1. Blood Basics and Other Body Fluids Objectives: Distinguish between blood types and their antigens and antibodies. Explain what happens in the process of agglutination. Use whole blood typing to type blood. Explain what tests are used to characterizing a stain as blood. Explain the significance of the precipitin test in serology. Discuss the contribution of polymorphic enzymes and proteins to blood’s individualization.

2. Define chromosomes and genes. Use Punnett squares to determine the probability of inheritance. Describe lab tests for seminal stains. Preservation of stains for lab examination. List the steps for collection of physical evidence in rape investigation.

3. History A. 1901- Karl Landsteiner discovered that people had different types of blood. From his work the ABO classification system of blood types was developed. B. By 1937 the Rh factor was discovered. C. At present over 100 different blood factors have been shown to exist, but the ABO factors are still the most important for properly matching a donor with a recipient. 1. If a forensic scientist individualized human blood by Identifying all of the known blood factors, the resulting evidence would be of the strongest kind for linking a suspect to the crime scene. D. With the advent of DNA technology, the blood typing has taken on a secondary role. The analysis of the DNA found in blood individualizes a blood sample with a very high degree of reliability.

4. 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.

5. Components of Blood Red Blood Cells (Erythrocytes) White Blood Cells (Leukocytes) Platelets (Thrombocytes) Plasma

6. Components of Blood Red Blood Cells (Erythrocytes) Red in color (hemoglobin is iron- carrying), made in the bone marrow, live 120 days, removed when damaged by liver and spleen (made into bile), carry O2, no nucleus, 30 trillion per body, … White Blood Cells (Leukocytes) Protect body from disease, have a nucleus, colorless, may diff. kinds, make antibodies, made in bone marrow and lymph nodes, 60 billion per body, … Platelets (Thrombocytes) Cell fragments, no nucleus, involved in blood clotting, made in bone marrow, formed by pinching off of cytoplasm from large cells, 1.5 trillion per body, produced 200,000 per day, live 7 days, … Plasma55% of the volume of blood, clear, straw colored liquid, 90% water, contains dissolved gases, nutrients, hormones, wastes, salts, enzymes, plasma proteins, …

7. Comparing Animal Blood

8. What is 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.

9. Blood Facts The average adult has about FIVE liters of blood inside of their body, which makes up 7-8% of their body weight. Blood is living tissue that carries oxygen and nutrients to all parts of the body, and carries carbon dioxide and other waste products back to the lungs, kidneys and liver for disposal. It also fights against infection and helps heal wounds, so we can stay healthy. There are about one billion red blood cells in two to three drops of blood. For every 600 red blood cells, there are about 40 platelets and one white cell.

10. Erythrocytes Transport oxygen from the lungs to the body tissues Transport carbon dioxide from the tissues to the lungs Red cells possess chemical structures on their surfaces called antigens or agglutinogens –impart blood type characteristics

11. Blood Type Your blood type is determined before you are BORN, by specific genes inherited from your parents. Three types (alleles) of blood type gene –A, B, O Each individual inherits one blood type gene from their mother & one from their father –6 possible combinations (genotypes) AA, BB, OO, AB, AO, and BO These genes determine your blood type by causing proteins (or carbohydrates) called ANTIGENS (or agglutinogens) to exist on the surface of all of you red blood cells.

12. Antibodies or Agglutinins Proteins that are present in the serum responsible for ensuring that the only blood cells that can survive in a person are cells of the correct blood type

15. Antigen-Antibody Reaction B-Antibodies produced by people with type A blood remove any red blood cells containing B antigens by clumping them together A- Antibodies produced by people with B blood remove any red blood cells possessing A antigens

17. Type O Blood Possessed by people whose genotype is OO –both parents passed on the O gene –have no antigens these cells can be introduced into a person with Type A or Type B because these cells are not attacked by the antibodies these people possess –have both A & B antibodies can only have other O type cells mixed with this blood

18. Type A Possessed by people with genotype –AA –AO A is dominant to O Possesses antibody B –will destroy any Type B red cells –compatible with A or O red cells

