired t test
for a typical reproducibility for dissection of tissues and
the expected difference as a result of treatment. This
approach was chosen because most investigators writing in
Nuclear Medicine and Biology perform such an analysis
after the experiment rather than carrying out a power
analysis before the experiment. The calculations were
carried out using the commercially available program
GraphPad Instat [2].
Unfortunately, the P value (the statistical significance) in
the April 2007 issue was stated as a two-tailed P value. In
fact, these analyses were run for a one-tailed P value.
Although the two-tailed P value is the recommended
approach, there are situations, such as blocking studies,
where the one-tailed P value is warranted. In order to better
reflect the results in blocking studies, percentage differences
between the control and treatment group were shown as a
decrease. This change from two-tailed P value to one-tailed
P value is instructive, because it changes the number of
animals needed to obtain a statistically significant result as
defined by Pb.05. For such a small P value, it is unlikely that
the difference is due to a coincidence of random sampling.
The investigator can reject the difference as a coincidence
and conclude instead that the populations have different
means [2]. In the first case in Table 1, where the change due
to treatment is the same as the variance of the assay (20%),

the number of animals increases from 8 to 11 for a one-tailed

P value vs. a two-tailed P value.
More complex programs to perform a power analysis are
also available [3]. The same Pb.05 can be chosen along with
the % (CV) due to biological variability. In addition, the
power can be chosen as well. This is a measure of the chance
of obtaining a statistically significant result at the Pb.05 level
for a given real difference between the control and the
treatment group. Eighty percent power is usually chosen as a
compromise to reduce the number of animals. It appears that
the number of animals dictated by the t test for situations
where the variance and the difference between control
and treatment are both 20% seems to correspond best
with 80% power.
The following editorial will explore the comparison of
these two approaches and go into detail concerning the
philosophy behind the Student's t test and the power
analysis [4].
Acknowledgment
We appreciate the comments of Drs. Timothy McCarthy
and David Raunig of Pfizer Global R&D who brought the
underestimation of the number of animals to our attention.
William C. Eckelman
Molecular Tracer LLC, Bethesda, MD, USA
Email-address: wceckelman@verizon.net
doi:10.1016/j.nucmedbio.2007.10.002

Table 1
Row in Ref [1]
1- or 2-tailed P value
% Difference between control and treatment group averages
% (CV) due to biological variability
Number of animals for Pb.05
Lower 95% CI control group
Upper 95% CI control group
Lower 95% CI treatment group
Upper 95% CI treatment group
80% Power analysis*

2
1
20
20
8
4.16
5.84
3.33
4.67
9

0969-8051/$ see front matter 2008 Elsevier Inc. All rights reserved.

2
20
20
11
4.33
5.67
3.46
4.54
12

3
1
20
15
5
4.07
5.93
3.25
4.74
5

2
20
15
5
4.07
5.93
3.25
4.74
8

4
1
25
20
5
3.76
6.24
2.82
4.68
9

2
25
20
6
3.95
6.05
2.96
4.54
11

5
1
30
20
5
3.76
6.24
2.63
4.37
8

2
30
20
5
3.76
6.24
2.63
4.37
10

Editorial / Nuclear Medicine and Biology 35 (2008) 12

References
[1] Eckelman WC, Kilbourn MR, Joyal JL, Labiris R, Valliant JF. Justifying
the number of animals for each experiment. Nucl Med Biol 2007;34:
22932.
[2] GraphPad InStat version 3.0a for Macintosh, GraphPad Software, San
Diego, CA, http://www.graphpad.com.

[3] GraphPad StatMate version 2.0 for Windows ,GraphPad Software, San
Diego, CA, http://www.graphpad.com.
[4] Schiebe P. Number of samples hypothesis. Nucl Med Biol 2008;35:
39.