Forensic Serology, Biology, and DNA

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
Whole Blood

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

• Hematocrit (45)
• Erythrocytes (99)
• Leukocytes
• Platelets

4
Serum

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

• Hematocrit (45)
• Erythrocytes (99)
• Leukocytes
• Platelets

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Serum

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

6
Serum

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

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
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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
Antigen on RBC
Remember
Antibody in serum
14
Forensic Serology

• Stain (diluted in distilled water)
• Add anti-A serum (/-)
• Add anti-B serum (/-)
• Add anti-D serum (/-)

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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?

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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

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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 (15 mil)
• Fluorescein 425-485 nm UV light (450 nm)
• Fluorescin to Fluorescein, with H2O2
• No DNA interference

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Is it Blood?

• Confirmatory tests
• Crystal tests
• DNA tests
• BCA phenolphthalein ( luminol)

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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)

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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

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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)

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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

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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 copieshigher 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)

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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

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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)

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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)

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Is it Human Hair?

• Scales (cuticle)
• Cortex
• Medulla

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human
non-human
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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