19. Type B Possessed by people with genotype –BB –BO B is dominant to O Possesses antibody A –will destroy any Type A red cells –compatible with B or O red cells

20. Type AB Possessed by people with genotype –AB A & B are co-dominant Possesses no antibody –can have A, B, AB, or O cells added –Can’t be added to any other blood type without being destroyed by an antibody

21. Blood typing is done by reacting whole blood with antibody A and antibody B What Type is it?

22. Blood Typing

23. How Common Is Your Blood Type? 46% 39% 11% 4%

24. Rh Factors Scientists sometimes study Rhesus monkeys to learn more about the human anatomy because there are certain similarities between the two species. While studying Rhesus monkeys, a certain blood protein was discovered. This protein is also present in the blood of some people. Other people, however, do not have the protein. The presence of the protein, or lack of it, is referred to as the Rh (for Rhesus) factor. If your blood does contain the protein, your blood is said to be Rh positive (Rh+). If your blood does not contain the protein, your blood is said to be Rh negative (Rh-). A+ A- B+ B- AB+ AB- O+ O-

25. Secretors An individual who secretes his or her blood type antigen(s) in all body fluids. Approximately 80% of the population are secretors.

26. Blood Enzymes and Proteins Enzymes: proteins that have important functions in regulating the body’s chemistry. Enzymes have the characteristic of existing in different forms (polymorphic) and can be separated into protein components called iso-enzymes. A common polymorphism is Hb, which causes sickle-cell anemia. Iso-enzymes can be separated by electrophoresis. Forensic serologists have studied these iso-enzymes with the goal of being able to individualize blood samples found at crime scenes. While there are many iso-enzymes in blood, only those that survive the drying and aging process are useful to the forensic serologist.

27. Iso-enzymes and Probability Each of these protein and enzyme variants, as well as all blood subtypes, have known distributions in a population. It's therefore a simple matter to calculate probability estimates that border on individualized blood typing. Probability is defined as the frequency of an event occurring. If there are several event that may occur, the overall probability will be the product of these probabilities. Probability is usually expressed in per cent (i.e., 25%), while the frequency of an event is expressed as a decimal value (i.e., 0.25) At a crime scene, a blood sample and the suspect’s blood had the following characteristics: A blood (42%), basic subtype A2 (25%), protein AK (15%) and enzyme PGM 2 (6.0%). What is the frequency of occurrence of these blood characteristics in the general population? Answer: 0.00094 (0.42 X 0.25 X 0.15 X 0.06) or 0.094% probability!

28. BLOODSTAIN CHARACTERIZATION Bloodstain analysis traditionally follows the following steps: –Is the stain blood? –Is the stain animal or human blood? –If human blood, what type? –Can the sex, age, and race of the source of blood be determined? Is the stain blood? –The benzidine test was long used until replaced by the Kastle-Meyer test. Benzidine was found to cause cancer. –Both tests are color tests based on the observation that hemoglobin will oxidize several classes of organic compounds. –Kastle-Meyer color test- when the reagent- phenolphthalein is mixed with hemoglobin in blood, the clear phenolphthalein will turn a deep pink. Not a specific test for blood, some vegetables react with the reagent in the same way, for example potatoes and horseradish. From a practical point of view, a positive Kastle-Meyer test indicates blood because how often will potatoes or horseradish contaminate a crime scene?

29. BLOODSTAIN CHARACTERIZATION Is the stain blood? Another test used is a Hemastix® strip, which will turn green in the presence of blood. Luminol is an important presumptive identification test for blood. The iron in hemoglobin reacts with the luminol and causes the emission of light (luminescence). Needs to be done in a dark room. –Extremely sensitive test- can detect bloodstains diluted to 300,000 times. –Spraying large areas is possible –Does not interfere with subsequent DNA tests.

