Information related to biological stains-semen.

1. EXAMINATION OF BIOLOGICAL STAINS
SEMINAL STAINS
Dr Sadia Qayyum
Department of Forensic Medicine
LNHMC

2. LEARNING OBJECTIVES
 Describe the composition of semen
 List the criteria for normal sperm count as per WHO
 Discuss the medico legal importance of seminal stains
 Enumerate the various methods of collection of seminal material and determination of motility of sperms
 Describe the physical, chemical, microscopic, electrophoretic, and immunological tests for the examination of
seminal stains.
 Explain the role of seminal stains in determination of blood groups

3. SIGNIFICANCE OF SEMINAL STAIN EXAMINATION
Civil / Criminal cases/ Medical
diagnosis
 Rape/attempted rape
 Disputed paternity
 Sodomy
 Legitimacy
 Bestiality
 Artificial insemination
 sterility

4. COMPOSITION
 In humans, seminal fluid contains several
components besides spermatozoa: proteolytic
and other enzymes as well as fructose are
elements of seminal fluid which promote the
survival of spermatozoa, and provide a
medium through which they can move or
"swim".
 Semen consists of two compartments

5. STRUCTURE
Sperm cell can be divided in 4 parts:
 HEAD
 NECK: It contains one typical centriole and one
atypical centriole such as the proximal centriole like.
 MID PIECE
 TAIL or "flagellum": it executes the lashing
movements that propel the spermatocyte.

6. NORMAL SPERM COUNT AS PER WHO
Total sperm count in
ejaculate
39–928 million
Ejaculate volume 1.5–7.6 mL
Sperm concentration 15–259 million per mL
Total motility
(progressive and non-
progressive)
40–81 percent
Progressive motility 32–75 percent
Sperm morphology 4–48 percent

7. MOTILITY AND LIFE SPAN
Full motility persist for about 3 hour, 50 percent are motile by 8
hour and 10 percent by 24 hour
In living persons – intact sperms may be found in vaginal washings
up to 12 hours after act, while sperm heads can be detected up to
24 hours.
In dead persons
1. Vagina –9 days
2. Cervix –12 days
3. Uterus –15 days
4. Anus –2 days
5. Mouth/oral cavity –9 hours

8. COLLECTION & PRESERVATION
 CLOTHING: Portion of cloth with the stain is cut,
dried in shade (not heated) and preserved.
 VAGINAL FLUID: Fluid from the vagina is
collected with a pipette or vaginal washing is done.
Swabs are taken with sterile gauze and smears are
prepared on sterile slides.
 DRIED STAINS
 MATTED PUBIC HAIR
 Stains on SMOOTH SURFACE:These are gently
scraped off into a glass container and preserved.
 Samples must be packed in containers that allow air
circulation; never in plastic bags or sealed
nonporous tubes, jars or boxes.

9. STEPS REQUIRED FOR EXAMINATION
1. Preliminary test
 Physical examination
 Chemical examination
2. Confirmatory test
 Microscopic examination
 blood grouping
 DNA testing

10. PHYSICAL EXAMINATION
 A dry seminal stain is grayish-white in color with
irregular map like appearance.
 The stain area appears stiffened.
 Seminal stains may be mistaken for starch stains.
 A fresh stain on a non-absorbent material appears
translucent.
 After a month, it becomes yellow to brown.

11. ULTRAVIOLET LIGHT EXAMINATION
 Seminal stains fluorescence bluish-white in color.
 Fluorescence is not specific for seminal stains because
other stains like body fluid, saliva, pus cell, milk, some
fiber whitener stains etc. may also give fluorescence.
 Invisible or old stains can be visible under ultraviolet
light.

12. PRESUMPTIVE CHEMICAL EXAMINATION
 Based on colorimetry and are qualitative in nature.
 Positive presumptive tests must be followed by a confirmatory test

13. 1. FLORENCETEST
 For detection of choline (seminal origin)
 The stain is extracted, dried on a glass slide and
covered with a coverslip and a drop of Florence
solution (8% w/v of iodine in water containing 5%
w/v of potassium iodide) is allowed to run under the
coverslip.
 If semen is present, dark brown rhombic crystals
resembling hemin (but larger) arranged in clusters
 A positive test is not proof of seminal fluid.
 A negative reaction proves that the stain is not
semen, IT may occur if choline content is low or the
stain is decomposed

14. 2. BARBERIO'STEST
 A saturated aqueous or alcoholic solution of picric acid
when added to dried stain extract on a glass slide
covered with a coverslip, produces yellow needle-shaped
crystals of spermine picrate
 The reaction depends on the presence of spermine from
prostatic secretions.
 This test is positive without the presence of
spermatozoa.

