1. The Practice of Statistics in the Life Sciences Fourth Edition
Chapter 8: Designing experiments
Copyright © 2018 W. H. Freeman and Company

2. Objectives Designing experiments Terminology of experiments
Randomized, comparative experiments
Completely randomized designs
Block designs
Matched pairs designs
Double-blind experiments
Ethics and experimentation

3. Terminology
The individuals in an experiment are the experimental units. If they are human, we call them subjects.
The explanatory variables in an experiment are often called factors.
A treatment is any specific experimental condition applied to the subjects. If an experiment has several factors, a treatment is a combination of specific levels of each factor.
The factor may be the administration of a drug.
One group of people may be placed on a diet/exercise program for 6 months (treatment), and their blood pressure (response variable) would be compared with that of people who did not diet or exercise.

4. Randomized, comparative experiments
Experiments compare the response to a given treatment versus:
another treatment
the absence of treatment (often called a control)
a placebo (a fake treatment)
Experiments randomize the assignment of subjects to treatments.
Experiments use replication: several or many individuals are studied.
When a potentially confounding variable cannot be randomized, it can be fixed, or controlled, instead. For instance, you can perform all your experiments on bacterial resistance to antibiotics at the same temperature. Temperature is controlled and will not influence results. However, your results may not apply to other temperatures.

5. Inventing experimental design (1 of 2)
Ronald Fisher was sent to a UK agricultural station to evaluate the effects of fertilizers. He found decades worth of bad data.
Fertilizer had been applied to a field one year and not in another to compare the yield of grain produced in the two years.
Fertilizer was applied to one field and not to a nearby field in the same year.

6. Inventing experimental design (2 of 2)
Satellite and soil recordings show that soil types and moisture vary, even within a single field.
Solution: Randomized comparative experiments.
He selected many fields and randomly assigned the fields to receive fertilizer or not. Grain yield was then compared for the two conditions. F

7. Importance of design (1 of 2)
Gastric freezing was once a recommended treatment for peptic ulcers. Patients would swallow a balloon through which a refrigerated liquid was pumped for an hour to cool the stomach. The treatment was shown to be safe and significantly reduce ulcer pain, and it was widely used for years (2,500 gastric freeze machines sold and 15,000 patients were chilled).
The initial study lacked a comparison group. The reported improvement was likely just due to the placebo effect.

8. Importance of design (2 of 2)
A randomized comparative experiment was later performed to compare the effect of gastric freezing with that of a placebo:
28 of the 82 patients subjected to gastric freezing improved
30 of the 78 patients in the control group improved
Because the rates were so similar, gastric freezing was then abandoned.
The initial study lacked a comparison group. The reported improvement was likely just due to the placebo effect.

9. Completely randomized designs
In a completely randomized experimental design individuals are randomly assigned to groups, then the groups are assigned to treatments, completely at random.
The assignment of individuals to treatments relies entirely on randomness.

10. Block designs (1 of 2)
In a block design, subjects are divided into blocks (groups sharing a given characteristic) before the randomization, in order to account for possible differences between the blocks.
The explanatory variables in an experiment are often called factors.
The blocks are NOT random. Randomization occurs inside each block.
A block design lets us choose how many individuals of each block will receive each treatment.

11. Block designs (2 of 2)
Main Outcome Measure: Mean number of symptom- free days per 2 weeks during the peak winter season (November-February), assessed by blinded interviews.

12. Matched pairs designs
Choose pairs of subjects that are closely matched (such as twins). Within each pair, randomly assign who will receive which treatment.
Or give the two (or more) treatments to each subject over time, in random order, so we have repeated measures for each subject.
Each subject is given two chili bowls: version A and version B. Subjects eat and rate both versions. Half of all subjects are given version A first then version B. The other half are given version B first then version A.

