Study of bodily fluids using antigen-antibody reactions Serology Study of bodily fluids using antigen-antibody reactions

Blood Components RBC – Erythrocytes WBC – Leukocytes Platelets – Contains fibrin which clots blood Plasma – Fluid portion of the un-clotted blood Serum – Fluid that is separated from clotted blood

Antigen – Antibody Reactions Antigens- proteins on surface of cells Antibodies- proteins that are produced in response to the presence of foreign antigens (Found in the serum of blood) Agglutination- reaction that occurs when specific antibodies attach to antigen sites and cause RBC to clump together >15 blood antigen systems have been identified, including A-B-O and Rh systems ( Saferstein, 2004, pg 330)

What Makes Blood Different?

Agglutination

ABO System Blood Type Antigens/RBC Antibodies/Serum A Anti-B B Anti-A Neither anti-A or B O Neither A nor B Both anti-A and B

Blood Typing Anti-A serum + blood Anti-B Serum + blood Antigen Present Blood Type + _

Immunoassay Techniques Antigen-Antibody reactions used for the detection of Drugs in blood, urine and semen EMIT : Enzyme-Multiplied Immunoassay Technique – are not totally specific Monoclonal Antibody Tests: antibodies are produced to react to ONE specific antigen Currently found in newest forensic test kits (pg333-335)

Standard Analysis of Blood Questions about crime scene blood: Is the substance blood? Is the blood human? Can the blood be associated with a particular individual?

Is the substance blood? Kastle-Meyer Color Test Phenolphthalein, hydrogen peroxide, and suspected blood stain are mixed; positive reaction indicated by a deep pink color Based upon observation that hemoglobin possesses peroxidase activity This test is a presumptive or preliminary test for blood. Substances other than blood may produce false positive results (pg 336)

Is the substance blood? Con’t Hemastix Color Test Hemastix—has been found to be very useful by field investigators—a dipstick moistened with distilled water is placed in contact with the suspect blood—a green color change indicates a positive result (pg 336)

Is the substance blood? Con’t Luminol Test A test that produces light when it reacts with blood rather than color Large areas can be quickly screened for blood, by spraying luminol reagent “Luminol test is extremely sensitive—it is capable of detecting blood stains that have been diluted up to 300,000 times.” Does not interfere with subsequent DNA testing! (pgs 336-337)

Is the blood human? Precipitin Test If you inject an animal with human blood, the animal will create antibodies that can then be recovered and used to test for various human antigens including human blood antigens The recovered antibodies are called human antiserum To test for human blood, human antiserum and an unknown blood are put together—if the blood is human a precipitate will form. (p338)

Can the blood be associated with a particular individual? A-B-O types and enzymes Used until the mid 1990’s, characterization of bloodstains included determining A-B-O types and polymorphic enzyme markers such as PGM (phosphoglucomutase) These tests added statistical information with which to reduce the number of possible sources of the bloodstain Because antigens, and enzymes occur independently of one another, the probability of a dried blood stain having a particular combination of these factors is determined by the product of their distribution in the population (pg 339)

Can the blood be associated with a particular individual? Con’t Statistical Example: Blood stain found to be of type A— corresponds to 42% percent of population Stain contains PGM-1—corresponds to 58% of population The combined effect of just these two factors, using the product rule is 42% x 58% or 24 % Clearly the more factors, the smaller the frequency of occurrence in the population

Can the blood be associated with a particular individual? Cont…. DNA Analysis Since the 1990’s DNA analysis has replaced forensic reliance on blood factors for the routine characterization of bloodstains DNA analysis gives extremely accurate individualization results.

Stain Patterns of Blood The location, distribution and appearance of bloodstains and spatters can be used to interpret and reconstruct the events that must have occurred to produce the bleeding. (pg. 342)

Stain Pattern Analysis General Conclusions Surface texture: The harder and less porous the surface, the less spatter results. Direction of Travel: The direction of travel of blood can be discerned by the stain’s shape — the pointed end always faces its direction of travel (pg 342)

Stain Pattern Analysis General Conclusions Cont… Impact Angle: A drop of blood striking a surface at a right angle gives rise to a nearly circular stain; as the angle decreases, the stain becomes more elongated in shape Origin of Blood Spatter: Can be determined by establishing lines through the long axis of several individual blood stains. The intersection of the lines represents the point from which the blood emanated. (pg 342) http://www.bloodspatter.com/BPATutorial.htm

