Orthotolidine in crime scene investigation
Orthotolidine is a chemical used in forensic science for presumptive testing of blood at crime scenes. When applied to a stain, a positive reaction indicates the probable presence of blood, which can aid in early crime scene reconstruction. The test relies on the interaction of orthotolidine with heme iron and hydrogen peroxide; upon this reaction, the solution turns blue, signaling a potential blood presence due to hemoglobin. However, it is important to note that this test cannot differentiate between human and animal blood, necessitating further laboratory analysis for specificity.
The procedure typically involves moistening a cotton swab with deionized water, applying orthotolidine, and then adding hydrogen peroxide to observe the color change. Established in 1912, orthotolidine was widely adopted for its sensitivity, although it poses some health risks compared to alternatives. To mitigate exposure, Hemastix—pre-impregnated sticks with orthotolidine—have been developed for easier and safer use. While Hemastix provides convenience and minimizes direct contact with the chemical, there is evidence that it may interfere with some DNA testing methods. Thus, orthotolidine remains a crucial tool in crime scene investigations, though its results should always be confirmed by more definitive testing.
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Orthotolidine in crime scene investigation
DEFINITION: Chemical used in presumptive tests for blood at crime scenes.
SIGNIFICANCE: A positive reaction to orthotolidine 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.
In the presence of heme iron and hydrogen peroxide, orthotolidine, clear in the reduced state, is converted to an oxidized state, which is blue. Because heme iron is present in hemoglobin, the protein that carries oxygen in red blood cells, a positive test can indicate the presence of blood. Such a 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, an orthotolidine test is only presumptive for blood; a positive result must be confirmed by laboratory tests.
Typically, a forensic investigator performs the test by moistening a cotton swab with deionized water and rubbing the swab on the stain, adding a drop of orthotolidine 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 an intense blue color within fifteen seconds. Often, a swab taken near the stain of interest is used as a control. If the swab turns pinkish before the hydrogen peroxide is added, the test is invalid. To ensure that the reagents have not deteriorated before use, the investigator validates the test using a known blood standard.
First introduced in 1912, orthotolidine, also known as o-tolidine, became almost as widely used as benzidine for testing suspected blood. It has a high sensitivity and is quite specific. It is less carcinogenic than benzidine but more so than phenolphthalein (also used for testing suspected blood), and it poses some risk to users. This risk has been minimized with the introduction of a product marketed as Hemastix: orthotolidine-impregnated sticks designed for use by medical professionals who need to identify the presence of blood in urine. Hemastix can also be used at crime scenes. A drop of deionized water is placed on the stain to be tested, and that drop is transferred to the stick. A resulting blue color suggests the presence of blood. Aside from the convenience this product offers, it allows the investigator to avoid direct contact with orthotolidine. However, whereas the standard orthotolidine test does not interfere with subsequent blood typing and DNA typing, the results of one study suggest that samples collected using Hemastix may interfere with some automated DNA testing procedures.
Bibliography
Geberth, Vernon J. Practical Homicide Investigation: Tactics, Procedures, and Forensic Techniques. 4th ed. Boca Raton, Fla.: CRC Press, 2006.
Gomes, Claudia, et al. "Presumptive Tests: A Substitute for Benzidine in Blood Samples Recognition." Forensic Science International: Genetics Supplement Series, vol. 6, Dec. 2017, doi.org/10.1016/j.fsigss.2017.09.213. Accessed 16 Aug. 2024.
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.