Chapter 8 Forensic Serology Courtesy of C Fanning

Chapter 8 Forensic Serology Courtesy of C. Fanning FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -1

History of Blood Typing n Karl Landsteiner (1901) o o Discovered blood typing Led to the A-B-O classification system FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -2

Unit Objectives for Serology n Understand the anatomy and physiology of blood. n Understand the concept of the antigen – antibody interactions and how they are applied to species identification and drug identification. n Contrast chromosomes and genes including Punnett squares, genotypes and phenotypes of offspring. n List and understand how whole blood is typed. List and describe forensic tests used to characterize a stain as blood. Describe the proper collection of physical evidence in a rape case. n n FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -3

Nature of Blood • Components of blood: q q plasma v red blood cells v white blood cells v platelets proteins FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -4

Nature of Blood Contents of Blood • Plasma q q is the fluid portion of blood and is composed of water. Contains red blood cells (erythrocytes), white blood cells (leukocytes), and platelets. • Serum q The liquid that separates from blood when a clot is formed FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -5

Nature of Blood FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -6

Erythrocytes: Red Blood Cells n Erythrocytes, also known as red blood cells (RBCs), function to transport oxygen in the blood. n RBCs also remove carbon dioxide from tissues by transporting it back to lungs http: //heart-disease. emedtv. com/blood-clotsvideo/what-is-blood-video. html FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -7

Antigens and Antibodies On the surface of each red blood cell are millions of antigens. • Antigen – a protein that stimulates the body to produce antibodies against it q q Are responsible for blood-type characteristics (A, B, AB). Blood antigens are grouped into systems FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -8

Antigens and Antibodies • 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. Cont. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -9

Antigens and Antibodies • People having the D antigen are Rh positive; those not having the antigen are Rh negative. • For every antigen there is a specific antibody https: //www. youtube. com/watch? v=JRna. Sfdjdl 0 FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -10

Antigens and Antibodies Serum also contains proteins known as antibodies • Antibodies – a protein in blood serum that destroys or inactivates a specific antigen • Each antibody contains the prefix anti- and is specific for one antigen • For example: Antibody Antigen anti-A A antigen anti-B B antigen FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -11

Antigens and Antibodies FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -12

Antigens and Antibodies The Reaction • For every antigen there is a specific antibody that will react with it to form clumps known as agglutination. • Agglutination – the clumping together of red blood cells by the action of an antibody. • Thus, an antibody reacts with its specific antigen and no other. Ø Example: anti-B (antibody) reacts only with antigen B FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -13

Antigen-Antibody Reaction The concept of specific antigen–antibody reactions • Pictured below on the left, anti-B (antibody) does not react with antigen A. • On the right, anti-B (antibody) reacts with antigen B. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -14

Antigens and Antibodies Video - http: //study. com/academy/lesson/blood-types-abosystem-red-blood-cell-antigens-blood-groups. html FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -15

Blood Agglutination Explained FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -16

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. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -17

Immunoassay Technique § The concept of antigen-antibody reaction is used in o blood typing o the detecting of drugs in blood and urine § An immunoassay is a test that uses antibodies to detect the presence of drugs and other substances in blood and urine FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -18

Immunoassay Technique n Thousands of individuals are subjected to urinalysis tests for the presence of drug-abuse. Such as: SMilitary personnel STransportation industry employees SPolice and correction personnel SPre-employment drug screenings n Note: Antibodies that react with drugs do NOT exist naturally but can be produced in animals such as rabbits. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein 19 © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -19

How are antibodies made from rabbits. . in order to help detect drugs? FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -20

Testing Urine for Drugs One-step immunoassay for detection of drug abuse in urine Read Supreme Court Ruling FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein 21 © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -21

Immunoassay Techniques cont. • A number of immunological assay techniques are commercially available for detecting drugs through antigen-antibody reaction. • Radioimmunoassay (RIA) uses drugs labeled with radioactive tags. • Enzyme-multiplied immunoassay technique (EMIT) is uses an enzyme tag for detecting drugs in urine. Ø is used by toxicologists because of its speed and high sensitivity for detecting drugs in urine. Video FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -22

Immunoassay Techniques cont. • Immunoassay is ONLY presumptive in nature and its result must be confirmed by additional testing. (not totally specific for any drug) FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -23

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 many different antibodies is known as polyclonal antibodies. • A specific collection of antibodies designed to combine with a single antigen site can be also be made and are known as monoclonal antibodies. Video FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -24

