Chapter_10_Forensic_Serology

Chapter_10_Forensic_Serology - 8/31/11 Forensic Serology...

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Unformatted text preview: 8/31/11 Forensic Serology History 1901 Karl Landsteiner discovered blood typing Blood is distinguishable by its group or type A-B-O system 1937: Rh factor discovered (a.k.a. D, +/-) Today: many different blood types known Until the early 1990's forensic scientist used blood typing High frequency of blood stains at crime scenes Rape, murder, assaults The more factors; the more individualized What is Forensic Serology? Forensic serologists... Determine type and characteristics of blood Blood stain and pattern analysis Examination of semen, saliva, other body fluids Trial testimony 1 8/31/11 What is blood? Blood is a mixture of cells, enzymes proteins, and inorganic compounds Plasma is the fluid portion of blood (55%) mostly water Suspended in the plasma are cells (45%) solid portion Erythrocytes (red blood cell) Leukocyte (white blood cells) Platelets Blood clots when fibrin (a protein) traps red blood cells Serum is the liquid that is separated from the blood when a clot forms Blood Composition 45% 2 8/31/11 Blood On the surface of red blood cells are antigens Antigens impart blood type characteristics to the red blood cell 15+ different types of antigen systems have been identified Ex: A, B, O, & Rh Rh is also known as antigen D, +/- Typing Type A = the person has A type antigens Type B = the person has B type antigens Type AB = both antigens Type O = has neither A or B antigens O is the universal donor (not lucky); AB is universal receiver (lucky) Rh (or D) = positive or negative, either has D antigen or not A+ = type A with D antigen B- = type B without D antigen 3 8/31/11 Blood Type A A+ A- B+ B- AB+ AB- O+ O- X X Antigens on RBC B Rh(D) X X X X X X X X X X More Blood Antibody: a protein that destroys or inactivates a specific antigen For every antigen there exists a specific antibody Agglutination Clumping together of red blood cells by the action of an antibody with its antigen Caused when antibody combines with its antigen, cross-linking red blood cells (e.g. A antigen with anti-A antibody) Test A+ blood with anti-A antibody = clump 4 8/31/11 Putting it together... Blood Antigens Antibodies in Reaction w/ Reaction w/ Type on RBC serum Anti-A serum Anti-B serum A A Anti-B + - B AB O B Anti-A - + - + + - A and B Neither Anti- A nor Anti-B Neither Anti-A and A nor B Anti-B AB has two clumps B clumps with anti-B A clumps with anti-A O clumps with nothing Clump Clump Clump Clump 5 8/31/11 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. Immunoassay Techniques Enzyme-multiplied immunoassay technique (EMIT) Detecting drugs in urine i.e., marijuana (THC-9-carboxylic acid) How does it work? 1. Protein-drug complex (antigen) injected in animal. Recovered serum contains antibodies. 2. Subject's urine is mixed with antibodies. Any drug present binds to antibodies. 3. Enzyme labeled drug is added in excess. It binds with free antibodies. 4. Unbound enzyme labeled drug is measured and related to original concentration of drug in urine. 6 8/31/11 Where do antibodies come from? 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. Manufacturing process uncontrolled in a biological may be of a different types with different strengths and expirations Where do antibodies come from? 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. 7 8/31/11 Blood Type Statistics Genetics of Blood Types Traits are demonstrated through an allele Allele: alternative form of a gene located at the same point (locus) on a particular pair of chromosomes In the A-B-O system, there are three alleles: A, B, and O In the Rh system, there are two alleles: Rh+ and Rh- A genotype contains all inherited alleles while a phenotype is the allele that is actually being expressed Ex: brown eyes versus blue eyes 8 8/31/11 Visualized Two alleles at a loci A and B are dominant; O is recessive Rh+ is dominant; Rh- is recessive Genotype AA BB AB AO BO OO Rh+/Rh+ Rh+/Rh- Rh-/Rh- Phenotype A B AB A B O Rh+ Rh+ Rh- Heredity and Blood Types What blood type will baby be? Father's Genotype O A Mother's Genotype O B Punnet square 9 8/31/11 Problems Variation in agglutination titer sensitivity can be poor Works best with blood less than five days old Dried stains require a different procedure Require a different antibody for every antigen Over 100 antigens What about mixtures? Forensic Application? Paternity Testing!!! Suppose Mom is type AB and Baby is type AB, could the male from the last example have fathered the child? (Dad was O,O) Suppose the Baby was type A or type B... What can we say about the male from the last example? 10 8/31/11 Forensic Characterization of Blood Stains 1. Is it blood? 2. From what species is the blood? 