Exam 2 - Biological Evidence

Question 1

Biological Evidence

Answer 1

Human, Animal, Others (vegetation, bacteria, viruses)

Question 2

Locating biological evidence

Answer 2

Visual (unaided eye, intense white light, HITWLS, UV illumination).
Tactile (touch).
Chemical tests.
Enzymatic tests.
Immunological tests.

Question 3

Blood

Answer 3

Cellular material;
- Red blood cells (erythrocytes) - no nucleus, no DNA.
- White blood cells (leukocytes) - contain nuclear and mitochondrial DNA.
- Platelets (thrombocytes) - contain only mitochondrial DNA (mtDNA)
- Plasma is 95% water, antibodies, vitamins, minerals, and 500+ proteins
[Serum is plasma without clotting proteins].

Question 4

Red Blood Cells

Answer 4

Contain hemoglobin:
- GLOBIN with 2 alpha subunits and 2 beta subunits (polypeptide chains]
- 4 HEME groups
Heme: Ferroprotoporphyrin
- Porphyrin is a class of pigments (including heme and chlorophyll) whose molecules contain a flat ring of 4 heterocyclic rings with Fe as central atom.
Ferro = Fe^+2 = Oxyhemoglobin (Fe^+2 attached to O=O)
Ferri = Fe^+3 = Methemoglobin (Fe^+3 attached to H2O or OH-)
[oxyHb –> metHb]

Question 5

Chemical tests for blood

Answer 5

Catalytic or presumptive tests 
- Heme possesses peroxidase/catalase activity 
Mechanism:
2 H2O2 (+ Heme) --> O2 + 
2 H2O 
[Oxygen cleaved from peroxide]
O2 + indicator (reduced) 
--> indicator (oxidized)
[Oxygen reacts with indicator - colorless to colored product]

Question 6

Chemical tests for blood

Answer 6

Catalytic tests perform better when blood has dried (older deposits) and not fresh
- Generalized electron transfer mechanism
H2O2 + 2 e- (donated from Heme) + 2H+ –> 2 H2O

indicator (reduced) - 2 e- (Heme is added)
–> indicator (oxidized)
[Phenolphthalin gives 2 electrons back to heme)

Question 7

Catalytic tests (very sensitive) for blood

Answer 7

Test - Positive Result

1. Phenolphthalin (Kastle-Meyer) - Pink color
2. Leucomalachite green (LMG) - Green color
3. Leucocrystal violet (LCV) - Violet volor
4. 3,3’,5,5’-tetramethylbenzidine (TMB) - Blueish-green or green
5. Luminol (BLUESTAR) - Blueish-violet chemiluminescence (emission: 425 and 485 nm)
6. Fluorescin - Green fluorescence under UV light (absorption: 490 nm emission: 520 nm)
7. 0-Tolidine [carcinogenic] - Greenish-blue or blue
8. Benzidine [carcinogenic] - blue color
9. Hydrogen peroxide (H2O2) - production of O2 (effervescence)

Question 8

Catalytic Testing Methods

Answer 8

- One-step application 
Combined reagent and hydrogen peroxide.
Many false positives (indicates blood when not present).
May be used for contrast enhancement and searching for latent blood.
- Two-step method
Eliminate false positives.
Fewer non-specific reactions.
- Multiple tests
Basic (KM) and acidic (LMG) tests.

Question 9

KM Reagent

Answer 9

Components

• Phenolphthalin; indicator
• Potassium hydroxide; (1) basic pH (2) phenolphthalein pink in basic pH.
• Ethanol (ethyl alcohol, EtOH); solvent enhances reaction
• Zinc dust; slows spontaneous oxidation of phenolphthalin.

Question 10

KM Reagent

Answer 10

Process
- KM reagent added to stain; color change indicates presence oxidizer
- 3% H2O2 added; color change to pink indicates positive result, blood may be present
Reaction:
- Heme catalyzes production of O2 from peroxide
- O2 reacts with phenolphthalin to yield it
- Phenolphthalin pink in basic pH

Question 11

False Positives

Answer 11

• Vegetable peroxidases (horseradish)
• Metallic salts
• Oxidizing agents
• Strong acids
• Bleach
• Copper and copper alloys (brass)

Question 12

False Negatives

Answer 12

• No indication of blood when present (some substances can inhibit reaction, blood is below the detectable limit of test.
• Rare
• Difficult to detect

Question 13

Blood

Answer 13

Absorbs UV and VIS, much less in near IR
Absorbs maximally at 415 nm (oxyHb) and 433 nm (deoxyHb)
- if there is a bloody fingerprint on a yellow wall, a 415 nm illumination with a yellow barrier filter can be used.

Question 14

Catalytic Test Sensitivity

Answer 14

• KM
  1 - 10 ppm blood (1 drop blood in 1.3-13 gallons of water)
• LMG and LCV
  10 - 100 ppm blood (1 drop in 0.13-1.3 gallons of water)
• Luminol
  0.2 - 1 ppm blood (1 drop blood in 13-66 gallons of water)

Question 15

Confirmatory Tests for Blood

Answer 15

• Microcrystal; crystal formation
• Chromatographic; paper or TLC (detect Hb)
• Spectral (UV-VIS spectrophotometry - detect Hb)
• Immunological; reaction with anti-human hemoglobin serum
• mRNA profiling; multiplex reverse transcription-PCR assay for body fluid-specific genes