Serology testing (assay) is largely used by forensic laboratories to analyze blood samples from suspects and bloodstains collected at the crime scene, in order to identify blood types of victims and assailants. The main objective of forensic tests, whether serological or other types, is to individualize samples through the identification of their sources.
Blood is the most common physical evidence in accidents, murder cases, and violent crime investigations. Besides blood, crime scene technicians may also find other stains and residues similar to blood in appearance at the scene, such as tomato sauce, red paint, or animal blood. To identify human blood, forensic scientists test samples at the crime scene with the chemical phenolphthalein, in an assay known as the Kastle-Meyer color test. Phenolphthalein releases hydrogen peroxide that reacts with an enzyme known as catalase in the blood. Catalase breaks down the hydrogen peroxide into water and oxygen, therefore releasing bubbles. However, as vegetables, animals, and some bacteria also produce catalase, this test only rules out the inorganic samples. Organic (plant or animal derived) samples are then collected for further serological analysis at the crime laboratory.
Body fluids such as blood, semen, saliva, and sweat, all contain serum. Serum is a liquid component of blood composed of water, trace minerals, several proteins including albumin, and immunoglobulins or antibodies. Albumin is the sticky protein that gives blood enough density for the water within it to remain inside the walls of arteries and veins. (Egg white contains high levels of albumin, which gives it the characteristic consistency.) When red and white blood cells are removed from blood, the resulting clear golden yellowish liquid is serum. Serology is therefore the study of the properties of serum. Serological tests have a wide range of applications in medicine, such as immunology and allergy assays, infection diagnosis, and blood typing. Serology can determine whether an individual was exposed in the past or if he is presently infected with a variety of pathogens (disease-causing organisms), such as hepatitis, measles, anthrax, syphilis, or HIV. Serology tests can also determine the presence of alcohol, illegal drugs, and poisons in the serum. Serological tests are also used in forensics to identify blood ABO groups, whose results, although not conclusive, may help to exclude or include suspects in the investigation process. If for instance, a suspect is blood type B and the samples from the crime scene are all types A and O, the suspect with type B blood can be excluded from the investigation.
Serology is such a convenient diagnostic tool because the immune system produces specific molecular tags in the blood for practically each foreign substance or invading microorganism. Each one specializes in binding to a specific molecule such as a viral, parasite, or bacterial protein, as well as to foreign substances such as poisons and drugs. For minutely small drug molecules against which the immune system is not very sensitive, special immune reagents were developed for the detection of drug abuse. An example is the Homogeneous Enzyme Immunoassays (EMIT), which is commercialized in kits ready for use.
To determine whether a blood sample is from a human or animal source, samples are tested with anti-human serum. This method was discovered by the German biologist Paul Uhlenhunth in the late 1870s. He injected protein from a chicken egg into a sample of rabbit's blood. After a few days, he extracted the rabbit's serum and mixed it with egg white, causing the separation of egg proteins from the solution to form a whitish clotting substance, precipitin. Precipitin is now a generic name for the resulting agglutinated complex formed when antibodies present in the serum of a species agglutinate the proteins in the blood of a different species. The forensic test consists of collecting the blood sample in a test tube containing serum from a rabbit containing antibodies against human blood, known as anti-human antibodies. If an insoluble complex of precipitin (clumping) occurs, the test is positive for human blood. This test can also be conducted using gel-electrophoresis, when a blood sample is put on a glass slide and covered by a layer of agar gel. The slide is positioned side by side with another containing the rabbit anti-human serum, inside a box filled with a solution that conducts electric current. As the current passes through, protein molecules are filtered into the gel and toward each glass slide. If precipitin is formed, the test is positive, and the blood sample is identified as human blood.
Electrophoresis is also used in typing the different groups of human blood, known as the ABO grouping system. After the discovery of antibodies and antigens (molecules to which antibodies bind), scientists identified four blood types among humans between 1875 and 1901. All human blood contains antigens in red cells that vary in type among individuals in accordance with inheritance (e.g., maternal and paternal inherited genes). Genes A and B (chromosome 9) encode enzymes that add specific sugars to an antigen at the ends of a complex sugar molecule (polysaccharide) that is present on the surface of erythrocytes (red blood cells). Individuals who inherit neither A or B genes have type O blood. As genes A and B are codominant (they do not dominate each other), individuals who inherit both genes (one from each parent) are type AB. The following other inherited combinations may occur: AA, BB, AO, BO, OO. Individuals AO or BO are respectively heterozygous type A and type B. AA or BB are homozygous types A or B.
Blood typing tests consist of mixing blood samples with anti-serum A on one side of the slide, and with anti-serum B at the other side. If the agglutination (clumping) occurs on both sides of the glass slide, the blood is AB. If it occurs only with anti-serum A, the blood is type A, or if it occurs only with anti-serum B, the blood is type B. If no agglutination occurs, the blood is type O. Because a person with type O blood does not present antigens to either A or B antibodies, they can donate blood to most blood groups. Carriers of gene A that have antibodies against B antigens in their blood plasma, and vice versa, can only receive transfusions of the same blood type or from type O blood. Individuals with AB blood type can receive transfusions from all donors. Type O carriers however cannot receive blood from the other types because their plasma contains antibodies against A and B antigens.
Population prevalence of blood types is approximately as follows: type A is more common in Caucasians and Europeans; type B among Africans, African descendents, and South Asia populations; AB type is predominant in China, Japan, and Korea; and Type O is predominant in Native Americans, Aborigines, and Latin American populations, and is common in Middle-Eastern populations as well. A small portion of the world population carries a rare variation of AB type subgroups that present weak reactions or no reaction at all to antibodies.
Another breakthrough of significance for both medical and forensic sciences was the discovery by Karl Landsteiner in 1940 that 85% of the human population carries erythrocytes that express the Rh(D) antigen, or Rhesus disease antigen (a protein also present in Rhesus monkeys). Blood is designated as being either Rh positive (+) or Rh negative (-). If an Rh- person receives blood from a donor who is Rh+, his immune system will develop antibodies against the antigen, causing disease or death, depending on the quantity of blood transfused. There are thirty possible combinations between ABO groups and Rh factors. Approximately two thirds of all people have an O+ or A+ blood type, with all other types comprising the remaining third. These variations allow the number of suspects in a crime investigation to be narrowed.
Another singular characteristic of proteins and enzymes is the presence of discrete variations in a single base pair of the genes that encode them, known as polymorphisms (or multiple forms of the same gene). More than 1% of any given population has polymorphisms in specific genes. Specific polymorphisms are also more prevalent in certain populations. For instance, the CYP enzymes of the gene Cytochrome P 450 show a specific polymorphed version in 40% of the Asian population, whereas another polymorph is more prevalent among Caucasians and Europeans. Several other enzymes also present a known prevalence among races, and are therefore, useful in forensic testing.
Genetic screening for polymorphisms in forensic samples is very helpful when combined with blood type and Rh factors, because it sharply reduces the probability the existence of two persons with the same blood characteristics being involved with the same crime to very insignificant odds. In addition, other serological tests can also be used to estimate age, sex, and race of suspects, such as hormonal levels in blood and other fluids, as well as genetic analysis such as chromosomal typing (or karyotyping), and DNA profiling.
SEE ALSO Animal evidence; Antibody; Antigen; Blood; Chromosome; Circumstantial evidence; Crime scene investigation; Cross contamination; DNA; Epidemiology; Fluids; Hemoglobin; Homogeneous enzyme immunoassay (EMIT); Illicit drugs; Immune system; Luminol; Parasitology; Paternity evidence; Saliva; Serum; Toxicological analysis.
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