Antigen-Antibody Reaction FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -25

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. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -26

Tests to Detect Blood n n Color Tests o Kastle-Meyer o Luminol o Bluestar Microcrystalline o Takayama test o Teichmann tests FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -27

How to determine if it’s blood… Color Tests n The Kastle-Meyer color test uses phenolphthalein to detect blood. ¡ Hemoglobin causes a deep pink color. Video n Luminol test can detect blood diluted up to 300, 000 times. ¡ Produces blue glow (luminescence) in a darkened area. Luminol Test * A new product called Bluestar can be used in place of luminol FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -28

n Luminol – the most sensitive chemical test that is capable of detecting bloodstains FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -29

Testing for Blood Microcrystalline tests § Microcrystalline tests that detect blood: o Takayama test o Teichmann test § depend on the addition of specific chemicals to the blood so that characteristic crystals will be formed. Cont. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -30

Tests used to detect Human/Animal Blood n n Precipitin Gel Diffusion FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -31

Animal or Human? Precipitin Test • Once the stain is determined to be blood, the precipitin test will determine whether the stain is of human or animal origin. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -32

Precipitin Test n blood is injected into a rabbit; n antibodies are formed; n the rabbit’s blood (containing antibodies) is extracted as an antiserum; n the antiserum is placed on sample blood. n The antibodies will react with human proteins, if human blood is present. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein 33 © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -33

Animal or Human? Precipitin Test • The precipitin test uses antisera derived from rabbits that have been injected with the blood of a known animal to determine the species origin of a questioned bloodstain. • Once blood is determined to be human, DNA analysis has allowed forensic scientists to associate blood to a single individual. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -34

Precipitin Test Why is this a good test for determining if it human blood? n very sensitive n requires only a small amount of blood n Blood can be old and still give a positive result ¡ Bloodstains from 4000 -5000 year old mummies have given positive results Video FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein 35 © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -35

Human vs Animal Blood Microscopic observation Frog Blood Larger nucleic red blood cells FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein Human Blood nonnucleated red blood cells 36 © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -36

Gel Diffusion n antibodies and antigens diffuse or move towards one another on an agar plate. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein 37 © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -37

A-B-O vs. DNA • Before DNA analysis, bloodstains were linked to a source by A-B-O blood typing and identifying 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. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -38

Heredity and 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. How its done. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -39

Heredity of Blood types • An individual’s blood type is determined by three genes, designated A, B, and O. • homozygous - A gene pair made up of two similar alleles Examples: AA and BB blood type A blood type B • heterozygous- A gene pair made up of two different alleles Examples: AO and BO blood type A FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein blood type B © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -40

Punnett Squares n Punnett Squares are used to show the crossing of the genes from the mother & father to produce the genotype of blood type. n The phenotype is the physical presentation of the organism after birth. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -41

Genotype and Phenotype § Phenotype — a person’s outward appearance § § Ex. You have B type blood Genotype — a person’s genetic makeup for a trait § § § Ex. You have BB or BO type blood. No blood test can determine your genotype. By studying the family history of an individual you may be able to determine their genotype Click for Web Extra 8. 4 FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -42

Blood Type Genetics Example n If a mother with type AB blood has children with a father who has homozygous type B blood, what will the children’s genotypes look like? AB AB BB BB FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -43

Blood Type Genetics Try These 1. If a mother with type AB blood has children with a father who has type O blood, what will the children’s genotypes look like? 2. A mother is heterozygous for type B blood and a father has type O blood, what will the children’s genotypes look like? FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -44

Learning Objectives Students should be able to : n List the laboratory tests necessary to characterize seminal stains. n Explain how rape evidence is collected. UV light makes seminal fluids glow brightly FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -45

Testing for Seminal Stains • Many criminal cases involve sexual offenses, making it necessary to search for the presence of seminal stains. • The best way to locate and 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. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -46

Testing for Seminal Stains Seminal fluid can also be identified by the presence of spermatozoa or of p 30, a protein unique to seminal plasma. Forensic scientists can successfully link seminal material to an individual by DNA typing. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -47

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. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -48

Rape Evidence • Items routinely collected from the suspect include all clothing, hair, and a blood sample or buccal mouth 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. FORENSIC SCIENCE: An Introduction, 2 nd ed. By Richard Saferstein © 2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 8 -49