Forensic analysis of seminal stains

1. Analysing Seminal Stains
Suneesh Nair Abhiram
Chenganassery Rajim Farhan
Forensic Medicine

2. Semen
• Semen is a fluid secretion containing cellular
elements
• Fluid formed in seminal vesicle and prostate
• The seminal vesicle contain
– High concentration of choline & lecithin
• The prostate secrete
– Acid phosphatase
– Spermine

3. • The cellular element; spermatozoa &
epithelial cells formed by testes
• Normal per ejaculation 2 to 5 ml semen
• Around 100 million sperm/ml, 80% motile
• Slightly alkaline & liquefy in short time due to
prostatic fibrinolysin

4. Need for Semen Detection
• In charges of rape & sodomy
• Murder case suspected, sexual assault
• To detect stains on cloth or body of
victim/accused
• Bed clothes or where offense has been
committed

5. Material Collection
1. The stain fabric is cut & dried in the shade
2. Vaginal & high vaginal swab taken on sterile
swab
3. Dried seminal fluid on thighs, anal & perianal
area taken by moist swab
4. Dry stain scraped into glass container by
blunt scalpel
5. Pubic hair cut & preserve for further
investigation

6. Semen Detection Method
• Physical
• Chemical
• Microscopic
• Electrophoretic

7. Physical Examination
• Garments often received, mud, blood, vaginal,
urine etc.
• Clothes thoroughly examine under filter UV
light
• Urine, leucorrheal discharge show bright
flourescence
• The flourescence of seminal stain is bluish
white color

8. Chemical Examination
• Acid Phosphatase Test:
–a quantitative test
–the amount of acid phosphatase is
estimated in a measured specimen, e.g. one
square centimeter of stained material give
positive reaction in old stain as well

9. • Florence Test:
–A drop of watery solution of suspected stain
when place on slide and let it dry, place
cover slip and a drop of florence reagent
(KI+H2O), it will give dark brown crystal,
choline periodide formed resembling
haemin crystal if seminal matter present
–False negative: low choline content or
decomposed stain
–False positive result: other body tissue
(except vagina) may contained choline

10. Florence Test

11. Chemical Examination
• Barberio’s Test
– The reaction depend upon spermine in semen
– When few drops of Barberio’s reagent
(alcoholic solution of picric acid) added to
spermatic fluid will produce yellow, needle
shape & rhombic crystal of spermine picrate
– Test is positive even without presence of
spermatozoa
– The limitation is pleomorphic nature of crystals

12. Spermine picrate crystal –
Barberio test

13. • Creatine Phosphokinase Test
–Spermatozoa contain 2x high concentration
of creatine phosphokinase compared to
other body fluids
–Level > 400 U/ml is almost diagnostic of
seminal stain

14. Microscopic Examination
• Microscopic examination can be done directly
or fixing and staining the smear
• In case of fabric, stained portion cut and
soaked in acidulated water 1 hour to 4 hour
• The fabric removed from solution and dabbed
several time on clean slide gently
• Slide can be seen directly under microscope or
fixed & stained

15. • Examine under low and high power objective
• Human spermatozoa:
– 50 micron long, 5 micron head, 3 micron breadth
– Short body long filamentous tail
– In stained specimen the hind part of head of
sperm acquire deep blue tint while rest of head
and tail deep red stained
– Hard to recognize sperm if its head fall off

16. • Disconnected head of sperm can be confused
with microorganism like yeast, Trichomonas
vaginalis
• As a rule a complete spermatozoa is necessary
before giving positive result
• The presence of Y chromosome in head of
spermatozoa under fluorescent microscope
help in identification

17. Electrophoretic Method
• Acid Phosphatase:
– Acid phosphatase isoenzyme help differentiate
seminal stain and stains from other sources on the
basis of mobility
– Semen can be detected even in absence of sperm,
i.e. in azoospermic or vasectomised person

18. • Lactate Dehydrogenase:
– the sperm specific iso-enzyme (LDHx), stable in
stain for over four weeks
– The iso-enzyme pattern of human semen is
different from that of common animals
– Positive results obtained in much larger number
than is possible with microscopic examination

19. Summary
• Best sensitive test I-tartarate inhabitable acid phosphatase
– Draw back, vaginal secretion also give positive test
• Florence test not sufficiently sensitive but can definitely rule
out vaginal secretion
• Microscopic examination of spermatozoa reliable & specific
– But may not be useful in very old stains
• LDH isoenzyme method is superior to microscopic detection
of spermatozoa
– But it has limitations to azoospermic & vasectomised person
• Therefore is evident, at least one confirmatory test should be
used in conjunction with I-tartarate acid phosphatase
screening test