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Chapter 6: Identification of Blood. Forensic Biology by Richard Li. 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

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biological properties of blood
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)

Hemoglobin: Transports oxygen from lungs to body tissues; helps with transport of CO2 out of the tissues and back to the lungs


Prosthetic group in hemoglobin; Binds oxygen; also has peroxidase activity

two types of assays
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
presumptive assays
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
presumptive assays7
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
presumptive assays8
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
presumptive assays9
Presumptive Assays
  • False positive results with luminol:
    • Bleach
    • Plants
    • Copper and copper-containing alloys
    • Feces
    • Urine (if blood is present, including menstrual blood)
presumptive assays10
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
presumptive assays12
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)

Emits (fluoresces) light here

Absorbs light here

confirmatory assays
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
confirmatory assays15
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