Forensic Biologyby Richard Li, with additions and edits by Ruth Ballard

Lecture 4: Identification of Semen

Outline

Biological characteristics of semenSpermatozoaDetection of semen

Presumptive vs confirmatory tests Presumptive tests for semen

Detection of sperm “Christmas Tree” stain Confirmatory test for semen

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Biological Characteristics of semen

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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Biological Characteristics of semen

Semen is an extremely good source of DNA The best! BUT…

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

possible5

Spermatozoa

Three distinct regions: Head – acrosome and nucleus (with

haploid DNA) Middle Piece (mitochondria) Tail (flagella; mobility)

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Presumptive vs Confirmatory Tests

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 Confirmatory tests

Not available for most body fluids▪ Main exceptions are semen and blood

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Presumptive Tests for Semen

Semen fluoresces under ALS UV light

▪ long-wave = “Woods Lamp” = 365 nm

Crime Lite (500 nm)Lots of false

positives

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Alternative light source

Presumptive Tests for Semen

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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Presumptive Tests for SemenAP assay

AP liberates naphthol from alpha-naphthol and the naphthol then reacts with brentamine to form a purple-colored dye

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sodium phosphate + naphtholα-naphthyl acid phosphate monosodium salt Acid

phosphatase

Coupling reaction

Purple azo dyenapthol + Brentamine

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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

Presumptive Tests for Semen

Positive acid phosphatase overlay

assay

Presumptive Tests for SemenSpot test method

▪ Wet sterile cotton swab with distilled water▪ Roll swab across stain▪ Saturate swab with AP solution

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Presumptive Tests for Semen 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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Presumptive Tests for Semen

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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Presumptive Tests for Semen

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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Presumptive Tests for Semen

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T

Positive RSID™ semen test

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 C

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T C

Negative RSID™ semen test

Detection of Sperm

Microscopic examination “Christmas Tree” stain

▪ Nuclear Fast Red stains nuclei red▪ Picroindigocarmine stains tails green

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Acrosomes don’t stain well in primate sperm

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