Blood is a fluid connective tissue of our body, also known as the fluid of life. As we know, it transports and circulates oxygen to different parts of our body and improves our immunity. Due to its abundant presence in the human body, when a violent crime such as murder has occurred, blood is the most found body fluid at the scene of a crime.
Analysis of bloodstains can help in the detection of species, identification of the victim, and even relates it to the suspect through blood detection assays and DNA sequencing. Hence blood as evidence is a very important field of forensic science, especially forensic serology.
Blood consists of red blood cells(RBCs) also known as erythrocytes which have a life span of about 3-4 months in human beings and mature erythrocytes do not have nuclei, therefore, lack nuclear DNA. White blood cells or leukocytes on the other hand are the main sources of nuclear DNA. It also provides the human body with the immunity it requires. It also consists of platelets also known as thrombocytes which help in blood clotting.
RBC’s consist of iron components in hemoglobin. Hence a molecule of hemoglobin is also known as ferroprotoporphyrin. The ferrous ion of heme forms four bonds with the nitrogens of protoporphyrin IX, along with a fifth bond with a hemoglobin chain and the sixth bond with a molecule of Oxygen.
Other chemicals such as carbon monoxide and cyanide also bind to the ferrous iron of the heme molecule and can cause chemical asphyxia. Heme groups are also present in the blood of various animals and other proteins such as myoglobin in muscles and neuroglobin in the brain.
Presumptive Tests For The Identification of Bloodstains
Traces of blood from a scene of the crime could be detected using presumptive assays. They are based on the principle of oxidation-reduction reaction catalyzed by the heme moiety of the hemoglobin molecules.
A colorless substrate when catalyzed by heme undergoes an oxidation reaction causing any of the three phenomena, chemiluminescence, change of color, or fluorescence. These are sensitive experiments and could be detected in blood samples with 0.00001-0.000001 folds of dilution.
Colorimetric Tests For Blood
The oxidation reaction causes a change of color on the substrate if it is a positive result.
Phenolphthalein Test: Phenolphthalein, a member of a class of indicators and dyes, is used in titrations of mineral and organic acids as well as most alkalis. The phenolphthalein assay for blood identification is also known as the Kastle–Meyer test. Kastle published a study in 1901, presenting the results of a reaction in which phenolphthalein, a colorless compound, is catalyzed by heme with hydrogen peroxide as the oxidant. The oxidized derivative is phenolphthalein, which appears pink under alkaline conditions.
Leucomalachite Green (LMG) Test: It is a triphenylmethane dye that is colorless and can be oxidized by the catalysis of heme to produce a green color. The reaction is carried out under an acidic environment and with hydrogen peroxide as the oxidant.
Benzidine: With oxidation resulting in blue color, benzidine reagent is added to the suspected stain to identify if it is blood or not. However, it is not a preferred usage because benzidine is a potential carcinogen.
Chemiluminescence And Fluorescence Tests
Other organic compounds whose oxidation products have chemiluminescent or fluorescent properties are utilized for testing. In the chemiluminescence assay, light is emitted as a product of a chemical reaction. In this category, luminol produces chemiluminescence when blood is present.
In contrast, a fluorescence assay requires the exposure of an oxidized product, such as fluorescein, to a particular wavelength of an excitation light source. The fluorescence is then emitted at longer wavelengths than that of the excitation light source.
One advantage of chemiluminescent and fluorescent reagents is that they can be sprayed over large areas where latent bloodstains are potentially located. A positive reaction identifies blood and also reveals the patterns of bloody impressions such as footprints and fingerprints. These methods are very sensitive and can pinpoint the locations of even small traces of blood. Additionally, they are useful for detecting blood at crime scenes that have been cleaned and shows no visible staining.
One disadvantage of chemiluminescent and fluorescent reagents is that precautions must be taken if stains are very small or have been washed. Luminol is the reagent used in the case of chemiluminescence assays and Fluorescein for fluorescent assay.
Confirmatory Tests For Bloodstains Identification
Hemochromogen Crystal Test: Also known as Takayama crystal assay. In this test, the suspected stain is treated with pyridine and glucose under alkaline conditions to form crystals of pyridine ferroprotoporphyrin if it is a positive result. This is due to the bonding of the reducing sugar with the ferroprotoporphyrin i.e., iron in hemoglobin. The crystals appear needle-shaped in pink color under a microscope.
Hematin Crystal Test: In this test, the Suspected stain is treated with glacial acetic acid salts and is heated simultaneously to form hematin chloride which appears as a prismatic or rhomboidal brown colored crystal under a microscope for a positive result.
Once the presence of blood is confirmed, the non-destructive part of the stain if required is taken for further analysis in DNA amplification and much more.