3. If it's human blood, how well can we associate it with a particular individual? Is it Blood? Hemoglobin tests: 1. Kastle-Meyer color test Turns pink 2. Hemastix Turns green 3. Luminol test Gives off a bluish-green glow under UV light (luminescence) 11 8/31/11 Blood 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. Presumptive Tests Do you have blood? Preliminary tests Benzidine Phenolphthalein (Kastle Meyer) Both tests rely on the fact that blood reacts with hydrogen peroxide (H2O2) in the presence of an indicator to produce a deep pink color Interferences include horseradish, potatoes, or any other substance with peroxidase activity 12 8/31/11 Hemastix/Hematrace tests originally for blood in urine Hemastix Blood Reagent Strips Hemastix are small 3" plastic test strips with a square of blood reagent material at the tip. Unknown substances may be absorbed onto a swab moistened with sterile water, and then transferred through contact to the square of blood reagent material. The yellow reagent material will then turn shades of green if blood is present. Used commonly to detect blood in urine. Sold in a vial of 50 test strips. Luminol Sprayed on a bloodstain and will omit light that can be observed in the light Interferences with metal Extreme sensitivity Needs darkness 13 8/31/11 Now that its blood... Is it human blood? Human anti-serum reacts with (antigens in) human blood Gives a precipitate at the interface Not me! What is anti-serum and how is it made? Blood serum that contains certain antibodies, in this case human Rabbit injected with human blood and anti- sera is collected Precipitin Test Once the stain has been characterized as blood, the precipitin test will determine whether the stain is of human or animal origin. The precipitin test uses antisera normally derived from rabbits that have been injected with the blood of a known animal to determine the species origin of a questioned bloodstain. Once it has been determined that the bloodstain is of human origin, an effort must be made to associate or dissociate the stain with a particular individual. 14 8/31/11 Precipitin Test Ring Precipitan Test Blood is placed in a capillary tube containing human anti-serum Ring = human blood; no ring = not human blood 15 8/31/11 16 8/31/11 Can we associate the blood? Can human blood be narrowed down to a particular human? Kinda... Prior to DNA analysis Blood typing and blood factors Use the multiplication rule...... Now: DNA analysis Another aspect...blood spatter Surface texture Harder, smoother, less- porous = less splatter Direction of travel Pointed end = direction of travel Impact Angle 90 = circle. As angle decreases, stain elongates Impact Splatter Intersection of lines along long-axis = emanation point 17 8/31/11 Semen Evidence 1. Locate stain 2. Characterize stain Presumptive test for seminal stain acid phosphatase enzyme color test Confirmatory tests for semen microscopic examination of spermatozoa p30 (prostate specific antigen, PSA) Acid Phosphatase test Seminal fluid residue can be detected with either: Sodium alpha naphthylphosphate and Fast Blue B dye: turns blue 4-methyl umbelliferyl phosphate (MUP): which fluroresces under UV light However, false positives may occur from Cauliflower and watermelon Fungi Contraceptive creams Vaginal secretions But take longer to react 18 8/31/11 Semen Stains Can be recovered from garments years later Can be visualized as a white or yellowish stain Can fluoresce under UV light Microscopic confirmation Immerse stained material in small amt of water Transfer drop to microscope slide and dry Examine under a compound microscope at 400X Potential issues: Spermatozoa brittle when dry Bind tightly to cotton Low sperm count or no sperm Tails fall off = time frame 19 8/31/11 Why PSA? Prostate specific antigen (PSA) is an enzyme produced in the ducts of the prostate and absorbed into the bloodstream It is proof of human sperm Will be found even in vasectomized males PSA Testing 20 8/31/11 PSA Testing Rape Evidence Victim rape collection kit Pubic combings Pubic hair and head hair reference samples Vaginal / rectal / oral / skin swabs Blood sample (DNA and tox) Fingernail scrapings All clothing Urine sample (tox) 21 8/31/11 Rape Evidence Suspect collection All clothing Pubic hair combings Head and pubic hair reference samples Penile swab Blood sample or buccal swab (DNA) Tests for Saliva Saliva consists of 99% water, mucin (protein that helps in swallowing), and amylase (enzyme that helps digest carbohydrates) To test for saliva mix starch, iodine, and the stain together. \ Starch mixed with iodine produces a blue color. If amylase is present it will turn a dark blue-purple. The color will fade in about 15 minutes at 37 C. 22 8/31/11 Differential Extraction Sperm Cells and Non-Sperm Cells Regular Cells are Broken Evidence swab is placed in EDTA, salt, buffer, and a detergent Proteinase K is added and heated for two hours Swab is centrifuged and removed Pellet contains the sperm and the fluid contains the broken cell DNA 23 8/31/11 Get out the Sperm DNA Tris-HCl, EDTA, salt, buffer, Sarkosyl, Proteinase K, DTT Wait two hours... Conclusions Forensic serology involves the detection of biological stains at a crime scene Serological analysis is performed prior to DNA testing and determines the type of stain and if it is human Serological tests consist of presumptive (chemical) and confirmatory (antigen/antibody)tests Presumptive tests for blood are based on peroxidase activity of hemoglobin Confirmatory tests are based on inherited factors in the blood ABO, Rh, PGM and are highly specific Serological tests exist for blood, (see above) semen (acid phosphatase and p 30) and saliva(amylase) Sperm can be identified microscopically by staining Victims of rape are processed by a sexual assault nurse examiner (SANE) in concert with a police officer and a victim's advocate. A rape kit is used in the collection of evidence. 24 ...
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This note was uploaded on 01/31/2012 for the course CHS 3501 taught by Professor Perr during the Fall '09 term at FIU.

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