2. Blood Stain Evidence
Blood is a type of biological evidence that can connect a suspect to a victim or object.
White blood cells contain DNA which may be used to identify an individual from a blood sample.
Lab testing can differentiate between human and animal blood by protein or DNA analysis.

3. Biological Properties of Blood
Blood delivers nutrients and oxygen to the cells and transports waste products away from cells.
Red blood cells
carry oxygen
and carbon
dioxide.

4. Biological Properties of Blood
White blood cells come in many varieties that fight off infection in different ways, such as by generating antibodies or breaking down intruder cells.
They also contain DNA.

5. Biological Properties of Blood
Platelets assist in clotting.

6. White blood cells play an important role in immune response and antibody production.
Platelets initiate and participate in clotting.
Red blood cells use hemoglobin to carry oxygen. They also remove carbon dioxide.
Plasma contains carbohydrates, lipids, hormones, inorganic salts, proteins (such as antibodies), and clotting elements.

7. 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).

8. 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.

9. 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.

10. 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.

11. Blood Type Frequency Type African American Asian Caucasian Hispanic O+
47%
39%
37%
53% O- 4% 1% 8% A+
24%
27%
33%
29% A- 2%
0.5% 7% B+
18%
25% 9% B-
0.4%
AB+ 3%
AB-
0.3%
0.1%
0.2%
Source: American Red Cross

12. Blood Origin Testing
Two categories of tests are available for investigators.
Presumptive tests are used to quickly determine whether an unknown substance is blood in the field. They are prone to false positives.
Confirmatory tests must be done at a lab and provide much more accurate results.

13. Blood Origin Testing Two categories of presumptive tests are:
Color changing tests
Luminol, Benzidine, Phenolphtalein, TMB / Hemastix
Glowing reaction tests
fluorescein

14. Phenolphthalein Presumptive Test
This test is better known as the Kastle-Meyer test.
In a positive reaction the reduced phenolphthalein will turn bright pink because the phenolphthalein is oxidized by hydrogen peroxide in the presence of hemoglobin.
Phenolphthalein reagents give false positives when vegetable materials are present.

15. TMB / Hemastix Presumptive Test
This test is performed with commercial plastic strips with a treated filter at one end.
A test swab is moistened with water and placed in contact with the stain. The swab is then placed onto the tip of the strip.
If blood is present the hemastix strip will turn green.

16. Luminol Presumptive Test
Luminol tests for latent bloodstains. It can detect blood that has been diluted up to 1 in 10,000,000.
When luminol and hydrogen peroxide are applied to the bloodstain the luminol produces a blue-white to yellow-green light.
Luminol can affect some testing processes but does not affect most blood typing or DNA analysis.
It produces a false positive with plant enzymes, oxidizers, metals, and chlorine.

17. Fluorescein Presumptive Test
Fluorescein tests for latent bloodstains by fluorescing under ultraviolet light.
It can even detect blood stains that have been cleaned with solvents such as bleach.
Fluorescein can also be applied to vertical surfaces, unlike luminol.
Copper and hypochlorite will cause false positives.

18. Presumptive Test Summary
Indication of Positive
Situation Used
False Positives
Phenolphtalein
Bright pink color
on visible stains
Vegetable material (potatoes and horseradish)
Tetramethylbenzidine (TMB) / Hemastix
Green to blue-green color
Oxidizing agents, catalyst, vegetable peroxidase, cosmetics
Luminol
Blue-white to yellow-green light
latent blood
Plant enzymes, oxidizing agents, metals, chlorine
Fluorescein
Fluoresce with UV light source
latent blood, vertical surface
Copper, hypochlorite

19. Blood Origin Testing
Once a stain has been characterized as blood through a presumptive test it is collected and stored for confirmatory testing.
The two most common confirmatory tests are the Takayama and Trichmann tests.

20. Takayama Confirmatory Test
The Takayama test is performed by adding an alkaline solution with a specific structure of hemoglobin to the stain on a microscope slide.
If blood is present, pink crystals will be observed as the slide is heated.

21. Trichmann Confirmatory Test
The Trichmann test is performed by adding a small amount of chloride containing acetic acid to the blood sample on a microscope slide.
Small crystals form on the slide as it is heated if blood is present.
Once the stain is confirmed as blood, it must be tested to determine if the source is human or animal.

22. Blood Origin Testing
The precipitin test will determine whether the stain is of human or animal origin.
The precipitin 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.
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.

23. Blood Properties
Since the 1990’s DNA found in the blood and other bodily fluids has been used to identify an individual, like a genetic fingerprint.

24. Blood Properties
DNA is unique to the individual, whereas blood typing can only eliminate an individual as a suspect. Blood type is class evidence and DNA is individual evidence.
The process of genetic fingerprinting is called DNA profiling or DNA typing.

25. Evidence Handling
It is crucial that bloodstains found at a crime scene are documented, collected, tested, preserved, and analyzed correctly.
Failure to do so will weaken or destroy potential evidence.

26. Evidence Handling
Proper evidence packaging is crucial to protect against:
Loss
Contamination
Deterioration
Cross-transfer
Suspect / scene / item / victim
Biohazards

27. Evidence Labeling Labeling Evidence description
Source (location, agency)
Chain of custody
Case and item numbers
Health hazards
Storage conditions (room temp, frozen, refrigerated)

28. Evidence Labeling
Biological material must be dried before packaging to prevent deterioration.
Dry at room temperature, do not use a heat gun, fan, or hair dryer.
Use paper containers for biological evidence.
Seal evidence with tape across the bag, initial and date over or under seal.

29. Evidence Packaging
Work on clean surfaces. Wear gloves, a mask, and eye protection – don’t contaminate with DNA.
Change gloves after handling each sample to eliminate cross-contamination.
Do not blow on samples to make them dry faster.
Do not touch blood samples with any non-disposable items.
Clean tools with 10% bleach solution and dry thoroughly.

30. Evidence Packaging Keep all stains separated.
Separate stains on the same garment with sheets of paper.
Package multiple garments in different bags.
Do not use any packaging that limits air exposure (tubes, parafilm, plastic baggies)

31. Evidence Storage
Biological evidence should be stored dry to prevent bacteria or mold growth.
Wet evidence should be stored frozen.
Some portion of samples should be preserved from destructive testing.