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 semenTypical 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- Spermatozoa3

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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

possible 5

Spermatozoa

Three distinct regions: Head – acrosome and nucleus (with

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

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Presumptive vs Confirmatory TestsPresumptive 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 courtConfirmatory 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

positives8

Alternative light source

Presumptive Tests for SemenAcid 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 assay15

Presumptive Tests for SemenSemenogelins

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 SpermMicroscopic 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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