Lecture 4: Identification of Semen
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Biological characteristics of semen
Spermatozoa
Detection of semen
 Presumptive vs confirmatory tests
 Presumptive tests for semen
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Detection of sperm
 “Christmas Tree” stain
 Confirmatory test for semen
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Typical ejaculation
 2-5 ml of semen, 160 million sperm
▪ 3 pg DNA/sperm = 480,000 ng DNA/ejaculate
▪ Only 1 ng DNA needed for STR typing!
 Seminal fluid
▪ Medium for ejaculation
▪ Enzymes and other proteins
▪ Acid Phospahatase (AP), Prostate Specific Antigen (PSA), and
semenogelin
 Sperm cells- Spermatozoa
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Semen is an extremely good source of DNA
 The best! BUT…
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Not all semen stains contain sperm
 Vasectomy- blocks sperm from being ejaculated
▪ Semen still produced
▪ DNA typing probably not possible
 Infertility
▪ Depending on severity, DNA typing may be possible
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Three distinct regions:
 Head – acrosome and nucleus (with haploid DNA)
 Middle Piece (mitochondria)
 Tail (flagella; mobility)
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Presumptive tests
 Fast, easy, inexpensive
 Great for screening evidence to find possible stains
 Usually detect enzymes specific to the body fluid
 False positives (hence “presumptive”)
▪ Open to attack in court
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Confirmatory tests
 Not available for most body fluids
▪ Main exceptions are semen and blood
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Semen fluoresces under
ALS
 UV light
▪ long-wave = “Woods Lamp”
= 365 nm
 Crime Lite (500 nm)
Alternative light source
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Lots of false positives
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Acid phosphatase enzyme
 Advantages
▪ High levels in fresh semen stains
▪ Very fast, inexpensive
▪ Can be done in the field
 Limitations
▪ Activity may be weak or absent in older stains
▪ Also present at low levels in vaginal fluid and bacteria
▪ Not species-specific
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AP assay
 AP liberates naphthol from alpha-naphthol and
the naphthol then reacts with brentamine to form
a purple-colored dye
α-naphthyl acid phosphate monosodium
salt
sodium phosphate + naphthol
Acid
phosphatase
napthol + Brentamine
Purple azo dye
Coupling reaction
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Overlay method
▪ Spray a Whatman paper circle
with distilled water
▪ Lay the paper down over the
suspected semen stain
▪ Leave in contact with stain 30-60
seconds
▪ Remove paper circle from stain
and spray with AP spot solution
▪ Look for a rapid color change to
purple
Positive acid
phosphatase overlay
assay
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Spot test method
▪ Wet sterile cotton swab with distilled water
▪ Roll swab across stain
▪ Saturate swab with AP solution
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MUP
 More sensitive than acid phosphatase
 AP catalyzes the removal of the phosphate
residue on the substrate 4-methylumbelliferone
phosphate (MUP), which generates fluorescence
under UV light
 Filter paper overlay
▪ Filter paper placed in contact with putative semen stain
and then removed and taken to dark room
▪ Sprayed with MUP
▪ Fluorescence detected with UV lamp
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Prostate-specific antigen (PSA)
 Major protein in seminal fluid
 Also detected in urine, fecal matter, sweat, milk
but at much lower levels
 Half-life of dried stain: 3 years
 Hydrolyzes semenogelins (seminal vesicle specific
antigens)
 Detected with immunochromatographic test strip
assay
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Semenogelins
 Higher concentration in seminal fluid than PSA
 Not found in urine, milk, sweat
 Greater specificity for semen than PSA
 Detected with immunochromatographic test strip
assay
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Immunochromatographic test strip assay for
semenogelin
 Rapid and simple
 Specificity still under debate
 Rapid Stain Identification (RSID-Semen)
▪ Independent Forensics
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human semenogenlin
monoclonal gold-labeled
murine anti human
semenogelin antibody to
epitope 1
monoclonal unlabeled
murine anti human
semenogelin antibody
to epitope 2
polyclonal unlabeled
goat anti murine
antiglobulin
T
Positive RSID™
semen test
C
T
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Negative RSID™
semen test
C
T
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Microscopic examination
 “Christmas Tree” stain
▪ Nuclear Fast Red stains nuclei red
▪ Picroindigocarmine stains tails green
Acrosomes don’t stain
well in primate sperm
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