Forensic Biologyby Richard Li, with additions and edits by Ruth Ballard

Lecture 5: Identification of Blood

Outline

Biological properties of bloodDetection of blood

Presumptive tests for blood▪ Colorimetric▪ Chemiluminescent▪ Fluorescent▪ immunological

Confirmatory tests for bloodABO typing

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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)

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Plasma and serum

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

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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)

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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

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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

Detection of Blood

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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

Detection of Blood

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!

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Presumptive Assays

Method: Spray area of

suspected stain with luminol

Turn off lights Look for blue

fluorescence No ALS required

Beware false positives! People v Dean

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Lights on Lights off

13Forensic Biology by Richard Li

Presumptive Assays

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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 425-485 nm (blue-purple) from an ALS

▪ Emits yellowish-green color (longer wavelength)

Presumptive Assays

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Immunological Example Seratec HemDirect

▪ Similar to RSID-semen assay for human semenogelin

▪ Targets human hemoglobin▪ We will perform this test in lab

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 16

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Positive Takayama confirmatory test for blood

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

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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

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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)

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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”

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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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