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

Lecture 4: Identification of Semen. Forensic Biology by Richard Li, with additions and edits by Ruth Ballard. Outline. Biological characteristics of semen Spermatozoa Detection of semen Presumptive vs confirmatory tests Presumptive tests for semen Detection of sperm

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

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  1. Lecture 4: Identification of Semen Forensic Biologyby Richard Li, with additions and edits by Ruth Ballard

  2. Outline • Biological characteristics of semen • Spermatozoa • Detection of semen • Presumptive vs confirmatory tests • Presumptive tests for semen • Detection of sperm • “Christmas Tree” stain • Confirmatory test for semen

  3. 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

  4. 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)

  6. 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

  7. Presumptive Tests for Semen • Semen fluoresces under ALS • UV light • long-wave = “Woods Lamp” = 365 nm • Crime Lite (500 nm) • Lots of false positives Alternative light source

  8. 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

  9. Presumptive Tests for Semen • AP assay • AP liberates naphthol from alpha-naphthol and the naphthol then reacts with brentamine to form a purple-colored dye sodium phosphate + naphthol α-naphthyl acid phosphate monosodium salt Acid phosphatase napthol + Brentamine Purple azo dye Coupling reaction

  10. Presumptive Tests for Semen • 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

  11. Presumptive Tests for Semen • Spot test method • Wet sterile cotton swab with distilled water • Roll swab across stain • Saturate swab with AP solution

  12. 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

  13. 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

  14. 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

  15. Presumptive Tests for Semen • Immunochromatographic test strip assay for semenogelin • Rapid and simple • Specificity still under debate • Rapid Stain Identification (RSID-Semen) • Independent Forensics

  16. T C 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

  17. T C Negative RSID™ semen test

  18. Detection of Sperm • 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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