1. Lecture 4: Identification of Semen
Forensic Biology by 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

5. 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

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

7. 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

8. 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

9. 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

10. 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

11. 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 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

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

13. 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

15. 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

16. 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

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

18. 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

19. Negative RSID™ semen testC
Negative RSID™ semen test

20. 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