Forensic Serology

Chapter 8

Nature of Blood• The word blood refers to a highly complex

mixture of cells, enzymes, proteins, and inorganic substances.

• Plasma, which is the fluid portion of blood, is composed principally of water.

• Red blood cells (erythrocytes), white blood cells (leukocytes), and platelets are the solid materials suspended in plasma.

• Antigens, usually proteins, are located on the surface of red blood cells and are responsible for blood-type characteristics.

Blood Typing• More than 15 blood antigen systems have been

identified, but the A-B-O and Rh systems are the most important.

• An individual that is type A has A antigens on his/her red blood cells, type B has B antigens, AB has both A and B antigens, and type O has neither A nor B antigens.

• Rh factor is determined by the presence of another antigen, the D antigen.

• People having the D antigen are Rh positive; those not having the antigen are Rh negative.

• For every antigen there is a specific antibody that will react with it to form clumps known as agglutination.

• Thus, if serum containing anti-B is added to red blood cells carrying B antigen, they will immediately react.

Ratios of Blood Types

• O+ 1 in 3 persons• O- 1 in 15 persons• A+ 1 in 3 persons• A- 1 in 16 persons• B+ 1 in 12 persons• B- 1 in 67 persons• AB+ 1 in 29 persons• AB- 1 in 167 persons

Serology• The term serology is used to describe a

broad scope of laboratory tests that use specific antigen and serum antibody reactions.

• The identity of each of the four A-B-O blood groups can be established by testing the blood with anti-A and anti-B sera.

• The concept of specific antigen-antibody reactions has been applied to immunoassay techniques for the detection of drugs of abuse in blood and urine.

Immunoassay• A number of immunological assay techniques are

commercially available for detecting drugs through antigen-antibody reaction.

• One such technique, the enzyme-multiplied immunoassay technique (EMIT), is used by toxicologists because of its speed and high sensitivity for detecting drugs in urine.

• In a typical EMIT analysis, antibodies that will bind to a specific drug are added to the subject’s urine.

• Other immunoassay procedures are also available, such as radioimmunoassay (RIA) which uses drugs labeled with radioactive tags.

In the EMIT assay, a drug that may be present in a urine specimen will compete with added labeled drug for a limited number of antibody binding sites. The labeled drugs are indicated by an asterisk. Once the competition for antibody sites is completed, the number of remaining unbound labeled drug is proportional to the drug’s concentration in urine.

Antigen-Antibody Reaction• When an animal, such as a rabbit or mouse, is

injected with an antigen its body will produce a series of different antibodies, all of which are designed to attack some particular site on the antigen of interest.

• This collection of antibodies is known as polyclonal antibodies.

• Alternately, a more uniform and specific collection of antibodies designed to combine with a single antigen site can be manufactured.

• Such antibodies are known as monoclonals.

 Stimulating production of drug antibodies.

Steps required to produce monoclonal antibodies.

Forensics of Blood• The criminalist must be prepared to answer

the following questions when examining dried blood: – (1) Is it blood? – (2) From what species did the blood originate? – (3) If the blood is of human origin, how closely

can it be associated to a particular individual?

• The determination of blood is best made by means of a preliminary color test.

• Wet blood has more value than dried blood because more tests can be run. – For example, alcohol and drug content can be determined from wet

blood only. Blood begins to dry after 3-5 minutes of exposure to air. As it dries, it changes color towards brown and black.

• Blood at the crime scene can be in the form of pools, drops, smears, or crusts.

• Pools of blood obviously have more evidentiary value in obtaining a wet sample.

• Drops of blood tell the height and angle from which the blood fell. The forensic science of blood spatter analysis says that blood which fell perpendicular to the floor from a distance of 0-2 feet would make a circular drop with slightly frayed edges.

• Drops from a higher distance would have more pronounced tendrils fraying off the edges (a sunburst pattern).

• A blood smear on the wall or floor tells the direction of force of the blow. 

• The direction of force is always in the direction towards the tail, or smaller end, of the smear, or splatter. In other words, the largest area of the smear is the point of origin (a wave cast-off pattern).

• Blood crusts need to be tested with crystalline methods to make sure it's blood.

• Refrigerated red blood cells have a shelf life of about 42 days, and the serum containing white blood cells can be refrigerated much longer, almost up to a year.  DNA can be extracted from blood (if white blood cells which always contain a nucleus are present), and also from sperm, bone marrow, tooth pulp, and hair roots. Blood, however, is commonly used in DNA testing, as per the following steps:

• 1. Blood samples are collected from the victim, defendant, and crime scene2. White blood cells are separated from red blood cells3. DNA is extracted from the nuclei of white blood cells4. A restrictive enzyme is used to cut fragments of the DNA strand5. DNA fragments are put into a bed of gel with electrodes at either end6. Electric current sorts DNA fragments by length7. An absorbent blotter soaks up the imprint; it is radioactively treated, and an X-ray photograph (called an autoradiograph) is produced 

The Tests• A positive result from the Kastle-Meyer color test is

highly indicative of blood. – Hemoglobin causes a deep pink color.

• Alternatively, the luminol test is used to search out trace amounts of blood located at crime scenes. – Produces light (luminescence) in a darkened area.

• Microcrystalline tests, such as the Takayama and Teichmann tests, depend on the addition of specific chemicals to the blood so that characteristic crystals will be formed.

• Precipitin test• Gel Diffusion

The precipitin test

Gel Diffusion

A-B-O vs DNA• Prior to the advent of DNA typing, bloodstains

were linked to a source by A-B-O typing and the characterization of polymorphic blood enzymes and proteins.

