Forensic Biology by Richard Li

1. Chapter 6: Identification of Blood Forensic Biologyby Richard Li

2. 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)

3. Plasma and serum

4. Hemoglobin: Transports oxygen from lungs to body tissues; helps with transport of CO2 out of the tissues and back to the lungs Heme: Prosthetic group in hemoglobin; Binds oxygen; also has peroxidase activity

5. 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

6. 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

7. 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

8. 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

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

10. 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

12. 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

13. 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

14. Positive Takayama confirmatory test for blood

15. 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