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erology

S. erology. By M. Hoffmeister & H. Montgomery. Identification & Character of Blood and Bloodstains. Forensic Science Uses blood analysis to determine the individual (source)

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erology

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  1. S erology By M. Hoffmeister & H. Montgomery

  2. Identification & Character of Blood and Bloodstains Forensic Science • Uses blood analysis to determine the individual (source) • Usually deals with a variety of fluids (blood, saliva, semen, and urine) in stain form and are often degraded/deteriorated. Environmental controls are essential ( uncontrolled heat and humidity can destroy a lot of biological information within a stain by enhancing degradation). Serology Chapter 11 & 12

  3. Identification & Character of Blood and Bloodstains Serology was very prominent in the 1950s to the 1980s, when DNA analysis became possible • It is still routinely done where DNA is analyzed. • Is done when DNA analysis is not feasible • Serology is being challenged by the reliability and the discrimination power of DNA analysis. • Important to understand preDNA serology f testimony in cases Serology Chapter 11 & 12

  4. Protocol for the Analysis ofBlood in Forensic Serology • Careful visualization of evidence to locate stains/ material visibly characteristic of blood. • Application of a suitable presumptive test. • Application of a confirmatory (specific and sensitive) test. • Determination of biological/species (animal or human) origin. • Characterization of blood using genetic markers or DNA. Serology Chapter 11 & 12

  5. Identification of Blood A visual observation coupled with positive chemical presumptive and confirmatory tests provide sound data to support the identification of blood. Two types of tests are given because ---- NO SINGLE TEST IS ABSOLUTELY SPECIFIC FOR BLOOD Serology Chapter 11 & 12

  6. Catalytic Color Tests Catalytic Color Tests - employ chemical oxidation of a chromogenic (color causing) substance by an oxidizing agent catalyzed by the presence of hemoglobin (in red blood cells). Two substances are needed for the color change: • Oxidizing agent (often hydrogen peroxide). • The heme group acts as a (peroxide – like) catalyst Serology Chapter 11 & 12

  7. Catalytic Color Tests Serology Chapter 11 & 12

  8. Catalytic Color Tests Common method of application: • Wet cotton swab with distilled water and sample the stain • Apply the reagents to the swab. • Note immediate observations – changes will take place quickly and may not appear the same after time has passed. False positives can usually be attributed to: • Chemical oxidants • Plant materials (with peroxidase-like enzyme). • Animal materials that may contain traces of blood. Serology Chapter 11 & 12

  9. Catalytic Color Tests Adler Test (Benzidine) • More extensively used than any other presumptive test for blood • Normally carried out in ethanol/acetic acid solution • Results in a characteristic blue color • Was deemed a carcinogen in 1974 and has been essentially discontinued for forensic use today Serology Chapter 11 & 12

  10. Catalytic Color Tests Adler Test (Benzidine) Serology Chapter 11 & 12

  11. Catalytic Color Tests Kastle-Meyer Test (Phenolphthalein) • Commonly used today • Uses phenolphthalein (acid/base indicator) • Results in hot pink color • Phenolphthalein (alkaline solution) becomes oxidized to phenolphthalein (pink in alkaline environment) • Phenolphthalein over zinc in KOH solution • False positives do not usually produce the pink color Serology Chapter 11 & 12

  12. Catalytic Color Tests Kastle-Meyer Test (Phenolphthalein) Serology Chapter 11 & 12

  13. Catalytic Color Tests O-Tolidine (ortho-tolidine) • Derivative of benzidine • Conducted under acidic conditions • Creates a blue color (similar to benzidine) • Reported as carcinogenic in rats in 1974, leading to its replacement by TMB Serology Chapter 11 & 12

  14. Catalytic Color Tests O-Tolidine (ortho-tolidine) Serology Chapter 11 & 12

  15. Catalytic Color Tests Tetramethylbenzidine (TMB) • Derivative of benzidine • Used in an acidic medium (acetic acid) • Resulting color change from green to blue/green • Hemastix (field test for blood) uses TMB and only requires distilled water and the questioned sample (yellow to blue/green) • Swabs with cometic materials may produce false positives Serology Chapter 11 & 12

  16. Catalytic Color Tests Tetramethylbenzidine (TMB) Serology Chapter 11 & 12

  17. Catalytic Color Tests Tetramethylbenzidine (TMB) Serology Chapter 11 & 12

  18. Catalytic Color Tests Leucomalachite Green (LMG) • Created in 1904 using the reduced form of the malachite green dye. • Produces a green color • Carried out in an acidic medium with hydrogen peroxide as the oxidizer Serology Chapter 11 & 12

  19. Catalytic Color Tests Leucomalachite Green (LMG) • Created in 1904 using the reduced form of the malachite green dye. • Produces a green color • Carried out in an acidic medium with hydrogen peroxide as the oxidizer Serology Chapter 11 & 12

