chapter 6: identification of blood. normal blood volume is 8% of body weight ▪ = 5-8 pints for...

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)

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Plasma and serum

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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

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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 electronTests result in:

Change of color (colorimetric assays) Release of photons▪ Chemiluminescence or fluorescence

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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 oxidized7

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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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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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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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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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

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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

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