Identification of Biological Fluids and Stains

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Identification of Biological Fluids and Stains

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Summary

• Semen
• Other Biological Fluids

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Semen Identification

• Why is semen identification important?

• Evidence in sexual assault cases
• Can help prove a crime was committed
• Can identify the perpetrator

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Semen Identification

• What is semen?

• Semifluid mixture of
• Cells
• Enzymes
• Other organic and inorganic materials

Sperm cells are most important they contain DNA
and are specific to semen.
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Semen Identification

• 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

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Semen Identification

• Sperm Cells

• Sperm cell spermatozoan
• Head contains DNA
• Tail (flagella) for movement
• Acrosome structure at front of head
• Contains enzymes to penetrate egg

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Semen Identification

• Sperm Cells

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Semen Identification

• Searching for Semen Stains

• Visual inspection yellowish-white

• May turn more yellow due to growth of bacteria

• Alternative light source look for fluorescence

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Semen Identification

• UV Light (Woods Lamp Technique)

• Due to non-protein substances in semen.

• Due to growth of bacterium Pseudomonas
  fluorescens (produces fluorescent substances

• Appearance can differ depending on background
  material. (bluish white vs. dark area)

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Semen Identification
UV light (350 nm) causes blue emission (460 nm).
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Semen Identification

• Presumptive Tests

• Seminal acid phosphatase (SAP)
• Spermine
• Choline
• Zinc

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Semen Identification

• 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

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Semen Identification
SAP is an enzyme secreted by the epithelial cells
of the prostate gland.
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Semen Identification
High levels of SAP in blood may indicate prostate
cancer.
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Semen Identification

• Seminal Acid Phosphatase

• Brentamine Fast Blue Test (the test for SAP)
• Will react with semen diluted 500x

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Semen Identification
Brentamine Fast Blue Test

• Breakdown of sodium-a-naphthyl phosphate by acid
  phosphatase
• Phosphate binds with o-dianisidine (Fast Blue B)
  to form a colored compound

sodium-a-naphthyl phosphate
o-dianisidine
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Semen Identification

• Seminal Acid Phosphatase

• Method (Brentamine Fast Blue Test)
• Substrate (sodium-a-naphthyl phosphate)
• Chromogen (o-dianisidine)
• Seminal acid phosphatase (in semen)

• SAP reacts with substrate.
• Product of this reaction reacts with the
  chromogen (causes color change)

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Semen Identification

• 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

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Semen Identification

• 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

Swabs of orifices and skin are often collected in
sexual assault cases.
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Semen Identification

• 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 paper
• Positive reaction is a purple color

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Semen Identification

• 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

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Semen Identification

• SAP False Positives

• 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.
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Semen Identification

• Confirmatory Tests

• Christmas Tree Stain (of sperm cells)

• Prostate-Specific Antigen (PSA)

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Semen Identification

• 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

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Semen Identification

• Christmas Tree Stain (Procedure)

• Collect portion of stain on moist swab or take
  cutting of stain

• Agitate swab/cutting in small tube with water

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Semen Identification

• Christmas Tree Stain (Procedure)

• Remove swab/cutting. If swab, cut tip off.

• Place cut swab or cutting in empty tube with hole
  in bottom (piggy-backing).

Two tubes
Hole in bottom

• Centrifuge. Sperm cells will pellet at bottom of
  bottom tube.

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Semen Identification

• Christmas Tree Stain (Procedure)

• Top liquid thrown away.

• Pellet can be reconstituted in water.

• Drop of this solution is placed on a microscope
  slide and semen cells are looked for under a
  microscope.

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Semen Identification

• Prostate-Specific Antigen (PSA or p30)

• PSA is a protein produced by the prostrate gland.

• Can confirm semen in SAP samples that do not
  contain sperm.

• Test relies on antigen (PSA) and antibody
  interaction

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Semen Identification

• 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

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Semen Identification

• 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)

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Identification of Saliva

• What is saliva?

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Saliva Identification

• 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

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Saliva Identification

• 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

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Saliva Identification

• 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)
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Saliva Identification

• Phadebas Reagent

• Starch linked to a colored dye
• Presence of Amylase releases the dye

• Two methods for using Phadebas Reagent
• Press Test
• Tube Test

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Saliva Identification

• 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

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Saliva Identification

• 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

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Saliva Identification

• 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.

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Saliva Identification

• 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)

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Saliva Identification

• 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

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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

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Urine Identification
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

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Urine Identification
Identifying Urine

• Urea
• Urease breaks down urea and releases ammonia.
  Ammonia detected using color changing chemical.

• Creatinine
• Addition of picric acid combined with creatinine
  to form colored product

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Urine Identification
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.

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Identification of 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.

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Feces Identification
DNA testing of Feces

• Low cell count and high bacteria content make
  testing difficult, but not impossible

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Identification of Vomitus

• No known test

• Possibly look at low pH, or undigested food