Describe the various tests prescribed for blood and seminal stains obtained during the examination of a victim of rape and their role in the investigation.

The examination of blood and seminal stains obtained from a victim of rape is a cornerstone of forensic investigation. These biological samples can provide definitive links between the victim and the assailant, corroborate accounts of the incident, and offer crucial insights into the sequence of events. The tests are broadly categorized into presumptive (screening) and confirmatory, followed by individualization techniques like DNA profiling.

Tests for Blood Stains

Bloodstains, if present on the victim's body or clothing, indicate potential injury during the assault or other associated violence. Their analysis helps determine the presence of human blood and subsequently, its origin.

1. Presumptive Tests (Screening Tests)

• Luminol Test:
  • Principle: Luminol reacts with the heme component of blood in the presence of an oxidizing agent (e.g., hydrogen peroxide), producing a blue-white luminescence in darkness. It is highly sensitive and can detect diluted or washed bloodstains.
  • Role: Used for preliminary screening of large areas to locate invisible or faint bloodstains at the crime scene or on clothing. It is non-destructive to DNA.
• Phenolphthalein Test (Kastle-Meyer Test):
  • Principle: This test involves a colorless phenolphthalein reagent which, in the presence of hydrogen peroxide and the catalytic activity of hemoglobin in blood, oxidizes to phenolphthalein, producing a pink color.
  • Role: A quick and sensitive test to indicate the probable presence of blood. It is often used to confirm positive luminol results on smaller, suspected stains.
• Tetramethylbenzidine (TMB) Test:
  • Principle: Similar to phenolphthalein, TMB reacts with hemoglobin to produce a blue-green color in the presence of hydrogen peroxide.
  • Role: Another sensitive presumptive test for blood.

2. Confirmatory Tests

These tests confirm unequivocally that the stain is indeed human blood.

• Teichmann Test (Hemochromogen Crystal Test):
  • Principle: Involves heating a small portion of the suspected bloodstain with glacial acetic acid and a halide (e.g., sodium chloride). Hemoglobin crystals (hemochromogen or hematin crystals) form upon cooling.
  • Role: Confirms the presence of blood by identifying its characteristic crystals.
• Takayama Test (Hemochromogen Crystal Test):
  • Principle: Similar to Teichmann, it forms characteristic pink rhomboid crystals of hemochromogen when heated with a pyridine-based reagent.
  • Role: A reliable confirmatory test for blood, often preferred due to cleaner crystal formation.
• Immunochromatographic Tests (e.g., ABAcard HemaTrace):
  • Principle: These tests detect human hemoglobin (Hb) by using anti-human Hb antibodies in a chromatographic strip format.
  • Role: Highly specific for human blood, offering a rapid and reliable confirmatory result. These are modern, easy-to-use tests commonly employed in forensic labs.

Tests for Seminal Stains

Seminal stains are critical evidence, directly linking the assailant to the act of sexual intercourse. Their detection, identification, and subsequent analysis are paramount.

1. Presumptive Tests (Screening Tests)

• Ultraviolet (UV) Light/Alternate Light Source (ALS) Examination:
  • Principle: Semen, particularly dried stains, often fluoresces under UV light (around 415-490 nm). This is due to the presence of substances like flavins and choline.
  • Role: Initial screening for invisible seminal stains on clothing, bedding, or body surfaces. It is non-destructive.
• Acid Phosphatase (AP) Test:
  • Principle: Seminal fluid contains high concentrations of acid phosphatase, an enzyme. The test uses a substrate (e.g., alpha-naphthyl phosphate) which, when hydrolyzed by AP, produces a colored product (e.g., purple with Fast Blue B salt).
  • Role: A highly sensitive and widely used presumptive test for semen. A rapid color change indicates the probable presence of seminal fluid. However, AP can also be found in other bodily fluids and plant materials, so it is not entirely specific.

2. Confirmatory Tests

These tests definitively confirm the presence of human semen, typically by identifying spermatozoa or seminal fluid components.

• Microscopic Examination for Spermatozoa:
  • Principle: Direct visualization of sperm cells (spermatozoa) under a microscope after staining (e.g., with Christmas Tree stain or Picro-Indigocarmine). Spermatozoa have characteristic head, midpiece, and tail structures.
  • Role: The most definitive confirmatory test for semen if intact spermatozoa are found. Even if the assailant is vasectomized or azoospermic, other tests are needed.
• Prostate Specific Antigen (PSA) Test / p30 Test:
  • Principle: PSA (also known as p30) is a protein exclusively produced by the human prostate gland and is present in high concentrations in seminal fluid. Immunochromatographic assays (similar to pregnancy tests) use antibodies specific to PSA.
  • Role: A highly specific confirmatory test for human seminal fluid, especially useful in cases where spermatozoa are absent (e.g., azoospermia, vasectomy, degradation). Examples include Seratec PSA and ABAcard p30 tests.
• Sperm-specific Lactate Dehydrogenase (LDH) Test (LDH-C4):
  • Principle: LDH-C4 is an isoenzyme of lactate dehydrogenase found specifically in spermatozoa. Its detection confirms the presence of sperm.
  • Role: Another specific confirmatory test for semen, particularly useful in challenging samples.

