It consists of preliminary and confirmatory methods of semen analysis, the biological fluid found at the sexual assault or rape cases.

1. SEMEN ANALYSIS
DR. ARCHANA MAHAKALKAR
DEPARTMENT OF FORENSIC BIOLOGY
GOVERNMENT INSTITUTE OF FORENSIC SCIENCE, NAGPUR

2. Introduction
 Semen is a massive, yellowish white, glairy, opalescent, secretion having a characteristic
odor known as seminal odor.
 The incandescent(emitting light) of the seminal stains gives bluish white Colour.
 Semen Identification is useful as evidence in sexual assault cases.
 It can prove that the crime was committed & help identify the criminal.
 The typical ejaculate consists of between 1.5 and 5 ml of semen.
 Prostate fluid of semen contains high concentrations of acid phosphatase (AP) and
prostate-specific antigen (PSA).
 Both are useful markers for the identification of semen in forensic laboratories.
 The epididymis and the bulbourethral secretions each account for approximately 5% of
the ejaculate.
 seminal vesicle fluid contains flavin that causes semen to fluoresce under ultraviolet light,
often utilized when searching for semen stain evidence.

3. .
Testes
sperms
Prostate
Acid
Phospatase
Prostate
Specific
Antigen(PSA)

4. Structure of sperm
 A normal spermatozoa consists of three main parts,
namely, Head, middle part and tail.
 Head consists of nucleus containing a haploid set of
chromosomes. It also possesses a tiny secretary cap
like structure called acrosome.
 Middle piece harbours mitochondria which provides
energy to the sperm to sustain motion.
 Flagellum is the name of the last segment, the tail.
 Tail provides the capability to pass through the
vaginal cavity towards the eggs. Without flagellum,
the sperm becomes nonmotile.

5. Acid Phosphatase (AP)
 The greatest forensic importance of AP is that the prostate is the most abundant
source of AP and contributes most of the AP activity present in semen.
 AP levels in semen are not affected by vasectomies.
 The half life of AP activity at 37oC is 6 months.
 However, the half life is decreased if a sample is stored in a wet environment.
 AP activity can be detected from dry seminal stains stored at –20oC up to one year.
 Low levels of prostatic AP are present in the sera of healthy males.
 Elevated levels of prostatic AP found in serum are useful in diagnosing and
monitoring prostate carcinoma.
 Many AP tests utilized in clinical testing may be used to identify semen for forensic
applications.

6. Prostate-Specific Antigen (PSA)
 PSA is a major protein present in seminal fluid at concentrations of 0.5 to 2.0
mg/ml.
 PSA is produced in the prostate epithelium and secreted into the semen.
 PSA can also be found in the paraurethral glands, perianal glands, apocrine sweat
glands, and mammary glands. Thus small quantities can be detected in urine, fecal
material, sweat, and milk.
 PSA can also be found at much lower levels in the bloodstream.
 An elevated plasma PSA is present in prostate cancer patients and it is widely used
as a screening test for this disease.
 PSA is also elevated in benign prostatic hyperplasia and prostatitis.
 The synthesis of PSA is stimulated by androgen, a steroid hormone.

7.  PSA is a protein that has a molecular weight of 30 kDa and is thus also known as
P30.
 It is responsible for hydrolyzing semenogelin (Sg) which mediates gel formation
in semen.
 PSA is a member of the tissue kallikrein (serine protease) family and is encoded
by the KLK3 locus located on chromosome 19.
 In addition to PSA, other tissue kallikreins encoded by KLK2 and KLK4 loci are
expressed in the prostate as well. The half life for PSA in a dried semen stain is
about 3 years at room temperature.
 The half life is greatly reduced when a sample is stored in wet conditions.

8. Seminal Vesicle-Specific Antigen (SVSA)
 SVSA includes two major semenogelins and constitutes the major seminal
vesicle-secreted protein in semen.
 Upon ejaculation, it forms a coagulum that is liquefied after a few minutes due
to the degradation of SVSA.
 In human semen, two major SVSAs are semenogelin I (SgI) and semenogelin II
(SgII).
 Both are present in a number of tissues of the male reproductive system
including the seminal vesicles, ductus deferens, prostate, and epididymis.
 They are also present in several other tissues such as skeletal muscle, kidney,
colon, and trachea.
 They have also been found in the sera of lung cancer patients.

9.  Use of Sg as a marker for semen identification instead of PSA presents certain
advantages.
 The concentration of Sg in seminal fluid is much higher than that of PSA and this
is beneficial for the sensitivity of detection.
 Sg is present in seminal fluid and absent in urine, milk, and sweat where PSA can
be found.
 Although Sg compounds are present in skeletal muscle, kidney, and colon, this is
not a great concern because tissue samples are not routinely collected for semen
detection in sexual assault cases.

10. Analysis

11. Presumptive Assays
1. Lighting Techniques for Visual Examination of
Semen Stains
 Lighting techniques are often used to search
for semen stains.
 Dried semen stains fluoresce when irradiated
with ultraviolet light, argon lasers, or ALSs.
 ALSs are most commonly used for visual
examination, 450- to 495-nm excitation
wavelengths can be used with colored goggles
or filters (530nm) that allow for the
visualization of fluorescence.

12. Acid Phosphatase Techniques
 2. Colorimetric Assays • The most common method for forensic
applications is the use of α-naphthyl
phosphate as a substrate coupled with
Brentamine Fast Blue B.
• Prostatic AP is water-soluble.
• Thus, a moistened cotton swab or piece of
filter paper can be used to transfer a small
amount of sample from a stain by brief
pressing onto the questioned stain area.
• The substrate reagent is added to the swab
or filter paper followed by the addition of
Brentamine Fast Blue B.
• If a purple coloration develops within 1 min,
the test is considered a positive test’

13. 3. Fluorometric Assays
Fluorometric methods are
more sensitive than the
colorimetric detection of AP
and are used for semen
stain mapping.
AP catalyzes the removal of
the phosphate residue on a
4-methylumbelliferone
phosphate (MUP) substrate,
a reaction that generates
fluorescence under
ultraviolet light.

14. Confirmatory Assays:1. Microscopic identification
 Microscopic identification of spermatozoa provides the proof of a
seminal stain.
 Histological staining can facilitate microscopic examination. The most
common staining technique is the Christmas tree stain .
 The red component known as Nuclear Fast Red (NFR) is a dye used
for staining the nuclei of spermatozoa in the presence of aluminum
ions.
 The green component, picroindigocarmine (PIC), stains the neck and
tail portions of the sperm.
 The acrosomal cap turns pink, the nucleus is red, the sperm tails stain
green, and the midpiece stains blue.
 Any epithelial cells present in the sample will appear blue-green and
have red nuclei.

15. 2. Laser capture microdissection (LCM)
 It was shown to be an effective technique for separating the spermatozoa from
non-sperm cells (i.e. epithelial cells from the victim) on a glass slide.
 This technique involves using a thin layer of a thermo-sensitive polymer which
is placed on the surface of an LCM cap.
 Once spermatozoa are identified on the slide under a microscope, a polymer-
containing LCM cap is placed over the spermatozoa on the slide.
 An infrared laser melts the polymer and causes it to adhere only to the
targeted spermatozoa.
 The spermatozoa are then lifted off the slide.

16. 3. Identification of PSA: Immunochromatographic Assays

17. 4. Enzyme-Linked Immunosorbent Assay (ELISA)

18. 5. Immunochromatographic Assays for Sg

19. 6. ELISA-Sg

20. 7. RNA-Based Assays

21. References:
 [Li, Richard] Forensic Biology- Second Edition

22. Thank You