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