PRESUMPTIVE TESTS FOR BLOOD
Presumptive tests for blood react with the hemoglobin present in blood. If hemoglobin is present, one of two general results occurs, depending on the test. Either a color less reactive substance changes to a colored form (from clear to pink, for example) or light of a specific wavelength is emitted (fluorescence or chemiluminescence) in the presence of hemoglobin. In the first type of test, the testing chemical is added to the suspected stain and then an oxidant is added, usually 3% hydrogen peroxide. The hydrogen per oxide reacts (oxidizes) with the hemoglobin and changes the color of the testing chemical; hemoglobin acts as the catalyst, speeding up the reaction. The most commonly used catalytic color tests are phenolphthalein, benzidine, leucomalachite green, and tetramethylbenzidine (TMB). For sensitivity and safety reasons among these tests, the phenolphthalein test is used more often than the other tests. The sensitivity of the phenolphthalein test can detect blood diluted down to 10−7 (1 part in 10 million) and even decades-old bloodstains can yield positive results. Phenolphthalein is cross-reactive with other substances, such as some vegetables. The test is performed by moistening a clean cotton swab with distilled water and rubbing it on the suspected stain. A drop of phenolphthalein solution is added to the swab’s tip; it should remain colorless. A drop of hydrogen peroxide is then added; if the tip turns pink, the test is presumptively positive for blood. If the swab tip remains colorless, then the result is negative for blood. The hydrogen peroxide would continue to react with the hemoglobin and degrade it if it were not for an enzyme called catalase found primarily in red blood cells that destroys hydrogen peroxide and frees water and oxygen; this makes the local pH more basic and turns the phenolphthalein pink. The color change must be within several seconds because the tip may turn pink through normal oxidation after several minutes of exposure to air. At times, it is not only the presence of blood that is of interest, but also the pattern or distribution of the blood. The area to be tested may be large or intricate, such as floors, walls, and automobile interiors. In these instances, the testing chemical is sprayed onto the surface(s) and then observed for any emitted light (glowing). Because the light output is faint, the treated surfaces must be viewed in the dark or with an alternate light source (ALS). Specialized photographic techniques must be used to capture the images because the effect of emitted light is temporary. These types of tests may affect subsequent tests; therefore, caution must be employed in their use (see Table 10.2). A noted expert in serology, Robert Spaulding (2002), has suggested that if the stain can be seen and collected, then this type of test should not be used. Two chemicals, luminol and fluorescein, are predominantly used for large scale serology testing. Luminol (3-aminophthalhydrazide) reacts in the presence of hemoglobin, much like phenolphthalein, when an oxidizer is applied. The reaction, however, results in a blue-white to yellow-green luminescence (light emitted as a by-product of a chemical reaction) if blood is present. Luminol is very sensitive to hemoglobin and will detect blood in dilutions of 1 in 5,000,000. Luminol, a suitable oxidant, and water are mixed and sprayed over the area of interest.
Table 10.2 The Effects of Various Presumptive Serology Tests on Subsequent Tests
The pattern will be visible for up to 30 s before additional treatment is required; overspraying, however, will result in “bleeding” of the patterns and a loss of detail. Fluorescein is another chemical that is used to check for the presence of blood and is prepared much in the same way as luminol except that the commercial preparation contains a thickener. This makes fluorescein stay on the surface better than luminol, making it easier to use on walls and other vertical surfaces. Unlike luminol, fluorescein produces fluorescence (light emitted as energy loss at a longer wave length than it is illuminated with) and must be illuminated at 450 nm via an ALS to be seen. Both luminol and fluorescein are characterized as irritants but are not known to be carcinogens. Nevertheless, safety precautions and protective equipment should be employed during their use.
