Chapter 6: Identification 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) 2

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

 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 5

 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 6

 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 7

 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 8

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

 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 10

11

 Fluorescin ▪ When oxidized by the peroxidase activity of heme in the presence of hydrogen peroxide, will fluoresce ▪ Must be exposed to wavelength nm (blue-purple) from an ALS ▪ Emits yellowish-green color (longer wavelength) 12 Absorbs light here Emits (fluoresces) light here

 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 13

14 Positive Takayama confirmatory test for blood

 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 15