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Forensic Serology, Biology, and DNA

Forensic Serology, Biology, and DNA. Lecture 4. Forensic Serology. Serology = the study of antigen and antibody reactions A, B, AB, O blood typing Rh+/- Karl Landsteiner (1901). Plasma (55%) Water (93%) Proteins (7%) Fibrinogen Albumins, Globulins, etc. Electrolytes Gases

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Forensic Serology, Biology, and DNA

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  1. Forensic Serology, Biology, and DNA Lecture 4

  2. Forensic Serology • Serology = the study of antigen and antibody reactions • A, B, AB, O blood typing • Rh+/- • Karl Landsteiner (1901)

  3. Plasma (55%) Water (93%) Proteins (7%) Fibrinogen Albumins, Globulins, etc. Electrolytes Gases Nutrients Lipids Waste Hormones Hematocrit (45%) Erythrocytes (99+%) Leukocytes Platelets Whole Blood

  4. Plasma (55%) Water (93%) Proteins (7%) Fibrinogen Albumins, Globulins, etc. Electrolytes Gases Nutrients Lipids Waste Hormones Hematocrit (45%) Erythrocytes (99+%) Leukocytes Platelets Serum

  5. Plasma (55%) Water (93%) Proteins (7%) Fibrinogen Albumins, Globulins, etc. Electrolytes Gases Nutrients Lipids Waste Hormones Serum

  6. Plasma (55%) Water (93%) Proteins (7%) Fibrinogen Albumins, Globulins, etc. Electrolytes Gases Nutrients Lipids Waste Hormones Serum

  7. Erythrocytes • Red blood cells • Hemoglobin (Hb) • Concave disk • High surface to volume ratio • Facilitates diffusion of gases • Proteins on surface (antigens)

  8. Hemoglobin • Quarternary protein • Heme groups (4) • Iron (Fe) binds one molecule of O2

  9. Carbon Monoxide • Carbon monoxide (CO) also will bind (245x) • Binds readily; leaves slowly • Anemic hypoxia

  10. CO Poisoning

  11. Antigens • Millions on red blood cells • A, B, and D antigens

  12. Antibodies • Highly specific binding proteins • Found in serum • Anti-A, Anti-B, Anti-D • Antiserum • Bivalent binding sites • Agglutination

  13. Quick Summary Remember: Antigen on RBC Antibody in serum

  14. Forensic Serology • Stain (diluted in distilled water) • Add anti-A serum (+/-) • Add anti-B serum (+/-) • Add anti-D serum (+/-)

  15. Forensic Serology • Stain (diluted in distilled water) • Add anti-A serum (+/-) = + • Add anti-B serum (+/-) = - • Add anti-D serum (+/-) = + Result = Antigen A and D = A, Rh+

  16. Forensic Characterization of Blood • Is it blood? • Is it human blood? • Can it be associated with blood from an individual?

  17. Is it Blood? • Hemoglobin: • Presumptive tests • Color tests (oxidation reactions) • Benzidine (Adler) color test: blue = + • Phenolphthalein (Kastle-Meyer) test: pink = + • O-Tolidine (deriv. of benzidine): blue = + • TMB (tetramethyl benzidine): blue = + • LMG (leucomalachite green): green = +

  18. Is it Blood? • Hemoglobin: • Presumptive tests (Chemiluminescence/Fluorescence) • Oxidation reactions (heme) • Luminol: blue/green luminescence in the dark • Sodium Bicarbonate/carbonate mix • Fades within 30 seconds • Dilutes stain, follow up with phenolph., DNA? • VERY SENSITIVE (1:5 mil) • Fluorescein: 425-485 nm UV light (450 nm) • Fluorescin to Fluorescein, with H2O2 • No DNA interference

  19. Is it Blood? • Confirmatory tests • Crystal tests • DNA tests • BCA: phenolphthalein (& luminol)

  20. Is it Human? • Precipitin test • Rabbits injected with human blood • Rabbits form anti-human antibodies (serum) • Anti-human serum added to suspected human blood • Ring Precipitin Test: test tube precipitate ring • Ouchterlony Double Diffusion Test: gel plate “wells” • Electrophoresis • Other species (from rabbits)

