Benzidine in crime scene investigation
Benzidine was historically a critical chemical used in crime scene investigations for presumptive blood testing. It reacts with heme iron in hemoglobin, turning from clear to deep blue in the presence of blood, thereby aiding in preliminary crime scene analysis. However, benzidine is not specific to human blood and can yield false positives from sources such as plant materials or cleaning agents, necessitating further confirmatory tests to identify the blood source accurately. Its significant carcinogenic properties led to a ban by the Environmental Protection Agency in 1974. Subsequently, 3,3′,5,5′ tetramethylbenzidine (TMB) was introduced as a safer alternative, although it is still considered a probable carcinogen. The TMB test functions similarly to the benzidine test, enabling investigators to identify blood stains without destroying the sample for further analysis. TMB can also be utilized in a spray form to reveal faint blood traces, but caution is required to preserve samples for DNA testing. Overall, while the use of benzidine has ceased, its legacy remains in modern forensic methodologies for blood detection.
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Benzidine in crime scene investigation
Definition: Chemical formerly used in the standard presumptive test for blood at crime scenes.
Significance: A positive reaction to benzidine or tetramethylbenzidine of a stain found at a crime scene suggests that the stain is probably blood; such information can facilitate an initial reconstruction of a crime and prompt follow-up.
For most of the twentieth century, benzidine was the standard chemical used in presumptive testing for blood at crime scenes. In the presence of heme iron and hydrogen peroxide, benzidine, which is clear in the reduced state, is converted to the oxidized state, which is deep blue. Because heme iron is present in hemoglobin, the protein that carries oxygen in the blood, a positive test can indicate the presence of blood. This test does not distinguish between human blood and animal blood, however; further testing is necessary to make that distinction and, if the blood is human, to determine whose blood it is. In addition, constituents of some plants, such as potatoes and horseradish, as well as oxidizing agents found in some cleansers, can catalyze the reaction. Accordingly, a benzidine test is only presumptive of blood; a positive result must be confirmed by laboratory test.
Developed in 1904, the benzidine test became the most popular presumptive test for blood because of its high sensitivity, specificity, and reliability. Benzidine, however, which was also used for the synthesis of dyes in the textile industry, proved to be highly carcinogenic, and its use and manufacture in the United States was banned by the Environmental Protection Agency in 1974. At that time, 3,3′,5,5′ tetramethylbenzidine (TMB) was developed as a presumptive test for blood. It is not as sensitive as benzidine, but it is much safer to use, although it is a probable carcinogen.
Typically, a forensic investigator performs the TMB test by moistening a cotton swab with deionized water and rubbing the swab on the suspect stain, adding a drop of TMB solution to the swab, waiting thirty seconds, and then adding a drop of 3 percent hydrogen peroxide to the swab. A positive reaction will turn the swab a blue-green color within fifteen seconds. Often a swab taken from near the stain is used as a control. If the swab turns blue-green before the hydrogen peroxide is added, the test is invalid. Validation of the reagents using a known blood standard is usually conducted.
The TMB reagent in a colloidal mixture can also be used to spray an area in order to raise faint bloodstains, such as might be left by handprints or shoe prints. Like luminol, this substance can allow investigators to see evidence of attempts to clean up blood from crime scenes. The standard TMB test does not destroy the sample, which can be subsequently tested for blood type and DNA, but the spray reagent, like luminol, fixes a stain so that it cannot be tested further; investigators must thus take care to limit the use of the reagent.
Bibliography
Lee, Henry C., Timothy Palmbach, and Marilyn T. Miller. Henry Lee’s Crime Scene Handbook. San Diego, Calif.: Academic Press, 2001.
Nickell, Joe, and John F. Fischer. Crime Science: Methods of Forensic Detection. Lexington: University Press of Kentucky, 1999.