General seminar on forensic semen examination

1. SEMEN EXAMINATION
• PRESENTED BY:-
• RAVI TANWAR
• M. SC. FORENSIC SCIENCE

2. SEMEN:
SEMEN IS MALE’S BODY FLUID CONTAINING
PROCREATIVE HAPLOID CELLS
SPERMATOZOON.
- SEMEN IS COMPOSED OF CELLULAR AND
FLUID PORTIONS.
- TOTAL QUANTITY PER EJACULATE – 2-5 ML

3. BIOLOGICAL CHARACTERISTICS OF
SEMEN
• Typical ejaculation
• 2-5 ml of semen, 160 million sperm
• Seminal fluid
• Medium for ejaculation
• Enzymes and other proteins
• Acid Phospahatase (AP), Prostate Specific Antigen (PSA), and semenogelin
• Sperm cells- Spermatozoa
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4. SEMEN
• Semen is a human body fluid present in human males. It is a
fluid with faint yellow colour and characteristic odour called
seminal odour.
• Volume is about 2-5 ml per ejaculate.
• No. of Spermatozoa= 50,000 to 3,50,000 per ml. (10% of
SP).
• It is a suspension of spermatozoa in seminal plasma.

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6. BIOLOGICAL CHARACTERISTICS OF
SEMEN
• Semen is an extremely good source of DNA
• Not all semen stains contain sperm
• Vasectomy- blocks sperm from being ejaculated
• Semen still produced
• DNA typing probably not possible
• Infertility.
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7. IMPORTANT TERMS
• Aspermia - complete lack of semen
• (Asthenozoo)spermia - reduced sperm motility
• (Azoo)spermia - absence of sperm cells in semen
• (Hyper)spermia - large semen volume
• (Hypo)spermia - small semen volume
• (Oligo)spermia - few spermatozoa in semen
• (Necro)spermia - dead or immobile sperms
• (Terato)spermia - sperm with abnormal morphology

8. • Semen consist of the following
• 1. Spermatozoa (10%)
• 2. Seminal Plasma (90%)
• 3. Epithelial Cell (< 1%)

9. SPERM:
- Taken from Greek word sperma (means =
seed).
- About 60 um in length.
- Consists of head, neck, and tail.
- Head is almost occupied by nucleus.
- Tail is longest part and consists of :- Middle
piece, Chief piece and End piece.

10. SEMINAL FLUID
• The seminal plasma is a mixture of secretion derived from the male
accessory reproductive organs like epididymis, seminal vesicles, the prostate,
vasa-deferantia, bulbourethral & urethral glands.
• The seminal plasma contains Citric Acid, Ascorbic Acid, Lactic Acid, Fructose,
potassium Choline Phosphate, Proteases, free Amino Acids, Ergothioniene,
Zinc, Calcium, Spermine, Lipids, Enzymes like Fibrinogenase, Diastase, Acid
& Alkaline Phosphatase, Glysidases, a & ß Mannosidases a & ß Glucosidases,
ß Givcouridases.

11. PROSTATIC FLUID
- ACID PHOSPHATASE
- SPERMINE
- CHOLESTROL
- PHOSPHOLIPIDS
- FIBRINOLYSIN
- ZINC
- THIS CONSTITUTES 30 % OF TOTAL
VOLUME OF SEMEN.

12. Step of Examination of Seminal stain:
1. Preliminary test
- Physical examination
- Chemical examination
2. Confirmatory test
- Microscopic examination

13. 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
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14. PHYSICAL EXAMINATION OF STAIN:
•Include Visual Examination.
•To naked eye seminal stains generally appear
translucent or opaque spots, at times with
yellowish tint and darker border depending on
colour and thickness of substrata, which, if
absorbent, also acquire stiffness due to dried
semen.

15. 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

16. CHEMICAL EXAMINATION:
- Florence test.
for detection of choline (seminal origin)
- Barberio's test
for deletion of spermine (prostates origin)
- Acid phosphatase test
for deletion of acid phosphate (prostates origin)

17. FLORENCE TEST
• Basis: Choline is detected in this method.
• Procedure: A few drops of watery solution of the stain
is extracted and taken on a slide and a drop of
Florence reagent (8%) W/V solution of Iodine in water
containing 5% W/V of Potassium Iodide) is poured &
allowed to mix slowly under a cover slip. Dark brown
crystals of choline periodide, generally needle
shaped, formed with a few minutes. Non-specifics &
false negative results are common.

