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Summary Table

Category	Detail
Purpose	Confirm recent sexual contact in medicolegal cases (sexual assault, rape, disputed paternity)
Specimen Sources	Vaginal swab (most common), oral swab, rectal swab, clothing, bedding, skin surface
Screening Tests	Acid phosphatase (AP) reaction, ultraviolet (UV) fluorescence
Confirmatory Tests	Microscopic identification of spermatozoa, Prostate-Specific Antigen (PSA/p30), DNA profiling
Critical Timeframes	Motile sperm in vagina: up to 6 hours; non-motile sperm in vagina: up to 72 hours (some sources extend to 120 hours); cervical canal: up to 5-7 days
Key Limitations	Azoospermic or vasectomized perpetrators produce semen without spermatozoa; absence of sperm does not exclude assault
Keywords	Acid phosphatase, Florence test, Berberio test, PSA/p30, secretor status, motile vs non-motile sperm, digital penetration

1. Principles and Context

Forensic sperm analysis is performed primarily to establish evidence of sexual contact in medicolegal investigations.

Semen is the composite fluid produced by the testes, seminal vesicles, prostate, and bulbourethral glands. A normal ejaculate contains approximately 2-6 mL of fluid with a sperm concentration of 15-250 million spermatozoa per mL. Understanding the composition matters because each component offers a different forensic marker. The prostatic fraction is rich in acid phosphatase and PSA, which is why these markers remain detectable even in the absence of spermatozoa (e.g., vasectomized individuals). The seminal vesicle fraction contributes fructose and the coagulum that initially traps semen in the vaginal vault, slowing drainage and extending the detection window.

A critical concept for exam questions: the presence of motile spermatozoa implies very recent ejaculation (within approximately 6 hours in the vaginal canal), while the presence of non-motile but intact spermatozoa extends the window to roughly 72 hours. Beyond that, only sperm heads or degraded components may be found, and the timeline becomes less reliable. This distinction between motile and non-motile sperm is frequently tested to determine the approximate timing of assault.

2. Detection Timeline by Anatomical Site

Site	Motile Sperm	Non-Motile Sperm (Intact)	Sperm Heads / Degraded
Vagina	Up to 6 hours	Up to 72 hours	Up to 5-7 days (some references cite longer)
Cervical Canal	Up to 12-24 hours	Up to 5-7 days	Up to 12-14 days
Oral Cavity	Up to 6 hours	Up to 12-24 hours	Rarely beyond 24 hours (salivary enzymes degrade rapidly)
Rectum	Up to 6 hours	Up to 24-48 hours	Up to 72 hours
Skin / Clothing	Variable	Variable (dried stains can preserve for years)	Dried stains on fabric are stable indefinitely if not washed

The oral cavity has the shortest detection window due to salivary amylase and the natural self-cleaning mechanism of the mouth. This is a commonly tested fact: a negative oral swab does not exclude oral assault if the examination was delayed. In contrast, the cervical canal has the longest window because cervical mucus acts as a protective reservoir.

3. Diagnostic Workup

Test	What It Detects	Limitation
UV fluorescence (Wood lamp)	Semen stains on skin/clothing fluoresce bluish-white	Non-particular: other substances (urine, fabric detergent, lubricants) also fluoresce
Acid phosphatase (AP) test	Prostatic acid phosphatase in semen	Present in vaginal fluid at lower levels; must interpret quantitatively
Florence test	Choline crystals form dark-brown rhombic crystals with iodine-potassium iodide	Not entirely particular to semen
Berberio test	Spermine reacts with picric acid to form yellow needle-shaped crystals	Confirms seminal plasma, not spermatozoa themselves
Microscopy (wet mount / stained)	Direct visualization of spermatozoa	Negative in azoospermic / vasectomized individuals
PSA / p30 (Prostate-Specific Antigen)	Glycoprotein produced exclusively by prostate	Detects semen even without sperm; highly particular
DNA profiling (STR analysis)	Perpetrator-linked genetic material	Requires adequate sample; degradation can be an issue

Step 1: Scene and Physical Examination. The first step is always a careful collection protocol. Swabs must be taken from the vaginal vault (posterior fornix yields the highest concentration), endocervix, rectum, and oral cavity as indicated by the history. Clothing and bedding are examined under UV light (Wood lamp) to locate potential semen stains, which fluoresce bluish-white. However, UV fluorescence is a screening tool only and is not particular because other biological fluids and non-biological substances produce similar fluorescence.

