The search for alternatives is not simply a literature search that strings a number of keywords together to demonstrate that use of a living animal in a particular study is required and that the study is unique. It is also not a search to establish the scientific validity of the study. While searches for scientific validity and non-duplication are required, these searches are inadequate for determining the possibilities for alternatives. The USDA web site on alternatives provides some examples of a successful search.
Once scientific validity has been established, the search for alternatives must focus on the techniques and procedures of the study. It is these that are the sources of potentially painful and stressful events that must be minimized or eliminated. The search for alternatives is conducted in an effort to find methods that will accomplish the goals of the research with less or no impact on animals. Medline includes the descriptor "Alternatives to Animal Testing" which covers the topic of alternatives (not specific alternatives) and is useful to find papers that may assist the investigator in understanding the approaches and concepts involved in alternative searching.
The search must concentrate on those procedures which have the potential to cause pain or distress. Since animals cannot verbally describe the sensations engendered by a procedure we must rely on a rule of thumb. Any procedure which would induce pain or distress in a human must be considered potentially painful or distressful in animals. In practice, there are many signs of pain that can be observed in animals by one who is familiar with their behavior. However, many animals will not show signs of pain until it is extreme - an adaptive behavior to prevent attack by predators when an animal is sick or injured. Some investigators believe that use of analgesics or anesthetics for a potentially painful procedure renders it "nonpainful" and thus relieves them of the responsibility for searching for alternatives. The USDA standards would define the procedure as "painful" (since analgesia or anesthesia was required) and therefore require that alternatives be considered. Under this standard, euthanasia of an animal to harvest tissue or fluids is considered a painful procedure and an alternative search must be conducted.