Presumptive tests for blood
Presumptive tests for blood are laboratory procedures utilized to detect stains that may indicate the presence of blood. These tests are essential in initial forensic investigations, particularly in violent crime scenes, as they help analysts identify and locate blood, which can provide crucial insights into the circumstances surrounding an event. When blood is shed, its appearance changes rapidly; it starts as a red liquid, then clots, and eventually darkens as it dries. Presumptive tests rely on the detection of hemoglobin, a protein unique to blood, using chemical reactions that result in a color change of specific dyes when oxidized.
However, the sensitivity of these tests—capable of detecting very diluted blood—also leads to potential false positives, where non-blood substances can trigger similar reactions. Forensic scientists must therefore confirm any presumptive results with additional tests that specifically identify blood proteins. This two-step process ensures a more reliable analysis, emphasizing the importance of accurate interpretation in forensic case management. Ultimately, presumptive tests are a critical initial step in the complex process of blood detection and forensic analysis.
Presumptive tests for blood
DEFINITION: Laboratory tests used to examine items for stains that may be blood.
SIGNIFICANCE: Presumptive tests are widely used in the initial screening of scenes and items for the presence of blood; thus, the correct use of such tests and correct interpretation of both positive and negative results is vital in analysis and case management.
Because blood is frequently shed during violent crimes, the search for and identification of blood is an important part of the work of forensic scientists both in the laboratory and at the crime scene. The presence of blood and its locations can provide information about the event and circumstances being investigated. Analysts may, if the case circumstances warrant it, subject any bloodstains identified to (deoxyribonucleic acid) to determine from whom the blood could or could not have come; this information can also shed light on the event and circumstances.
The Appearance of Bloodstains
Although the detection of bloodstains may seem like a simple process, in reality it can be quite complex. Immediately after it is shed, blood is a red liquid. It starts to clot within a few minutes, and in relatively large quantities of liquid blood, a separation of blood into clotted red material and a light-colored liquid can be noted. In smaller amounts, blood dries before clotting is complete, and a shiny, slightly translucent red stain is observed. Bloodstains darken and change color to brown with time. Any treatment of the stain with water or smearing of the stain will dilute the blood, or decrease the amount present, which results in a color change. Any treatment of the blood with a cleaning agent or other chemical will alter the stain’s color to a greenish shade.
The material on which a bloodstain is present also affects the stain’s appearance. The texture and absorbency of some materials can obscure the physical appearance of blood, and the color of the background material affects the analyst’s perception of the color of a stain.
The visual identification of a stain as possibly being blood is a useful start in a forensic examination, but additional work needs to be done before the stain can be identified as blood. To confirm the presence of blood, a forensic scientist will usually carry out a presumptive test.
The Chemical Basis of Presumptive Tests
Blood is a unique and complex biological fluid containing many different cell types and a wide range of circulating proteins, glycoprotein, and ions. One of these cell types is the red blood cell, or erythrocyte. The major protein component of the red blood cell is hemoglobin, which contains an iron ion and is responsible for binding oxygen and transporting it around the body. Hemoglobin is found only in blood, so being able to identify this protein in a stain allows the forensic scientist to identify the stain positively as blood.
When hemoglobin binds oxygen, it effectively acts as a type of oxidizing enzyme called a peroxidase, and this enzymatic activity is used in presumptive tests. Certain dyes are colorless but can be oxidized to yield a colored product. When such a dye is added to a possible bloodstain in the presence of an oxidant, the change in color shows that an oxidizing agent, such as hemoglobin, is present. Phenolphthalein is the dye component of the Kastle-Meyer presumptive test for blood; the dye changes from colorless to dark pink. Another example is tetramethylbenzidine, which is the dye component of a commercial test for blood in urine, the Combur-Test, which can also be used to detect bloodstains. The dye in this test changes from colorless to dark green. The test is also a presumptive test, although one with a far more complex reaction pathway than that of more basic presumptive tests; the action of hemoglobin on luminol produces light.
Practical Limitations of Presumptive Tests
Sometimes, substances other than blood can cause the dye to change color in a presumptive test. These substances fall into two categories. First, any chemical that can react directly with the dye to oxidize it—that is, one that does not require the oxidizing action of hemoglobin on the oxidant—can give a positive result. Examples of these are metal salts, such as some copper salts or rust, hypochlorites, and bleaches. Forensic scientists refer to results like these, where positive results are given by substances other than blood, as false positives.
This sort of false positive can be excluded—that is, not thought of as coming from a possible bloodstain—by the application of the dye to the stain in the absence of the oxidizing agent. A positive result under those conditions means the stain is not blood, as the oxidizing agent is needed for hemoglobin to give a positive result in presumptive tests.
The other kinds of substances that give false positives are enzymes such as catalases or peroxidases, which can break down the oxidizing agent in the same way as hemoglobin. The most common of these are plant peroxidases, which are widespread in the plant kingdom and are found in cabbage, horseradish, and other fairly common plants.
Presumptive tests are very sensitive, giving positive results to blood dilutions of between 1 in 105 and 1 in 106 under ideal conditions. Given that forensic samples are usually not tested under ideal conditions, because most are on substrates that are dirty or contaminated to some extent, the actual sensitivity in the field is probably lower. The sensitivity means that the tests can be used to screen items for small amounts of blood or diluted blood, as well as to test stains that look like blood to see if they might be blood.
As other substances can give positive results in such tests, it is not possible for a forensic scientist to say that a stain that gives a positive result in a is definitely blood. Such a stain can be described as a probable bloodstain or similar. Confirmatory tests need to be conducted before an analyst can say definitively that any given stain is blood. These are typically immunological tests that are capable of detecting proteins specific to blood; most scientists agree that crystal and spectroscopic tests for hemoglobin can be regarded as confirmatory as well.
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