30. BLOODSTAIN CHARACTERIZATION microcrystalline tests- Takayama and Teichmann tests –Both depend on the addition of specific chemicals to the blood so that the characteristic crystals with hemoglobin derivatives will be formed. –Crystal tests are far less sensitive than color tests and they are more susceptible to interference from contaminants that may be present in the stain.

31. BLOODSTAIN CHARACTERIZATION Is the stain animal or human blood? To answer this question, forensic serologists use antiserum tests. The standard test is called the precipitin test This involves forming antibodies to human blood in an animal. The animal serum thus treated will cause a precipitate to form when reacted with human blood. Precipitin test is very sensitive and requires only a small amount of blood for testing. Extracts from mummies 4-5 thousand years old have given positive reactions to this test. So the age of the blood tested can be very old. Also, bloodstains diluted by washing in water still give positive precipitin tests

32. BLOODSTAIN CHARACTERIZATION Gel diffusion- takes advantage of the fact that antibodies and antigen will move toward each other on an agar gel- coated plate forming a line of precipitate in between the two. Electrophoretic method- an electric potential is applied to the gel medium; the antibodies and antigens move toward each other forming a line of precipitate in between the two, but the process is quickened.

33. Principles of Heredity Transmission of Traits –1.Antigens, proteins and enzymes that have been previously discussed are genetically controlled traits. –Accomplished by genes, which is the basic unit of heredity. –Genes are on chromosomes (46 in 23 pairs). Each chromosome in a pair has the same genes in the same order at the same spot. –The human egg and human sperm contain 23 chromosomes, which combine during fertilization.

34. A female has XX chromosome, and male has XY chromosome. Genes and chromosomes come in pairs. The position of a gene on the chromosome is called the locus. Alleles are alternative forms of genes that influence an inherited characteristic. An example of allele genes is the A-B-O blood type system. Genotype- the exact allelic combination Phenotype- the physical appearance of the organism as a result of the genotype.

35. Principles of Heredity Transmission of Traits A-B-O blood types: –When a gene is made of two similar genes, they are said to be homozygous. Examples include AA,BB, or OO. –If the gene is made up of two different genes, it is said to be heterozygous. An example would be AB, AO, or BO. –A and B genes are dominant, while O genes are recessive. –A pair of A-B-O allele genes together are the genotype of an individual. –The phenotype of an individual is the outward characteristic of the individual. –There is no lab test to determine a person’s A-B-O genotype. –If the genotypes of both parents are known, a Punnett square may be constructed to determine potential genotype of offspring.

36. Paternity Testing Blood typing can be used to resolve cases of disputed paternity. The more blood groups tested, the more certain one can be that the suspect is indeed the father. –HLA (human leukocyte antigen) test relies on identifying a complex system of antigens that exist on the surface of white blood cells. If a suspect cannot be excluded as fathering a child after this test is performed then there is a 90% chance that he is the father. –DNA testing has raised the odds of establishing paternity to beyond 99%. ( Do the punnett square in your notes- OO x AB and then go to the last page of your notes to do the practice sheet on paternity.)

37. Forensic Characterization of Semen Tests used to locate seminal stains- –Acid phosphatase color test- acid phosphatase is an enzyme secreted by the prostate into seminal fluid. Its concentrations in seminal fluid are 400 times greater than in any other body fluid. Moistened filter paper is rubbed on suspect fabric or surfaces and then Fast Blue B solution is applied. This enzyme will turn purple in the presence of Fast Blue B solution. A reaction time of less than 30 seconds indicates the presence of semen. (Other substances will react but not as fast.) –MUP test- Moistened filter paper is pressed against a suspected garment and then MUP solution is sprayed onto the paper. Under a UV light semen stains will fluoresce.

38. Acid Phosphatase Test The more semen, the deeper the purple

39. Forensic Characterization of Semen Microscopic identification of spermatozoa. Sperm are elongated structures 50-70 microns long. The criminalist would immerse the stained material in distilled water, stir rapidly, dry the fluid on a slide and examine under 400x. it is difficult to locate sperm this way due to several reasons: Sperm bind tightly to cloth materials They are extremely brittle when dry and easily disintegrate. Many sex crimes involve individuals with low sperm counts, a condition known as oligospermia, or they may involve individuals with no sperm at all, a condition known as aspermia. Today, forensic scientists can link seminal material to one individual with DNA technology.