Principles of Heredity The gene is the basic unit of heredity. They found on chromosomes. All human cells contain 46 chromosomes (23 pairs). Only exception: reproductive cells During fertilization, a sperm and egg combine so each contributes chromosomes to form the new cell (zygote)

Principles of Heredity Cont.. Inheritance Rules A gene pair of two similar genes—is homozygous—no problem determining a person’s outward characteristics A gene pair of two different genes—is heterozygous—one gene is dominant, the other recessive—person’s outward characteristics depend on the dominant gene

Paternity Testing Many cases of disputed paternity can be resolved when suspected parents and offspring are related according to their blood group systems Type O blood is considered a recessive gene so……

Paternity Example O O A AO AO B BO BO Father’s Genotype Mother’s If the Child in question is type AB would the suspect father be cleared?

Paternity Testing Con’t Routine paternity testing involves characterizing many blood factors other than A-B-O. Using these other factors the probability of establishing paternity becomes better than 90%. Once DNA analysis is done the odds become better than 99%. (pg 348)

Forensic Characterization of Semen A large number of cases involve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains. The forensic examination of semen is a two-step process: Semen must be located Tests must be performed to prove its identity—it may even be tested for blood type Note: Semen is checked for blood type because many individuals are secretors

Secretors 80% of individuals are classified as secretors This means that their blood-type antigens are also found in high concentrations in most body fluids Ex- Saliva, semen, vaginal secretions, and gastric juices **Saliva & semen have higher concentrations of A & B antigens than blood!!

Semen Location and Identification There are two methods of locating semen: Semen is often readily visible on a fabric because it has a stiff crusty appearance- unreliable The acid phosphatase color test can locate and at the same time identify semen.

Acid Phosphatase Color Test Acid phosphatase is an enzyme secreted by the prostate gland into seminal fluid that is in far greater concentrations than those found in any other body fluids. Its presence can be detected using different tests: 1. Contact with a special acidic solution and Fast Blue B dye shows a purple change 2. Contact with a solution of MUP fluoresces under UV light. (p.349)

Acid Phosphatase Test, Con’t Large areas or fabrics can be quickly searched by moistening filter paper with the detector solutions and rubbing over the suspect area Some substances will give a false-positive result (fruit juice, contraceptive creams, etc), but none of these substances will react with the speed of seminal fluid. Note: A reaction of less than 30 seconds is a strong indication of semen. (p 349)

Microscopic Examination of Semen Rapid stirring of a fabric-stain-water mixture will transfer a small amount of sperm into the water. A drop is then taken, dried, stained and looked at under the microscope. The presence of sperm indicates the presence of semen.

Microscopic Problems Locating sperm with the microscope is not that easy for three reasons: 1. Sperm are very brittle when dried and easily disintegrate. They are also hard to remove from cloth material. 2. Sexual crimes may involve males who have an abnormally low sperm count, condition called oligospermia. 3. Males who have no sperm in their seminal fluid, condition called aspermia—often due to increasing numbers of vasectomies.

Detecting Semen, Cont… A positive result for acid phosphatase and a negative result for semen give ambiguous results—In this case, p-30 or Prostate Specific Antigen (PSA) is used to detect seminal plasma – Immunoassay tests

p30 Prostate Specific Antigen Test Formation of a visible line between the wells of anti p-30 and semen extract–proves the stain is seminal

Collection of Rape Evidence Victim Outer and undergarments are placed in separate paper bags, while victim stands on a piece of paper. The paper is then folded and collected. Bedding or the object upon which assault took place should be submitted to the lab. Rape victim must be subjected to a medical examination Collected items include pubic hairs, vaginal swabs, blood, fingernail scrapings, clothing and urine (p 352)

Rape Suspect Suspect must also undergo an exam. Routine items collected include clothing, hairs, penile swab and blood samples

Timeline Living or motile sperm may survive 4 to six hours in the vagina of a living person. Non-motile sperm may be found in a living female for up to three days and occasionally up to six days Intact sperm (sperm with tails) are not normally found 16 hours but have been found as late as 72 hours after intercourse.

Important Considerations Based Upon Timeline How long ago was the person raped? When did voluntary sexual activity last occur prior to the sexual assault? Blood is taken from any consensual partner having sex with the victim within 72 hours of the assault.

What are these?