Biological Evidence: Goals

1. Identify a stain/tissue

2. Identify species of origin

- is it human?

3. ‘Individualize’ it – i.e. get a DNA profile?

4. Compare it to a known sample

Serology and DNA Profiling

Serology – identification of body fluids

Blood, semen, saliva, urine, feces.

DNA profiling – genetic identification

Serology – is that stain a body fluid?

Presumptive Tests – could that stain be a body fluid?

Easy, quick, on-site tests

Low detection limits (i.e. you have to have a lot of

what you are testing for)

Not very specific (can get false positives)

Serology – is that stain a body fluid?

Confirmatory Tests – is that stain a body fluid?

Done in the lab (takes longer)

Very specific

High detection limit (i.e. do not have to have much of

what you are testing for)

Blood – how do we know it’s blood?

Components of Blood

Cellular Components

Erythrocytes (Red Blood

Cells)

Leukocytes (White Blood

Cells)

Platelets

Fluid (plasma) •Water

•Salts

•Proteins (albumin and

antibodies)

Presumptive Tests for Blood

Based on blood’s peroxidase activity

Peroxidase – an enzyme that catalyzes the reduction of

hydrogen peroxide into water and oxygen

The oxygen then reacts with a chemical to produce a

color of give off light

H2O2 H2O + [O] Heme

Presumptive Tests for Blood

Luminol

Tetra-methylbenzidine Test (TMB)

Reduced phenolphthalein (Kastle-Meyer) Test

Leuco-crystal violet

Luminol

Fluoresces if blood is present

Tetramethylbenzidine Test (TMB) 1. Wet a swab and rub on stain

2. Add 1-2 drops of benzidine reagent

3. If there is an immediate color change – false positive

4. If no color- add a drop of hydrogen peroxide

5. If test is positive a blue-green color will appear

Reduced Phenolphthalein Test (Kastle-Meyer)

Turns pink if positive for blood

Leuco-crystal Violet Test

Turns dark purple in the presence of blood

Remember these are presumptive tests…

Sources of false positives

Certain metals (corroded copper or nickel)

Oxidizing agents (bleach)

Plant peroxidases

Controls

Help determine if your test is valid

i.e. reagents are working, you did it properly

Positive control – are the reagents working?

Negative control – are the reagents contaminated?

Substrate control – does the substrate (e.g. sheet,

carpet, etc.) give the expected result?

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Biological Properties of Blood

• Main elements used in forensic labs:

– Red blood cells

– Serum proteins

• Red blood cells have structures called antigens on their

surfaces. They are grouped into classification systems

determined by their relationship to one another (blood

type).

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Biological Properties of Blood

• Serum proteins such as antibodies are used to test the

sample to confirm that it is blood.

• An antibody activates or destroys a specific antigen

which allows for particular reactions to occur when

specific groups of antigens and antibodies are mixed.

• These reactions allow for determination of blood type.

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Biological Properties of Blood

• The ABO group system for blood typing was first used in the early 1970’s to link blood to an individual.

• An individual that is type A has A antigens on their red blood cells.

• Type B has B antigens on their red blood cells.

• Type AB has both A and B antigens.

• Type O has neither A or B antigens.

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Biological Properties of Blood

• In addition to the A and B antigens there is also another

antigen, the Rh factor.

• Its presence is indicated by a positive sign and its

absence is indicated by a negative sign.

• This is appended after the A/B/O indicator.

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Blood Type Frequency

Type African

American Asian Caucasian Hispanic

O+ 47% 39% 37% 53%

O- 4% 1% 8% 4%

A+ 24% 27% 33% 29%

A- 2% 0.5% 7% 2%

B+ 18% 25% 9% 9%

B- 1% 0.4% 2% 1%

AB+ 4% 7% 3% 2%

AB- 0.3% 0.1% 1% 0.2%

Confirmatory Tests - Blood Identification

Based on the presence of hemoglobin

It is a protein found in red blood cells

Red blood cells are anucleated (they do not have

a nucleus) and are biconcave disks

Confirmatory Tests for Blood

Microscope tests Takayama test

Teichmann test

Takayama Test

Microscopic test for hemoglobin

Red/pink leaf-like crystals are formed

Teichmann Test

Red/brown crystals form in the presence of blood

Confirmatory Tests for Blood

Immunological Tests

Ouchterlony

ABAcard - Hematrace

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Blood Origin Testing

• The Ouchterlony test will determine whether the stain is

of human or animal origin.

• The test uses an antigen that is designed to destroy

human blood.

• This test takes advantage of the fact that antigens and

antibodies naturally move toward each other on a gel

plate.

Ouchterlony (Diffusion) test

The extracted blood stain (antigen) and the human

antiserum (antibody) are placed in separate holes

opposite each other on the gel. If the blood is

human, a line of precipitation forms where the

antigens and antibodies meet.

ABAcard - Hematrace Sample (antigen) is placed in sample well

Antibodies are found on the test strip

If the antigen and antibody agglutinate (clump) you get

a pink line on the test strip…indicated the sample

contains blood!!

There is also a positive control that should produce a

line if the test worked properly

Next Steps…

1. Read the investigation background

information

2. Work on the pre-lab questions