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