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 “Christmas Tree” stain Confirmatory test for semen

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

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

Spermatozoa Three distinct regions: Head – acrosome and nucleus (with haploid DNA) Middle Piece (mitochondria) Tail (flagella; mobility)

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

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

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

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 Coupling reaction Purple azo dye napthol + Brentamine

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

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

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

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

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

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

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 C

Negative RSID™ semen test C Negative RSID™ semen test

Acrosomes don’t stain well in primate sperm 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