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Lecture 5: Identification of Blood

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1 Lecture 5: Identification of Blood
Forensic Biology by Richard Li, with additions and edits by Ruth Ballard

2 Outline Biological properties of blood Detection of blood ABO typing
Presumptive tests for blood Colorimetric Chemiluminescent Fluorescent immunological Confirmatory tests for blood ABO typing

3 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)

4 Plasma and serum

5 Detection of Blood Presumptive assays
Several methods; all based on the oxidation-reduction reaction catalyzed by heme Heme is found in hemoglobin Peroxidase (catalase) activity

6 Detection of Blood In oxidation-reduction reactions:
One molecule loses an electron (is oxidized) Another gains that electron (is reduced) Transfer is often via a hydrogen atom Reduced atoms gain hydrogen atoms The catalase activity in heme oxidizes (strips electrons off) chemicals used in the assays This results in a color change (colorimetric assay) or the release of light (chemiluminesce or fluorescence)

7 Detection of Blood Colorimetric presumptive assays
Example: Phenolphthalein Peroxidases break down hydrogen peroxide to water and oxygen free radicals (O-) Oxygen free radicals are strong oxidants and strip hydrogens off phenolphthalein (Kastle-Meyer reagent) The reduced form of phenolphthalin is colorless but the oxidized form is bright pink Not the same dark red color of blood 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


9 Detection of Blood Method Moisten Q-tip swab in distilled water
Lightly touch suspected blood stain with tip of Q-tip Add one drop K-M reagent Add one drop hydrogen peroxide Look for fast color change to bright pink

10 Detection of Blood Limitations False positive reactions
Strong oxidizing agents (e.g. prolonged exposure to air) Any substance with natural peroxidase activity Some bacteria and plant materials Rusty metal Not species-specific Reacts with blood from any animal Results can be misleading in forensic casework Will detect blood in urine, saliva, and other body fluids if trace amounts are present

11 Detection of Blood Chemiluminescent assays
Light is emitted as a product of the chemical reaction Example: Luminol Peroxidase activity of heme acts on the luminol chemical Reaction causes luminol to fluoresce Useful when stain has been cleaned up (not visible) Can detect blood spatter patterns Really cool “CSI” test!

12 Presumptive Assays Method: Beware false positives!
Spray area of suspected stain with luminol Turn off lights Look for blue fluorescence No ALS required Beware false positives! People v Dean Lights on Lights off

13 Forensic Biology by Richard Li

14 Presumptive Assays Fluorescent assays Example: 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)

15 Presumptive Assays Immunological Example Seratec HemDirect
Similar to RSID-semen assay for human semenogelin Targets human hemoglobin We will perform this test in lab

16 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

17 Positive Takayama confirmatory test for blood

18 ABO Typing ABO System A, B, AB, O Type A: have A antigen
Type B: have B antigen Type AB: both A and B antigens Type O: neither A nor B antigens Can be found in many tissues other than blood Saliva, semen are most important for forensics

19 ABO Typing System controlled by three genes:
FUT1 encodes a fucosyltransferase Adds H antigen to glycolipid on surface of red blood cells Almost everyone is homozygous for normal form of FUT1 Rare “Bombay phenotype” does make the enzyme FUT2 encodes a related fucosyltransferase Adds H antigen to glycoprotein on surface of cells of other body tissues 80% of population has at least one functional FUT2

20 ABO Typing Human ABO locus (9q34.2) Encodes galactosyltransferase
Adds a second sugar group onto H antigen ABO gene has three alleles: O allele = null (non-functional) A allele = A-transferase (adds N-acetylgalactosamine to H antigen) B allele = B-transferase (adds galactose to H antigen)


22 ABO Typing Secretors and non-secretors
Almost everyone has a functional copy of FUT1 ABO type expressed in blood 80% have functional copy of FUT2 ABO type also expressed in other body tissues E.g semen, saliva 20% do not have a functional copy of FUT2 Homozygous for a nonsense mutation in FUT2 resulting in a truncated protein “Non-secretors”

23 ABO Typing Non-secretors caused problems in early forensic serology
ABO type could not be detected in semen or saliva stains If semen or saliva stain tested “O” Assailant could be a non-secretor A, B, AB, or O blood type possible Assailant could be a Type O secretor

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