• This approach has now been supplanted by the newer DNA technology.

• DNA analysis has allowed forensic scientists to associate blood and semen stains to a single individual.

Stain Patterns of Blood• The crime scene investigator must remember

that the location, distribution, and appearance of bloodstains and spatters may be useful for interpreting and reconstructing the events that produced the bleeding.

• Surface texture and the stain’s shape, size, and location must be considered when determining the direction, dropping distance, and angle of impact of a bloodstain.

Stain Patterns of Blood• Surface texture is of paramount importance. In general,

the harder and less porous the surface, the less spatter results.

• The direction of travel of blood striking an object may be discerned because the pointed end of a bloodstain always faces its direction of travel.

• The impact angle of blood on a flat surface can be determined by measuring the degree of circular distortion. At right angles the blood drop is circular, as the angle decreases, the stain becomes elongated.

• The origin of a blood spatter in a two-dimensional configuration can be established by drawing straight lines through the long axis of several individual bloodstains. The intersection or point of convergence of the lines represents the origin point.

Illustration of stain convergence on a two-dimensional plane. Convergence represents the point from which the stains emanated. Courtesy Judith Bunker, J. L. Bunker & Assoc., Ocoee, FL

Categories of Blood Stains

Passive Transfer

Projected

Transfer Bloodstains

• A transfer bloodstain is created when a wet, bloody surface comes in contact with a secondary surface.

• A recognizable image of all or a portion of the original surface may be observed in the pattern, as in the case of a bloody hand or footwear.

Passive Bloodstains

• Passive bloodstains are drops created or formed by the force of gravity acting alone.

Projected Bloodstains• Projected bloodstains are

created when an exposed blood source is subjected to an action or force, greater than the force of gravity. (Internally or Externally produced)

• The size, shape, and number of resulting stains will depend, primarily, on the amount of force utilized to strike the blood source.

Projected Bloodstains

• Arterial Spurt/Gush– Bloodstain pattern(s) resulting

from blood exiting the body under pressure from a breached artery

• Cast-off Stains– Blood released or thrown from

a blood-bearing object in motion

• Impact Spatter– Blood stain patterns created

when a blood source receives a blow or force resulting in the random dispersion of smaller drops of blood.

– Velocity affects stain pattern

Impacted Spatter

Low Velocity

Medium Velocity

High Velocity

Directionality of Bloodstains• When a droplet of blood strikes a

surface perpendicular (90 degrees) the resulting bloodstain will be circular.That being the length and width of the stain will be equal.

• Blood that strikes a surface at an angle less than 90 degrees will be elongated or have a tear drop shape.

• Directionality is usually obvious as the pointed end of the bloodstain ( tail ) will always point in the direction of travel.

SIN < = Width (a) 1.5cm Length (c) 3.0cm

SIN < = 0.5

< = 30 degrees

Heredity & Paternity• The transmission of hereditary material is

accomplished by means of microscopic units called genes, located on chromosomes.

• Alternative forms of genes that influence a given characteristic (such as eye color or blood type) are known as alleles.

• Paternity testing has historically involved the A-B-O blood typing system, along with blood factors other than A-B-O.

• Currently, paternity testing has implemented DNA test procedures that can raise the odds of establishing paternity beyond 99 percent.

Testing for Seminal Stains• Many of the cases sent to a forensic laboratory involve

sexual offenses, making it necessary to examine exhibits for the presence of seminal stains.

• The best way to locate and at the same time characterize a seminal stain is to perform the acid phosphatase (an enzyme secreted into seminal fluid) color test.– A purple color indicates acid phosphatase enzyme.– A screening test for semen by determining acid phosphatase

content; because seminal fluid contains high concentrations of acid phosphatase, while other body fluids and extraneous foreign materials have very low concentrations, high values of acid phosphatase on vaginal aspirate or lavage, or on wash fluid from stains, render positive identification of semen, even if the male is aspermic.

Testing for seminal stains

• Semen can be unequivocally identified by either the presence of spermatozoa or of p30, a protein unique to seminal plasma.

• Forensic scientists can successfully link seminal material to an individual by DNA typing.

An antibody–antigen–antibody sandwich or complex is seen as a colored band. This signifies the presence of PSA in the extract of a stain and positively identifies human semen.

PSA testing by electrophoresis.

Rape Evidence• The rape victim must undergo a medical

examination as soon as possible after the assault. • At that time the appropriate items of physical

evidence including clothing, hairs, and vaginal and rectal swabs can be collected for subsequent laboratory examination.

• All outer and undergarments should be carefully removed and packaged separately in paper (not plastic) bags.

• Bedding, or the object upon which the assault took place, may also be carefully collected.

Physical Evidence from the Victim

• Pubic combings• Pubic hair

standard/reference sample

• External genital dry-skin areas

• Vaginal swabs and smear

• Cervix swab• Rectal swabs and smear

• Oral swabs & smear• Head hairs• Blood sample• Fingernail scrapings• All clothing• Urine specimen

Rape Evidence• If a suspect is apprehended within 24 hours of

the assault, it may be possible to detect the victim’s DNA on the male’s underwear or on a penile swab of the suspect.

• Items routinely collected from the suspect include all clothing, pubic hair, head hair, penile swab, and a blood sample or buccal swab for DNA typing.

• The forceful physical contact between victim and assailant may result in a transfer of such physical evidence of blood, semen, saliva, hairs, and fibers.