18. BARBERIO’S TEST
• Basis: Detection of Spermine
• Procedure: A few drops of Berberio’s reagent when
added to spermatic fluid produces crystals of sperm
in picrate (needle shaped, rhombic & of yellow
colour).
• For various valid reasons, like non-specificity and lack
of reproducibility, the florence and berberio’s tests
have not been accepted universally.

19. 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
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20. 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
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21. PRESUMPTIVE TESTS FOR SEMEN
• Spot test method
• Wet sterile cotton swab with distilled water
• Roll swab across stain
• Saturate swab with AP solution
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22. 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
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23. 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
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24. Confirmatory tests:
Microscopic examination:
- Demonstrable sperm in full size and
shape is confirmatory test for presence of
human semen in sample.
Age of sperm. 24 – 48 hours (in vitro).
up to 72 hours (in utero)

25. Microscopic examination:
Microscopic examination

26. • The Microscopic detection of the Seminal stains is based in morphology
of spermatozoa.
• Microscopic detection of spermatozoa. Cloth pieces from different stains
are taken in 0.5 ml of 0.01 N HCL in small test tubes placed in a beaker
containing water. After sonication for 5 minutes the extracts and the cloth
pieces are transferred to separate micro scope slides and cloth pieces
delicately teased with a needle. Threads are removed and the residual
liquid is gently evaporated to dryness. Residue obtained is stained
haematoxylin and eosin.

27. FLUORESCENCE MICROSCOPY:
• Is also used for detection of spermatozoa. It is based
on the principle that Y Chromo some is fluorescent to
quinacrine. With this method it is possible to detect
both intact spermatozoa as well as the disconnected
heads.

28. MEDICOLEGAL SIGNIFICANCE OF
DETECTION OF SPERM & SEMEN
-RAPE
-SODOMY (Anal intercourse)
-BESTIALITY Sexual intercourse by a human being with a lower animal like
dogs, calves, sheep etc.),
-ATTEMPTED RAPE
-LUST MURDERS
-AZOOSPERMIC ASSAILANTS – CHEMICAL TESTS
-In case of FALSE ACCUSATION by a women
-INCEST (Sexual intercourse in blood relation)
-CIVIL CASES LIKE DISPUTED PATERNITY

29. RAPE EVIDENCE
• The rape victim must undergo a medical examination
as soon as possible after the assault.
• At that time the appropriate items of physical
evidence including clothing, hairs, and vaginal and
rectal swabs can be collected for subsequent
laboratory examination.
• All outer and undergarments should be carefully
removed and packaged separately in paper (not
plastic) bags.
• Bedding, or the object upon which the assault took
place, may also be carefully collected.

30. PHYSICAL EVIDENCE FROM
THE VICTIM
• Pubic combings
• Pubic hair standard/reference
sample
• External genital dry-skin areas
• Vaginal swabs and smear
• Cervix swab
• Rectal swabs and smear
• Oral swabs & smear
• Head hairs
• Blood sample
• Fingernail scrapings
• All clothing
• Urine specimen

31. METHOD OF COLLECTION
• Handling of articles bearing stains should be done
very carefully to avoid damage to spermatozoa.
• Vaginal / anal / penile swabs should be sent along
with their smears on slides. Swabs should be taken on
sterile gauze / cloth and their smears prepared on
sterile slides. These should be dried in air at room
temperature and swabs dispatched in sterile test tube
and slides in clean wrappers.

32. RAPE EVIDENCE
• If a suspect is apprehended within 24 hours of the
assault, it may be possible to detect the victim’s DNA
on the male’s underwear or on a penile swab of the
suspect.
• Items routinely collected from the suspect include all
clothing, pubic hair, head hair, penile swab, and a
blood sample or buccal swab for DNA typing.
• The forceful physical contact between victim and
assailant may result in a transfer of such physical
evidence of blood, semen, saliva, hairs, and fibers.

33. THANK YOU