15. 3. BRENTAMINE FAST BLUETEST
 It is the most common presumptive test for seminal acid
phosphatase.
 Acid phosphatase activity is 500-1000 times greater in
human semen than in any other bodily fluid
 An enzyme substrate, sodium -naphthyl phosphate is
converted to sodium phosphate and naphthol by the acid
phosphatase enzyme in the semen and a coupled reaction
with bentamine fast blue dye takes place, forming a purple
color.
 It can produce false positives because similar enzyme activity
is found in other body fluids (e.g. vaginal secretions and fecal
stains), human red cells, semen of higher apes as well as in
presence of fungi, bacteria and even plants (juice of
cauliflower), pregnancy, menstruation, bacterial vaginosis may
also elevate its level
 Dried and old seminal stains which have not undergone
putrefaction give positive reaction.

16. CONFIRMATORY METHODS
 the sample is contaminated by other bodily fluids
(saliva, vaginal secretions), epithelial cells, cellular debris
wherein selective degradation may be done by treating the
cell extract with a mixture of proteinase k and sodium
dodecyl sulfate before staining and microscopic
examination.
 Most commonly used confirmatory test for semen is
visualization of one or more intact spermatozoa after
staining with dyes such as hematoxylin and eosin and
‘christmas tree’ stain.
 Fluorescence in situ hybridization (fish): this cytogenic
analysis uses a y chromosome specific dna probe to identify
y-bearing (male) cells.This technique identifies not only
spermatozoa, but also cells of male origin and also confirms
male-female contact

17. 1. MICROSCOPY
 Posterior half to one-third of head and the tail takes eosin and is
stained deep red or pink, while anterior half to two-third takes
very light or faint basic or blue stain or may appear unstained
 Size of human spermatozoa is about 55 µ length.
 Head is ovoid and flattened, 5 × 3.5 in dimensions.
 It has a short neck and a long filamentous tail (50 µ) which tapers
to a fine point
 Older the stain, lesser is the chance of finding intact sperms.

18. 2. NON-CELLULAR SEMEN MARKERS
1. Markers are specific and unique to seminal plasma but
independent of spermatozoa.
2. The two most commonly employed constituents are Acid
phosphatase and the prostate-specific glycoprotein p30 (PSA).
3. These tests are conclusive even in the absence of demonstrable
sperms, azoospermia or vasectomized individuals.

19. ACID PHOSPHATASETEST (QUANTITATIVE)
 Finding a significantly elevated acid phosphatase level is
consistent with the presence of semen.
 Undiluted semen has an acid phosphatase level of 340-360
Bodansky units/ml.
 A value of > 100 Bodansky units with/without motile sperms
indicate that ejaculation occurred within 12 h of examination.
 Isoenzymes can be detected using polyacrylamide gel
electrophoresis followed by staining with methyl umbelliferyl
phosphate reagent which can distinguish the acid phosphatase
present in other substances

20. PROSTATE SPECIFIC ANTIGEN ( HUMAN SEMINAL
PLASMA (P30)
 The glycoprotein p30 is derived from prostrate and is found in
seminal plasma, male urine and blood and has not been found in
any female body tissue or fluid.
 p30 in sample reliably identifies semen regardless of whether acid
phosphatase is elevated or spermatozoa are detected.
 It is determined serologically using antiserum that is specific for
the p30 antigen.
 It can also be done using immunochromatographic strip test using
antibodies raised against the human PSA and enzyme-linked
immunoassay (ELISA).
 Its normal range in semen is 300-4200 g/ml with mean of 1200
g/ml and is detectable in vaginal fluid up to 27 h (range 13-47 h)
after intercourse as compared to 12 h for acid phosphatase.
 p30 can be detected in dried and old stains (> 10 years in material
stored at room temperature) and in cadavers

21. HUMAN ORIGIN?
 Determination of species can be done by:
1. Microscopy
2. Precipitation reaction

22. DETERMINATION OF BLOOD GROUP
 If the seminal stain is from secretor, absorption-
elution method is used to determine the ABO
blood group.
 In sexual assault cases, it requires the
comparison of blood group substances
recovered in the evidence material with those of
the victim and the suspect
 Traditional grouping is cheap, fast and universally
available.

23. INDIVIDUALITY
 Each sperm is an intricate motile cell, rich in DNA with a
head that is made up mostly of chromosomal material.
 DNA profiling (fingerprinting) can be done to identify the
offender.
 The primary advantage of DNA profiling is its ability to
accurately individualize semen that contains only minimal
numbers of spermatozoa.
 It has a high degree of sensitivity and discrimination.

24. LETS SUMMARIZE……….

25.  Study this topic from your text book also
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