It consists of preliminary and confirmatory examination methods of vaginal fluid and menstrual fluid analysis.

1. Vaginal Fluid Analysis
Dr.Archana Mahakalkar
Department of Forensic Biology
Government Institute of Forensic Science, Nagpur

2. Introduction
• The identification of vaginal secretions and menstrual blood is important for the
investigation of sexual assault cases.
• Such identification can help to corroborate allegations of sexual assault. For example,
in a sexual assault investigation, a stain was observed after examining the suspect’s
clothing.
• Subsequently, forensic DNA analysis revealed that the DNA of the stain originated
from the victim, establishing a link between the victim and the suspect.
• However, the defendant may assert that the victim’s DNA originated from a sweat
stain as a result of casual contact and deny any criminal act.
• If vaginal secretions were found in the stain, the evidence has probative value to
corroborate an allegation of a sexual act.

3. Methods of Analysis
Dane’s
Staining
Method
Vaginal Acid
Phosphatase
Histological
Identification
Lugol’s Iodine
Staining
RT-PCR
Identification
of Vaginal
Bacteria
D-dimer
Assay
RNA-
Based
Assays
Lactate
Dehydrog
enase
Assay

4. Identification of Vaginal Stratified Squamous Epithelial Cells
• Histological structure of human
vaginal tissues.
a)The squamous mucosa and
submucosa
b)Multiple layers of cells in the
squamous mucosa
(Source-Rechard Li)

5. Lugol’s Iodine Staining and Periodic Acid–Schiff Method
• Lugol’s iodine solution, named after the
French physician Jean Lugol, is originally
used as an antiseptic that is applied to
skin or tissue to prevent infection.
• In forensic applications, it is utilized for
the identification of glycogenated vaginal
epithelial cells.
• The technique is based on the principle
that iodine reacts with intracellular
glycogen to exhibit a color.

6. • Lugol’s stock solution is an aqueous solution of 5% iodine
(I2) and 10% potassium iodide (KI).
• Potassium iodide allows the iodine to be soluble in water
through the formation of the triiodide ion.
• For staining the vaginal epithelial cells, 5% of the stock
solution is usually used as the working solution.
• Iodine atoms fit into the helices of glycogen to form a dark
brown glycogen–iodine complex .
• Vaginal epithelial cells can also be stained using the periodic
acid–Schiff method. The cytoplasm of the vaginal epithelial
cells is stained magenta, and the nucleus is stained purple.
• Diagram of a glycogen–iodine complex. In the complex, the
glycogen chain forms a helix structure with six
monosaccharide residues (red) per turn. Iodine molecules
(gray) fit in the helix to form the glycogen–iodine complex,
exhibiting a color. (© Richard C. Li.)

7. Epithelial cells stained with Lugol’s iodine solution
(a) Vaginal (b) buccal epithelial cells ( Rechard C.Li)

8. Dane’s Staining Method• Skin, buccal, and vaginal
epithelial cells belong to
stratified squamous
epithelium.
• Differentiated skin epithelial
cells are keratinized and are
classified as keratinizing
squamous epithelial cells,
while buccal and vaginal
epithelial cells are
nonkeratinizing squamous
epithelial cells.
• Additionally, the skin
epithelial cells lose nuclei
and other cellular organelles
during differentiation. In
contrast, buccal and vaginal
cells contain nuclei.
Skin Buccal Vaginal epithelial

9. Identification of Vaginal Acid Phosphatase

10. Identification of Vaginal Bacteria
• The most frequently occurring Lactobacillus species found in
the vagina are L. iners, L. crispatus, L. gasseri, and L. jensenii.
Among them, L. iners is the most common species of
Lactobacillus in women.
• Lactobacillus can be identified based on the sequences of
DNA markers such as the 16S rRNA gene and the intergenic
spacer region between the 16S rRNA and 23S rRNA gene.
• In forensic applications, L. iners, L. crispatus, L. gasseri, and L.
jensenii can be detected in vaginal secretions.
• Thus, the identification of Lactobacillus taxa can potentially
be utilized for the forensic identification of vaginal fluid.

11. Confirmatory Assays of Vaginal Secretions
• Recently, the analysis of tissue-specific gene expression has been utilized for the
identification of vaginal secretions.
• Using the reverse transcription polymerase chain reaction (RT-PCR) technique, the
mRNAs of the tissue-specific genes of vaginal epithelial cells can be detected.
• For example, two commonly used markers for vaginal secretion identification are MUC4
and HBD1.
• MUC4 encodes a mucin protein that is a major component of vaginal mucus, and HBD1,
the human β defensin 1, encodes a vaginal antimicrobial peptide.
• Both MUC4 and HBD1 are expressed in vaginal epithelial cells and are considered
reliable markers of vaginal fluid.
• Additional mRNA and miRNA markers are described in table..

13. D-dimer Assay for Menstrual blood Identification

14. Lactate Dehydrogenase Assay

15. RNA-Based Assays
• In menstrual blood samples, among all of the MMP genes tested, MMP11 is the most
sensitive and specific marker for distinguishing menstrual blood from peripheral blood.
Using the
• RT-PCR technique, MMP11 mRNA can be detected in menstrual blood from the first to
the eighth day of menstruation but it is absent in peripheral blood and vaginal
secretions.
• Therefore, MMP11 can be used as a marker for the identification of menstrual blood.
• Likewise, MMP7 is also a useful marker for menstrual blood identification. However, the
MMP7 mRNA level in menstrual blood is less than that of MMP11.

16. References:
• Li, Richard, Forensic Biology Identification- Second Edition

17. Thank You