Forensic Biology by Richard Li

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Forensic Biologyby Richard Li

• Chapter 6 Identification of Blood

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Biological Properties of Blood

• Normal blood volume is 8 of body weight
• 5-8 pints for average adults
• Fatal if lose 40 or more of blood volume
• Two portions
• Fluid portion
• Plasma- fluid portion of blood that can clot
• Serum- remaining fluid after clot is removed
• Cellular Portion
• Red blood cells (erythrocytes hemoglobin No
  DNA)
• White blood cells (Leucocytes fight infection
  DNA present)
• Platelets (Thrombocytes blood clotting No DNA)

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Plasma and serum
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Two Types of Assays

• Presumptive
• Very sensitive, fast, and easy to perform
• Depend on oxidation-reduction reaction catalyzed
  by heme group of blood
• Result in color change or release of photon by
  chemiluminescence or fluorescence
• Confirmatory
• Need a lab to perform greater specificity
• Depend on crystal formation, primary serological
  reactions, spectrophotometry, or RNA-based assays

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Presumptive Assays

• Detect traces of blood
• Oxidation-reduction reaction catalyzed by heme
• Oxidation- lose electron
• Hydrogen peroxide used as an oxidant
• E.g. K-M test described in Lecture 5
• Reduction- gain electron
• Tests result in
• Change of color (colorimetric assays)
• Release of photons
• Chemiluminescence or fluorescence

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Presumptive Assays

• Colorimetric Assays
• Phenolphthalein (Kastle-Meyer)
• -Introduced in Lecture 5
• We will perform this test in lab
• Leucomalachite green (LMG)
• Colorless in reduced state green when oxidized
• Benzadine and Derivatives
• Benzadine colorless in reduced state dark blue
  when oxidized
• Tetramethylbenzidine (TMB) colorless in reduced
  state blue-green when oxidized

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Presumptive Assays

• Chemiluminescent assays
• Light is emitted as a product of the chemical
  reaction
• Luminol- emits light blue color
• Useful when blood has been cleaned up
• Performed in darkness
• Can detect small traces of blood
• Can detect patterns
• May dilute sample

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Presumptive Assays

• False positive results with luminol
• Bleach
• Plants
• Copper and copper-containing alloys
• Feces
• Urine (if blood is present, including menstrual
  blood)

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Presumptive Assays

• Fluorescence assays
• Absorption of UV or visible radiation kicks
  electrons up to a higher orbitial (higher energy
  state)
• When electrons drop down to original ground
  state
• Energy released is transferred to vibrational and
  rotational energy of molecular bonds (most
  common)
• Energy released as a photon of lower energy
  wavelength (less common) fluorescence

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Presumptive Assays

• Fluorescin
• When oxidized by the peroxidase activity of heme
  in the presence of hydrogen peroxide, will
  fluoresce
• Must be exposed to wavelength 425-485 nm
  (blue-purple) from an ALS
• Emits yellowish-green color (longer wavelength)

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Confirmatory Assays

• Microcrystal assays
• Hemochromagen crystal assay (Takayama)
• Hematin crystal assay (Teichmann)
• Method
• Small amount of putative blood added to a slide
• Chemical solution added
• Slide heated to form crystals (if blood present)
• Crystals viewed under the microscope

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Positive Takayama confirmatory test for blood
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Confirmatory Assays

• Other
• Chromatographic and electrophoretic methods
• Identify human hemoglobin based on mobility on
  columns or in gels
• Spectrophotometric methods
• Identify human hemoglobin based on light spectra
  absorbed by hemoglobin and its derivatives
• Immunological methods
• Anti-human hemoglobin antibodies (see Lecture 5)
• RNA-based methods
• Assay for presence of mRNAs found only in human
  blood