40. Collection of Rape Evidence Finding evidence of rape: –Physical injuries such as bruises or bleeding tend to confirm the fact that a violent crime took place. –Forceful physical contact will result in transfer of physical evidence- blood, semen, hairs, and fibers. This evidence will help forge a link between the between the crime and the criminal. Protection of Physical Evidence- –Place a clean bed sheet on the floor and lay clean paper over it. –The victim removes their shoes and stands on the paper. –All outer and inner garments of the involved parties should be carefully removed and placed in separate paper bags one item at a time to avoid cross contamination. The paper collects any loose foreign material that falls from the clothing. –The paper should be then folded so that all foreign materials will be contained inside.

41. –The bedding or object upon which the assault took place should be submitted to the lab for processing. Items suspected of containing seminal stains must be handled carefully to avoid the loss of material. –If it is not possible to transport the stained article, the stained area should be cut out and submitted with an unstained piece as a substrate control. –The rape victim must be examined by medical personnel as soon as possible after the assault. At this time the appropriate items of physical evidence should be collected by trained personnel. The following evidence should be collected: Pubic combings Hair- pubic and head for standard reference Smear- vaginal, rectal and oral- for DNA typing Blood sample- for toxicology testing Fingernail scrapings Urine- for drug testing (GHB, Rohypnol) Saliva residues for DNA typing

42. Collection of Rape Evidence If the suspect is apprehended the following items are routinely collected: –all clothing –Pubic hair combings –Pulled head –hair and pubic hair for standard references –Penile swab –A blood sample

43. Vocabulary: DNA: abbreviation for deoxyribonucleic acid- the molecules carrying the body’s genetic information. DNA is double-stranded in the shape of a double helix Plasma: the fluid portion of unclotted blood Serum: the liquid that separates from the blood when a clot is formed Antigen: a substance, usually a protein that stimulates the body to produce antibodies against it

44. Antibody: a protein that destroys or inactivates a specific antigen. Antibodies are found in the blood serum Antiserum: blood serum in which there are specific antibodies Agglutination: the clumping together of red blood cells by the action of an antibody Serology: the study of antigen-antibody reactions Hemoglobin: a red blood cell protein responsible for transporting oxygen in the bloodstream and the red coloring of blood

45. Precipitin: an antibody that reacts with its corresponding antigen to form a precipitate Secretor: an individual who secretes his or her blood-type antigen(s) in body fluids. Approximately 80 percent of the population are secretors Enzyme: a type of protein that acts as a catalyst for certain specific reactions Polymorphism: the existence of more than one form of genetic trait

46. Iso-enzymes: multiple molecular forms of an enzyme, each having the same or very similar enzyme activities Gene: a unit of inheritance consisting of a DNA segment located on a chromosome Chromosome: a rodlike structure in the cell nucleus, along witch the genes are located. It is composed of DNA surrounded by other material, mainly proteins Egg: the female reproductive cell Sperm: the male reproductive cell

47. Zygote: the cell arising from the union of an egg and a sperm cell X-chromosome: the female sex chromosome Y-chromosome: the male sex chromosome Locus: the physical location of a gene on a chromosome Allele: any of several alternative forms of a gene located at the same point on a particular pair of chromosomes. For example, the genes determining the blood types A and B are alleles

48. Homozygous: having two identical allelic genes on two corresponding positions of a pair of chromosomes Heterozygous: having two different allelic genes on two corresponding positions of a pair of chromosomes Genotype: the particular combination of genes present in the cells of an individual Phenotype: the physical manifestation of a genetic trait such as shape, color, and blood type

49. Acid phosphatase: an enzyme found in high concentration in semen Oligospermia: a condition describing an abnormally low sperm count Aspermia: the absence of sperm; sterility in males