Forensic Serology: Blood Analysis

• Forensic serology involves laboratory tests using specific antigen and serum antibody reactions.

Forensic Serology

• Karl Landsteiner discovered blood typing in 1901, leading to the A-B-O system and revolutionizing blood transfusions.
• The Rh factor was discovered in 1937, adding another layer to blood differentiation.
• Before DNA technology, blood factors were the best way to link blood to an individual in forensics.
• DNA technology has changed the field, allowing the individualization of bloodstains and other biological evidence.

The Nature of Blood

• Blood is a complex mixture with cells, enzymes, proteins and inorganic substances.
• Plasma is the fluid component, mainly water comprising 55% of blood volume.
• Erythrocytes (red blood cells), leukocytes (white blood cells), and platelets are the solid materials in plasma.
• Serum is the pale yellowish liquid left after blood clots.

Antigens and Antibodies

• Red blood cells transport oxygen and remove carbon dioxide, and have characteristic chemical structures called antigens on their surface that determine blood type.
• More than 15 blood antigen systems have been identified, with A-B-O and Rh being the most important.
• Type A individuals have A antigens, type B have B antigens, type AB have both, and type O have neither.
• The Rh factor (D antigen) determines Rh positive or negative status.
• Serum contains antibodies that react with specific antigens.
• Anti-A antibodies react only with A antigens, anti-B with B antigens, and anti-D with D antigens.
• Antiserum is serum containing specific antibodies.
• Antibodies have two reactive sites (bivalent), allowing them to attach to antigens on two different red blood cells, causing agglutination (clumping).

Blood Typing

• Serology includes typing whole blood for its A-B-O identity.
• Only anti-A and anti-B antiserums are needed to determine the A-B-O blood type.
• Type A blood is agglutinated by anti-A serum, type B by anti-B serum, type AB by both , and type O by neither.
• The presence of natural antibodies in blood can identify blood type: type A has anti-B, type B has anti-A, type O has both, and type AB has neither.
• Distribution of blood types varies, with a typical distribution in the US being 43% O, 42% A, 12% B, and 3% AB.

Immunoassay Techniques

• The antigen-antibody reaction has been applied to drug detection in blood and urine.
• Animals are injected with a drug-protein complex, acting as an antigen to produce specific antibodies.
• Testing laboratories use commercially prepared sera from these animals to detect various drugs.
• Immunoassay results for drugs are presumptive and require confirmation by additional testing, such as gas chromatography-mass spectrometry.

Forensic Characterization of Bloodstains

• Criminalists must determine if a stain is blood, the species of origin, and if human, how closely it can be associated with an individual.
• Preliminary color tests are used to determine the presence of blood.
• Phenolphthalein test (Kastle-Meyer test) is a commonly used color test that indicates peroxidase-like activity in blood hemoglobin.
• Hemastix strips can be moistened with distilled water and placed in contact with a blood stain.
• A positive reaction is the appearance of a green color on the strip.
• Luminol reacts with blood to produce light, allowing large areas to be screened for bloodstains in darkened conditions.
• Bluestar is a newer product with similar uses to Luminol, though it does not require complete darkness.
• Luminol and Bluestar are sensitive and do not interfere with subsequent DNA testing.
• Microcrystalline tests are more specific and involve adding chemicals to blood to form characteristic crystals with hemoglobin derivatives.
• The Takayama and Teichmann tests are two popular microcrystalline tests.
• The precipitin test determines if a bloodstain is of human or animal origin.
• Animals are injected with human blood, causing them to form antibodies, which are then collected as human antiserum.
• Human antiserum reacts specifically with human antigens.
• The classic method involves layering a bloodstain extract on top of human antiserum in a capillary tube.
• Gel diffusion involves antibodies and antigens diffusing towards one another on a gel, creating a precipitation line where they meet.

Principles of Heredity

• Hereditary material is transmitted via genes, which are positioned on chromosomes.
• All nucleated human cells contain 46 chromosomes in 23 pairs.
• Reproductive cells (egg and sperm) contain 23 unmated chromosomes.
• During fertilization, sperm and egg combine to form a zygote with 23 mated chromosome pairs.
• The egg cell contains an X chromosome, while the sperm cell contains either an X or a Y chromosome.
• An X-carrying sperm results in an XX (female), while a Y-carrying sperm results in an XY (male).
• A gene's location on a chromosome is its locus.
• Alleles are alternative forms of genes that influence a specific trait and are aligned with each other on a chromosome pair.
• Homozygous gene pairs are made up of two similar genes.
• Heterozygous gene pairs are made up of two different genes.
• Dominant genes mask recessive genes, while codominant genes both express their characteristics in an individual.
• A pair of allele genes together constitutes the genotype of the individual.
• The phenotype refers to the outward characteristic such as blood type.
• Genotyping is important in disputed-paternity cases in civil courts.

Forensic Characterization of Semen

• Many forensic lab cases involve sexual offenses, requiring the examination of exhibits for seminal stains.
• A normal male ejaculation contains 2.5 to 6 milliliters of seminal fluid with 100 million or more spermatozoa per milliliter.
• The examination involves first locating the stain and then testing it.
• Seminal stains often have a stiff, crusty appearance, but the acid phosphatase test is more reliable for detection of a stain.
• Acid phosphatase, an enzyme from the prostate gland, is present in high concentrations in semen.
• The acid phosphatase test involves applying a filter paper moistened with water in the suspect area, then applying sodium alpha naphthylphosphate to filter paper.
• A purple color indicates acid phosphatase and also reacts positively with 4-methylumbelliferyl phosphate (MUP).
• Semen can be identified by the presence of spermatozoa through microscopic examination.
• Spermatozoa are slender, elongated structures 50-70 microns long with a head and tail.
• An immunology fluorescence staining kit uses a monoclonal antibody to detect sperm heads.
• A low sperm count is called oligospermia, and the absence of sperm is called aspermia.
• Prostate-specific antigen (PSA or p30), a protein, can be used to identify semen, though its levels may be detectable in other human tissues.
• A positive PSA (p30) combined with an acid phosphatase test indicates semen.

Collection and Preservation of Rape Evidence

• Seminal constituents are important evidence that show that sexual intercourse took place, however their absence does not refute rape and physical injuries aid in examination.
• Garments should be carefully removed and packaged separately in paper bags.
• The victim must disrobe while the clothing falls on a clean paper sheet to collect loose foreign materials.
• Clothing needs to each be contained in a separate paper bag to help prevent cross contamination.
• Suspected seminal stains must be handled carefully; do not fold through the stain or rub it against the surface.
• Wear disposable latex gloves when touching such evidence to minimize direct personal contact.
• The rape victim must undergo a medical examination as soon as possible with trained personnel.
• An evidence-collection kit from the local crime laboratory should be used.
• Collect pubic combings, pubic hair standard/reference samples (cut 25 hairs), external genital dry-skin areas (swab), vaginal swabs and smear (using two swabs), cervix Swabs, rectal swabs and smear.
• Additional steps include: oral swabs and smear, swabs of body areas (such as breasts), head hairs, blood sample, buccal Swab, Fingernail scrapings, all clothing, urine specimen.