  20. Catalytic Color Tests Tests using Chemiluminescence & Fluorescence • The observed results often enables one to determine the limits, shape and details of the original bloodstained area (and patterns if present) • By nature, they are potential sources of contamination of the blood • If the blood can be seen and collected, these probably should not be used • Luminol and Fluorescein are irritants but not carcinogens • Chemiluminescence is the process by which light is emitted as product of a chemical reaction • Fluorescence occurs when a chemical substance is exposed to a particular wavelength of light (usually a short wave like, UV light) and light energy is emitted at longer wavelengths Serology Chapter 11 & 12

  21. Catalytic Color Tests Tests using Chemiluminescence Serology Chapter 11 & 12

  22. Catalytic Color Tests Tests using Fluorescence Serology Chapter 11 & 12

  23. Catalytic Color Tests Luminol (3-aminophthalhydrazide) • Luminesces after oxidation in acid or alkaline (aqueous) solution. • The catalytic activity of the heme group accelerates the oxidation of the luminol • Produces a blue-white to yellow-green light if blood is present • Outlines & details are often visible up to 30 second before additional spraying is required • Doesn’t affect these tests: presumptive, confirmatory, species origin, ABO tests, RFLP and PCR tests • Interferes with several enzyme and protein genetic marker systems • Sensitivity: detects dilutions up to 1 in 10,000,000 • Results should be photographed b/c transient (temporary) • False positive with bleach (flash) Serology Chapter 11 & 12

  24. Catalytic Color Tests Luminol (3-aminophthalhydrazide) Serology Chapter 11 & 12

  25. Catalytic Color Tests Fluorescein • Prepared like phenolphthalein—reduced in an alkaline (basic) solution over zinc to fluorescin. • Fluorescin is applied to the sample and the heme group catalyzes the oxidation by hydrogen peroxide to make fluorescein. • When treated with UV light, it will fluoresce • Includes a commercial thickener which causes it to adhere to the surface (good for vertical surfaces). • Does not fluoresce with bleach • Shows no interference with STR testing of blood for DNA. Serology Chapter 11 & 12

  26. Catalytic Color Tests Fluorescein Serology Chapter 11 & 12

  27. Confirmatory Test for Blood (Crystal tests are often used) Teichmann Test • First described in 1853 • Heating dried blood in the presence of glacial acetic acid and a halide salt (usually chloride) to form a hematin derivative. Serology Chapter 11 & 12

  28. Confirmatory Test for Blood (Crystal tests are often used) Teichmann Test Serology Chapter 11 & 12

  29. Confirmatory Test for Blood (Crystal tests are often used) Takayama Test • First used in 1912 • Involves heating dried blood in the presence of pyridine, a glucose (reducing agent), and sodium hydroxide (basic) solution to form a pyridine derivative. Serology Chapter 11 & 12

  30. Confirmatory Test for Blood (Crystal tests are often used) Takayama Test Serology Chapter 11 & 12

  31. Confirmatory Test for Blood (Crystal tests are often used) Teichmann Test • First described in 1853 • Heating dried blood in the presence of glacial acetic acid and a halide salt (usually chloride) to form a hematin derivative. Takayama Test • First used in 1912 • Involves heating dried blood in the presence of pyridine, a glucose (reducing agent), and sodium hydroxide (basic) solution to form a pyridine derivative. Serology Chapter 11 & 12

  32. Species Determination in Bloodstains • Microscopy uses a visual comparison of the blood cell morphology (shape/appearance) Serology Chapter 11 & 12

  33. Species Determination in Bloodstains • Microscopy uses a visual comparison of the blood cell morphology (shape/appearance) • Protein analysis(3 different techniques are used.) • Immunoprecipitation reactions (most use) • If an animal is injected with a human serum protein, the animal’s immune system will recognize it as foreign and release produce antibodies against the antigen. • When the antibodies and antigen come in contact and usually a precipitation is formed (precipitin reaction) Serology Chapter 11 & 12

  34. Species Determination in Bloodstains Immunoprecipitation reactions Serology Chapter 11 & 12

  35. Species Determination in Bloodstains • Protein analysis (continued) • Electrophoresis—a technique in which charged molecules (i.e. proteins) are caused to migrate in an electric field in a suitable support medium under controlled conditions of temp, pH, voltage, and time. • Support media: starch gels, agarose gels, polyacrylamide gels • Positively charged molecules migrate toward the cathode (- electrode)& negatively charged molecules migrate toward the anode (+ electrode). • Bands are visualized by a stain or chemical rxns. • In FS, it is usually done in a gel on a glass plate w/ samples (i.e. blood soaked thread or stain extracts) are placed in the gel. Serology Chapter 11 & 12