13. The placebo effect (1 of 2)
Improvement in health or perceived condition may be due not to any active treatment, but only to the patient’s belief that he or she is being cared for or helped.
therapeutic results on up to 35% of patients
famous placebo: kiss/blow/hug/Band-aid to help kids cope with minor injuries
neural response to the placebo effect seen as early as the spinal cord
“negative placebo effect” also observed; labeling an active drug a placebo lowers its effectiveness

14. The placebo effect (2 of 2)
Science (2009), DOI: /science
Sci Transl Med (2014), DOI: /scitranslmed

15. Double-blind experiments
Bias, conscious or unconscious, from the placebo effect (subjects) or the experimenter is a challenge. “Blinding” can help against bias.
A double-blind experiment is one in which neither the subjects nor the experimenter(s) know which individuals received which treatment until the experiment is completed.
However, subjects must be informed that they will get one of a number of treatments, and must consent to that condition (it would be unethical otherwise).
Double-blind studies typically involve a third party taking charge of the random assignment of subjects to treatment, and of the delivery of the correct treatment to each patient.

16. Examples of blinded studies (1 of 2)
The Clinical Efficacy of Kinesio Tape for Shoulder Pain: A Randomized, Double-Blinded, Clinical Trial
doi: /jospt
Efficacy of acupuncture for migraine prophylaxis: A single-blinded, double-dummy, randomized controlled trial
doi: /j.pain
A fake treatment (placebo) is also sometimes called a sham procedure.
Top: Correct versus sham application of sport tape “kinesio.”
Bottom: To compare the efficacy of acupuncture versus a drug in pill form, a double-dummy was designed: the combined use of sham acupuncture and placebo pill.

17. Examples of blinded studies (2 of 2)
A fake treatment (placebo) is also sometimes called a sham procedure.
Top: Correct versus sham application of sport tape “kinesio.”
Bottom: To compare the efficacy of acupuncture versus a drug in pill form, a double-dummy was designed: the combined use of sham acupuncture and placebo pill.
FIGURE 2. Therapeutic Kinesio Tape application
FIGURE 3. Sham Kinesio Tape application

18. Experimental design examples
Six male runners participated in two running trials separated by one week. In random order, they consumed either chocolate milk or a Gatorade-like sports drink after their run. Protein synthesis was assessed three hours later.
In a study of sickle-cell anemia, 152 patients were given the drug hydroxyurea, and 147 were given a placebo (dummy pill), based on random assignment. The researchers counted the episodes of pain in each subject at the end of the study.
Top: Matched pairs design: Only one random sample of six male runners, each examined twice, once after drinking chocolate milk and once after drinking a sports drink (doi: /MSS.0b013e ).
Bottom: Completely randomized design: Patients were randomly assigned to the two treatments, hydroxyurea and placebo (doi: /NEJM ).

19. Lack of realism (1 of 2)
Is the sample representative of the target population? Random sampling is meant to gain information about a larger population. Is the treatment appropriate for the response you want to study?
Men and women do not necessarily have the same physiology. If a drug will be approved for use in both genders, it should be studied using a sample of both genders and possible differences between the sexes should be investigated.
High saccharin intake was found consistently to lead to bladder cancer in the males of one particular breed of rats. However, the results were never reproduced in other species or even in other breeds of rats. It was later found that this particular breed metabolizes saccharin differently, explaining the peculiar bladder cancer cases.

20. Lack of realism (2 of 2)
FDA requiring lower recommended dose for certain sleep drugs containing zolpidem (Jan. 10, 2013): “Since women eliminate zolpidem from their bodies more slowly than men, the FDA has notified the manufacturers that the recommended dose should be lowered for women.”
Carcinogenicity studies administer high doses of a potential carcinogen to lab rats. Results don’t always apply to humans (e.g., saccharin delisted in 2000).
Men and women do not necessarily have the same physiology. If a drug will be approved for use in both genders, it should be studied using a sample of both genders and possible differences between the sexes should be investigated.
High saccharin intake was found consistently to lead to bladder cancer in the males of one particular breed of rats. However, the results were never reproduced in other species or even in other breeds of rats. It was later found that this particular breed metabolizes saccharin differently, explaining the peculiar bladder cancer cases.

21. Ethics and experimentation
Biology deals with life. Experimentations have an impact on live subjects and ecosystems. What rights do human subjects, animals, and ecosystems have?
There is a difference between what can physically be done and what can be done ethically. When is it okay/not okay to include a placebo group? When should an experiment be interrupted?
Personal standards vary, and extreme experimentations have occurred. Committees have been established to review all research proposals. Subjects must give “informed” consent.