1. Identification of Biological Fluids and Stains

2. Summary
Semen
Saliva
Urine
Feces
Vomit

3. Identification of Semen
Why is semen important in an investigation?
Evidence in sexual assault cases
Can help prove a crime was committed
Can identify the perpetrator

4. Identification of Semen
What is semen?

5. Identification of Semen
What is semen?

6. Identification of Semen
What is semen?
Semifluid mixture of:
Cells
Enzymes
Other organic and inorganic materials
Sperm cells are most important component as these are specific to semen.

7. Identification of Semen
What is semen?
Ejaculate volume = 2 – 6 mL
100 – 150 million sperm cells per mL
Sperm count may be much lower
Disease
Genetics
Drugs/Alcohol
Vasectomy

8. Identification of Semen
Sperm Cells

9. Identification of Semen
Sperm Cells
Sperm cell = spermatozoan
Head contains DNA
Tail (flagella) for movement
Acrosome – structure at front of head
Contains enzymes to penetrate egg

10. Identification of Semen
Searching for Semen Stains
May be on clothing, skin, bedding, etc.
Visual inspection; yellowish-white
Alternative light source: look for fluorescence

11. Identification of Semen
Presumptive Tests
Test for seminal acid phosphatase (SAP)

12. Identification of Semen
Seminal Acid Phosphatase
Acid phosphatases are a class of enzymes found in various living tissues
Mammalian liver to cauliflower stem juice
Semen contains very high levels of seminal acid phosphatase
Levels high until 40; gradual decrease
Levels not relation to sperm count

13. Identification of Semen
Seminal Acid Phosphatase
Brentamine Fast Blue Test
is the test for SAP
Will react with semen diluted 500x

14. Identification of Semen
Seminal Acid Phosphatase
Method (Brentamine Fast Blue Test):
Substrate
Chromogen (color changing reagent)
Seminal acid phosphatase (in semen)
SAP reacts with substrate.
Product of this reaction reacts with the chromogen (causes color change)

15. Identification of Semen
Seminal Acid Phosphatase
Method for Stain (Brentamine Fast Blue Test):
Moist swab or filter paper is rubbed over stain
Reagent containing substrate and chromogen is dropped on swab/paper

16. Identification of Semen
Seminal Acid Phosphatase
Method for Stain (Brentamine Fast Blue Test):
Moist swab or filter paper is rubbed over stain
Reagent containing substrate and chromogen is dropped on swab/paper
Positive reaction is a purple color

17. Identification of Semen
Seminal Acid Phosphatase
Method for Swab (Brentamine Fast Blue Test):
Moist filter paper is rubbed over swab
Reagent containing substrate and chromogen is dropped on filter paper
Positive reaction is a purple color
Swabs of orifices and skin are often collected in sexual assault cases.

18. Identification of Semen
Seminal Acid Phosphatase
Method for Large Areas (Brentamine Fast Blue):
Lay large strips of filter paper over area
Mark paper and item so paper can be replaced after test
Spray with water and firmly press
Remove and spray paper with reagent
Positive reaction is a purple color

19. Identification of Semen
Seminal Acid Phosphatase
Fresh stains will produce a strong color change reaction.
Old stains may produce a weak reaction
Other materials (urine, vaginal secretions, perspiration) may produce weak reactions
Experience of analyst will determine how to proceed.

20. Identification of Semen
Confirmatory Tests
Christmas Tree Stain (of sperm cells)
Prostate-Specific Antigen (PSA)

21. Identification of Semen
Christmas Tree Stain
Microscopic examination for sperm cells
Staining helps distinguish sperm cells from epithelial cells
Two Dyes used:
Green – Stains tails
Red – Stains heads

22. Identification of Semen
Christmas Tree Stain (Procedure)
Collect portion of stain on moist swab or take cutting of stain.
Agitate swab/cutting in small tube with water.
Smear water on slide.
Stain with red and green dyes.

23. Identification of Semen
Prostate-Specific Antigen (PSA or p30)
PSA is a protein produced by the prostate gland.
Can confirm semen in SAP+ samples that do not contain sperm cells.
Test relies on antigen (PSA) and antibody interaction

24. Identification of Semen
Prostate-Specific Antigen (PSA or p30)
Swab or cutting agitated in water
Drop of this solution added to test well
Liquid moves through result well

25. Identification of Semen
Prostate-Specific Antigen (PSA or p30)
Antibody is already bound to membrane
Visible test line (T) means PSA is present
Other lines are controls (show test was done properly)

26. Identification of Saliva
What is saliva?

27. Identification of Saliva
What is saliva?
Water, mucus, enzymes, other stuff
Humans produce 1 – 1.5 L of saliva a day
Aids in digestion by lubricating food and begins breakdown of starches

28. Identification of Saliva
How is saliva detected?
Look for the presence of Amylase
Enzyme that breaks down starch
Found in many body fluids; very high in saliva
Two tests:
Starch-iodine test
Phadebas Reagent

29. Identification of Saliva
Starch-Iodine Test
Iodine and starch turn blue
As amylase breaks down starch, color subsides
Procedure:
Gel containing starch is stained blue with iodine
Sample is added to well in gel. If amylase (saliva) is present, the blue color begins to vanish or
Before
Negative
Positive (saliva)

30. Identification of Saliva
Phadebas Reagent
Starch linked to a colored dye
Presence of Amylase releases the dye
Two methods for using Phadebas Reagent:
Press Test
Tube Test

31. Identification of Saliva
Phadebas Reagent (Press Test)
Phadebas reagent (tablets dissolved in water) sprayed on filter paper, then allowed to dry
Phadebas paper placed on area to be tested
Paper is sprayed with water and pressed against area

32. Identification of Saliva
Phadebas Reagent (Press Test)
Paper is left on area and watched for blue color change
Can be watched for up to 40 minutes for color change

33. Identification of Saliva
Phadebas Reagent (Tube Test)
Small piece containing stain is placed in tube
Water and Phadebas tablet added. Heated.
Centrifuged to pellet tablet remnants and piece of cloth.
Amount of color in top liquid is measured by a spectrophotometer.

34. Identification of Saliva
Saliva Tests (summary)
Since amylase is present in other body fluids, these tests do not confirm saliva.
The tests are not very sensitive (low levels of saliva may not be detected)

35. Identification of Saliva
DNA in Saliva
With some biological stains, DNA testing is more important than identifying the stain.
If a test destroys cells, it may not be done before DNA testing
Example: stamps, envelope, bite marks; saliva is inferred

36. Identification of Urine
Searching for Urine
Cases of harassment, mischief, sexual assault, etc.
Stains identified through visual examination (alternate light may help)
Rarely used; difficult to identify as urine and difficult to get DNA profile

37. Identification of Urine
Identifying Urine
Look for urea or creatinine
These are in other body fluids in lower concentrations
Difficult to detect; Liquid nature of urine allows chemicals to spread out and become diluted over a large area

38. Identification of Urine
Identifying DNA in Urine
Some epithelial cells (from urinary tract lining) and other cells
In stains, cells are spread out; typing difficult
Bacteria also makes typing difficult.
Cells may be concentrated from liquid samples; DNA typing possible.

39. Identification of Feces
Identifying Feces
Feces – end product after digestion
Undigested food, mucosal cells, bacteria
Identified by greenish brown color, odor, undigested food
Can test for urobilinogen, but stain must be… apparent.

40. Identification of Feces
DNA testing of Feces
Low cell count and high bacteria content make testing difficult, but not impossible

41. Identification of Vomitus
No known test
Possibly look at low pH, or undigested food