Identification of Biological Fluids and Stains

Summary Semen Saliva Urine Feces Vomit

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

Identification of Semen What is semen?

Identification of Semen What is semen?

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.

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

Identification of Semen Sperm Cells

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

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

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

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

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

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)

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

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

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.

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

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.

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

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

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.

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

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

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)

Identification of Saliva What is saliva?

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

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

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)

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

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

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

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.

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)

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

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

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

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.

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.

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

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