  36. Species Determination in Bloodstains Electrophoresis Serology Chapter 11 & 12

  37. Species Determination in Bloodstains • Protein analysis (continued) • Isoelectric Focusing (IEF) - an electrophoretic method that takes advantage of the fact that at a certain pH, a protein in aqueous solution will exhibit a point of no net charge (isoelectric point - IEP). • The buffer in the gel controls the pH throughout the system • A pH gradient is set up in the gel w/ the low end at the anode. • When current is applied, the proteins migrate where they encounter their IEPs and form a band • The bands here can sometimes be more easily observed that with electrophoresis. Serology Chapter 11 & 12

  38. Species Determination in Bloodstains Isoelectric Focusing (IEF) Serology Chapter 11 & 12

  39. Species Determination in Bloodstains • Serum Protein Analysis- serum proteins are a large collections of proteins in the serum • Ring Precipitin Test • 2 liquids placed in a test tube: antiserum and extract of questioned bloodstain • As the anti-human serum comes in contact with human blood sample via diffusion, a small layer of a precipitate will form between these two liquids. • No reaction will occur if the blood is animal and used with anti-human serum. Serology Chapter 11 & 12

  40. Species Determination in Bloodstains Ring Precipitin Test Serology Chapter 11 & 12

  41. Species Determination in Bloodstains • Serum Protein Analysis- serum proteins are a large collections of proteins in the serum • Ring Precipitin Test • 2 liquids placed in a test tube: antiserum and extract of questioned bloodstain • As the anti-human serum comes in contact with human blood sample via diffusion, a small layer of a precipitate will form between these two liquids. • No reaction will occur if the blood is animal and used with anti-human serum. • Outerlony Double Diffusion Test • Carried out in a gel on a glass plate or petri dish • Wells are punched into agar, the antiserum is placed in the center well and the extracts are in the surrounding wells. • Immunoprecipitate lines form between the wells to show a positive response. Serology Chapter 11 & 12

  42. Species Determination in Bloodstains Outerlony Double Diffusion Test Serology Chapter 11 & 12

  43. Species Determination in Bloodstains Serology Chapter 11 & 12

  44. Species Determination in Bloodstains • Serum Protein Analysis(continued) • Crossed-Over Electrophoresis • Rows of opposing wells are cut in an agarose gel plate. • Once current is applied, the extract proteins (antigen) move toward the anode. • Antibodies move via electroendosmosis—cations and water of hydration move toward the cathode. • When the antibodies meet with the extract protein (antigen) precipitin bands form between the two rows of wells • Non-Serum Analysis includes using antihuman Hemoglobin • Hemoglobin is a protein in blood that is species-specific for immunological properties. Serology Chapter 11 & 12

  45. Genetic Markers in Blood Blood group- a group of antigens produced by allelic genes at a single locus and inherited independently of other genes.) Serology Chapter 11 & 12

  46. Genetic Markers in Blood Antigen-based Markers and Protein Markers • Antigen-Based Markers: Blood Groups • ABO system • Discovered by Landsteiner in 1900 • Types (A, B, AB, O) refer to antigens on the surface of the RBC • Antibodies for the antigens (anti-A, anti-B) are present in the plasma. • Agglutination would occur between anti-A serum and types A and AB blood • Most individuals (80%) secrete their ABO characteristics in other body fluids (they are called secretors). Serology Chapter 11 & 12

  47. Genetic Markers in Blood Antigen-based Markers and Protein Markers • Antigen-Based Markers: Blood Groups • ABO system Serology Chapter 11 & 12

  48. Genetic Markers in Blood Blood Types Serology Chapter 11 & 12

  49. Genetic Markers in Blood Antigen-based Markers and Protein Markers • Antigen-Based Markers: Blood Groups • ABO system • The Lewis (Le) System • Le antigens are absorbed from the plasma onto the surface of RBCs. • Provide secretor status (whether the ABO type can be determined from bodily fluids). • Rhesus (Rh) system (+ or -) • Discovered by Landsteiner and Wiener (1940) • Antigens are on the surface of the RBC membrane • Not present in non-blood body fluids • Natural Rh antibodies are not common in serum Serology Chapter 11 & 12

  50. Genetic Markers in Blood Antigen-based Markers and Protein Markers • Protein Markers: Blood Groups • Hemoglobin (Hb) • Major protein in RBCs • Phenotypes can be identified w/ electrophoresis or IEF • Haptoglobin (Hp) • Enzyme Markers • uses electrophoresis and IEF (Isoelectric Focusing) • Phosphoglucomutase (PGM) • Most well known enzyme • Found in many tissues of plants, animals, & microorganisms • In humans, it exists in significant concentrations in blood, semen & in small amounts in vaginal secretions & cervical mucus. • Can place within 10 subtype population groups (most discriminatory of all enzyme systems used in Forensic Science) Serology Chapter 11 & 12

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