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

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

2. Outline • Biological properties of blood • Detection of blood • 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: • 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 425-485 nm (blue-purple) from an ALS • Emits yellowish-green color (longer wavelength)

15. Presumptive Assays • Immunological • Example SeratecHemDirect • 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