Step 2: Presumptive Chemical Testing. The acid phosphatase (AP) test is the best initial screening test for semen. Seminal acid phosphatase concentration is 400-fold higher than in any other body fluid, which is why a strongly positive AP reaction is highly suggestive. The Florence test (choline detection) and Berberio test (spermine detection) are older chemical methods that confirm the presence of seminal plasma components but not spermatozoa themselves. These are tested as factual recall items.

Step 3: Confirmatory Testing. The gold standard for confirming sexual contact is microscopic identification of spermatozoa using differential staining techniques. The most widely used is the Christmas tree stain (nuclear fast red + picroindigocarmine), which stains the sperm head red/pink and the tail blue/green. Alternative methods include the Baecchi stain (acid fuchsin + methylene blue), which stains the head red and the tail blue, and the malachite green + eosin yellowish combination, which stains the head red and the tail green. All three achieve the same goal: definitive visualization of spermatozoa under light microscopy, even when sperm are non-motile or partially degraded.

Staining Method	Components	Sperm Head	Sperm Tail
Christmas Tree Stain	Nuclear fast red + picroindigocarmine	Red/pink (acrosomal cap pink)	Blue/green
Baecchi Stain	Acid fuchsin + methylene blue	Red	Blue
Malachite Green Stain	Malachite green 1% + eosin yellowish 1%	Red (eosin yellowish)	Green (malachite green)

In cases involving vasectomized or azoospermic perpetrators, spermatozoa will be absent regardless of staining method. In these cases, PSA/p30 detection becomes the confirmatory test of choice because it is produced by the prostate regardless of sperm presence.

Step 4: Perpetrator Identification. DNA profiling using Short Tandem Repeat (STR) analysis is the standard for linking a semen sample to a particular individual. Mixed DNA samples (victim and perpetrator) can be separated using differential extraction, where epithelial cells lyse under milder conditions while sperm heads, protected by disulfide bonds, require stronger lysis with DTT (dithiothreitol).

4. Secretor Status and Blood Group Determination

Summary Table

Category	Secretor (approx. 80% of population)	Non-Secretor (approx. 20%)
ABO antigens in body fluids	Present (saliva, semen, vaginal fluid)	Absent
Forensic utility	Blood group can be determined from semen stain	Blood group cannot be determined from semen alone
Governed by	FUT2 gene (Se gene)	Homozygous recessive (sese)

Approximately 80% of individuals are secretors, meaning they express ABO blood group antigens in their body fluids including semen. This allows forensic examiners to determine the blood group of the perpetrator from a semen stain without needing a blood sample. If the perpetrator is a non-secretor, ABO typing from semen is not possible, and the case depends entirely on DNA analysis.

5. Medicolegal Considerations in Examination

Aspect	Detail
Consent	Written informed consent from the victim (or guardian) required before examination
Examiner	Should be same gender as victim when possible; two witnesses present
Chain of custody	Every sample must be labeled, sealed, signed, and documented at every transfer
Negative findings	Do not exclude assault (digital penetration, condom use, vasectomy, delayed reporting)
Timing of exam	Ideally within 72 hours, but collection can be attempted later depending on site

6. Traps and High-Yield Pearls

The single most common trap in forensic sperm analysis questions is the false equivalence between absence of evidence and evidence of absence. Test-writers deliberately construct vignettes where the forensic exam is negative (no sperm, no injury, no semen detected) and then ask whether sexual assault can be excluded. The answer is always no. A negative exam in the setting of a consistent history does not rule out assault.

The second major trap involves timeline interpretation. If a vignette describes motile sperm found on vaginal swab, the exam is testing whether you recognize this as evidence of contact within the last 6 hours, not within 72 hours. Conversely, if only sperm heads are found, the contact may have occurred several days prior.

Third, students frequently confuse screening vs. confirmatory hierarchy. Acid phosphatase is the best initial test, microscopy is confirmatory for the presence of sperm, and PSA/p30 is confirmatory for semen even without sperm. DNA profiling identifies the perpetrator but does not by itself prove assault occurred. The core competency being tested is your ability to sequence these tests logically and interpret each result within its proper limitations.

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