  21. Associate with an Individual • ABO typing • Blood enzymes and proteins • PGM (phosphoglucomutase) (1, 2-1, 2) • Hp, Hb, etc. each with varying frequency • Frequency (0.1 x 0.43 O type) = .043

  22. DNA • DNA profile or DNA typing • Deoxyribonucleic acid • Cells contain chromosomes (nucleated) • Chromosomes have genes • Genes are DNA strands • DNA double helix strands • Nucleotides (4) are basic molecules

  23. Base Pairing • Nucleotides (bases) • Adenine (A) • Guanine (G) • Thymine (T) • Cytosine (C) • A = T • G = C • 100 million b.p. on avg. chromosome

  24. DNA Typing • RFLP (Restriction Fragment Length Polymorphisms) • PCR (Polymerase Chain Reaction) • STR (Short Tandem Repeats)

  25. Restriction Fragment Length Polymorphisms (RFLP) • Enzyme added to cut DNA in fragments • Length of fragments will vary • Separated (by weight) electrophoretically • Treated with radioactive tags • Visualized with X-ray photography • Outdated, long, not as highly discrim., etc.

  26. Polymerase Chain Reaction (PCR) • “Grow” new DNA • DNA replication • Primer (known sequence) added • Cycles (32 cycles, billions of strands) • 20-30 cycles (each about 2 minutes) • 1/50th of RFLP amount (nanogram) • Known sequences of primers allow for typing

  27. Short Tandem Repeats (STR) • Loci = locations on the chromosome • Loci have repeating segments • Very short (3-7 bases / 400 b.p. strand) • Frequency of repeat strands: • Type (13 commonly used) • Length • Number of repeats • Numbers generated (2 = 1 from each parent) • Amelogenin gene (found on X and Y)

  28. Mitochondrial DNA • DNA in nucleus or mitochondria • Nuclear = chromosome = both parents • Mitochondria = only maternal DNA • BUT! More copies…higher sensitivity • All maternal descendants have the genes • Hair, teeth, charred remains, nails, bones • Application: • PCR • Sequencing (base pair order) • Slow, costly, not frequently done

  29. DNA databases • CODIS: Combined DNA Index System • 13 STR database • Kits (Pro-Filer Plus, Co-Filer, etc.) • Warrants (John Doe)

  30. Other Considerations • Degradation of DNA • Contamination of DNA • Chemicals • Dilution • Mixed profiles • Controls • Commercial kits (primers, loci, frequency) • Population differences • Statistical identification

  31. Forensic Characterization of Semen • Semen: seminal fluid + spermatazoa • Fluid • Nutrients, buffers, mucous, proteins, sugars • Acid phosphatase (400x greater in semen) • Sperm • Tail (flagellum) and head (nucleus) • Morphology • Count

  32. Forensic Characterization of Semen • Presumptive test • Acid phosphatase • Sodium alpha naphthylphosphate + Fast Blue B dye • Swab area, apply test • Blue = + for AP • Sensitivity 1/500 dilution • Confirmatory test • PSA (prostate specific antigen) test • Apply anti-PSA serum to stain • Presence of sperm cells

  33. Other Biological Issues • Secretors (ABO antigens in other fluids) • Saliva (amylase tests) • 2 loci (AMY1, AMY2) • Sweat also • Urine (urea and creatinine) • Feces (bile byproducts, cells, blood)

  34. Obtaining Samples • Sexual Assault Evidence Kits • Swabs, blood, hair • Buccal swabs (knowns) • Cheek cells • Bloody evidence • DRY!!! • Packaged separately

  35. Hair Analysis • Traditionally: microscopy • Occasionally: root with blood or cells • Race, body origin • Now: DNA • Typing from skin cell, blood (PCR, STR) • From hair cells (mitochondrial)

  36. Is it Human Hair? • Scales (cuticle) • Cortex • Medulla

  37. human non-human

  38. Medulla • Types: • Absent, fragmented, interrupted, continuous • Humans (head hair) • Cylindrical • Absent or interrupted very common • Exception: Asians (continuous) • Medullary index • Ratio of: • Diameter of medulla • Diameter of hair shaft • In humans, typically .33 • In most other animals, .50 or greater

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