Advanced Techniques for Both Blood and Seminal Stains: DNA Profiling

After presumptive and confirmatory tests, the ultimate step for individualization and linking evidence is DNA analysis.

• DNA Extraction: Genetic material (DNA) is extracted from the detected blood or seminal stains. Modern techniques are sensitive enough to extract DNA from minute or degraded samples, including "touch DNA" (trace DNA).
• Quantification: Determines the amount of human DNA present and assesses its quality.
• Amplification (PCR - Polymerase Chain Reaction): Specific regions of DNA, particularly Short Tandem Repeats (STRs), are amplified. STRs are highly polymorphic and vary between individuals.
• DNA Profiling/Typing: The amplified STR fragments are separated and detected, generating a unique DNA profile for each individual. Y-STR analysis is particularly useful in sexual assault cases involving multiple male perpetrators or when high levels of female DNA mask male DNA.
• Comparison: The DNA profile obtained from the stains is compared with reference samples from the victim and suspected assailants. It can also be uploaded to national databases like CODIS (Combined DNA Index System) or its Indian equivalents to search for potential matches.
• Role in Investigation:
  • Positive Identification: Can conclusively link a suspect to the crime, providing irrefutable evidence of physical contact.
  • Exclusion: Can definitively exclude innocent suspects.
  • Corroboration: Supports or refutes statements made by the victim or suspect.
  • Multiple Perpetrators: Y-STR analysis helps in identifying DNA from multiple male contributors, even in mixed samples.
  • Cold Cases: DNA can be preserved and used years later to solve previously unsolved cases.

Role of these Tests in Investigation

These tests are integral to sexual assault investigations in several ways:

• Establishing the Crime: The presence of seminal fluid confirms sexual contact, which is a crucial element in proving sexual assault. The presence of the victim's blood or assailant's blood can indicate a struggle or injury.
• Linking Suspects to the Victim/Crime Scene: DNA profiles generated from blood and seminal stains found on the victim can be matched to a suspect, thereby establishing their presence and involvement. Similarly, the victim's blood on the suspect's clothing can link them.
• Corroborating Statements: Forensic findings can either support the victim's account of the assault or contradict a suspect's denial of involvement. For instance, finding a suspect's semen on a victim, coupled with injuries, strongly corroborates a non-consensual act.
• Excluding Individuals: If the DNA profile from the stain does not match that of a suspect, it can help exclude them from the investigation, focusing resources on other leads.
• Providing Investigative Leads: In cases where a suspect is unknown, a DNA profile can be entered into a database, potentially leading to a match with a previously convicted offender or from other crime scenes.
• Determining the Number of Assailants: Advanced DNA techniques like Y-STR profiling can differentiate DNA from multiple male contributors, which is crucial in gang rape cases.
• Time Since Assault: While not precise, the state of degradation of biological fluids can sometimes provide a rough estimate of the time elapsed since deposition, though this is highly variable.
• Medico-legal Documentation: The systematic collection and analysis of these stains, along with detailed documentation, form a critical part of the medico-legal report, which is admissible in court as scientific evidence.

In India, guidelines like those from the Directorate General of Health Services and the National Human Rights Commission (NHRC) issued SOPs (2020) emphasize the importance of proper collection and handling of forensic evidence, including blood and seminal stains, to ensure effective prosecution and conviction in sexual assault cases. The DNA Technology (Use and Application) Bill 2019 further underscores the legal framework for DNA profiling in criminal justice.

Type of Stain	Presumptive Tests	Confirmatory Tests	Role in Investigation
Blood Stains	Luminol, Phenolphthalein (Kastle-Meyer), TMB	Teichmann, Takayama, Immunochromatographic (e.g., HemaTrace)	Indicates injury/struggle, presence of human blood. Source identification via DNA.
Seminal Stains	UV Light/ALS, Acid Phosphatase (AP) Test	Microscopic examination (spermatozoa), PSA/p30 Test, LDH-C4	Confirms sexual contact, identifies presence of seminal fluid/sperm. Source identification via DNA.
Both (Post-Confirmatory)	N/A	DNA Profiling (STR, Y-STR, mtDNA)	Individualization, linking suspect to victim, exclusion of innocent, database searches.