Forensic biology and serology

In short

Forensic biology and serology is the examination of biological evidence of human and animal origin — blood, semen, saliva, urine, vaginal secretion, hair, bone, diatoms, insects and plant material — to establish *what* the stain is, *which species* it came from, and *how* it got there. It answers the questions that come before identity: is this stain blood at all, is it human, is it semen, was the victim alive when wounded, was the body moved. It is examined in B.Sc/M.Sc Forensic Science papers, UGC-NET Forensic Science, and recruitment tests for Scientific Assistant/Scientific Officer posts in CFSLs (DFSS, MHA), State FSLs, NFSU and NCRB-linked units. In practice it is the daily work of the Serology and Biology divisions of Indian FSLs, admitted in court as expert opinion under Section 39 of the Bharatiya Sakshya Adhiniyam, 2023 (old Section 45, Indian Evidence Act).

Explained

Scope, sequence and the presumptive-confirmatory logic

• Three-tier examination: (1) screening/presumptive — fast, cheap, sensitive, not specific; (2) confirmatory — specific for the fluid or species; (3) individualisation — grouping or DNA (covered separately).
• A negative presumptive test effectively excludes the fluid; a positive presumptive test only *permits* the next step. Never report a presumptive result as identification.
• Order of work is fixed: document and photograph → collect (dried stains lifted on sterile swab moistened with saline/distilled water, or cut the substrate) → air-dry → pack in paper/khaki envelope, never polythene (moisture causes bacterial and fungal degradation) → seal, label, chain of custody → non-destructive tests first, DNA-consuming tests last.
• Search aids: oblique/grazing light, UV and ALS (alternate light source) — semen, saliva and urine fluoresce around 450 nm with orange goggles; luminol/fluorescein for washed or diluted blood in darkness.
• Indian statutory frame: BNSS, 2023 Section 176(3) requires forensic investigation and videographed collection for offences punishable with seven years or more (being phased in by States); Sections 51–52 BNSS cover medical examination of the accused and Section 184 the examination of a rape survivor, with the report to the investigating officer within seven days; the MoHFW *Guidelines and Protocols: Medico-legal Care for Survivors/Victims of Sexual Violence* (2014) prescribe the SAFE kit; the "two-finger test" was held unlawful by the Supreme Court in 2022. Labs seek NABL accreditation to ISO/IEC 17025:2017.

Blood: identification, species and grouping

• Composition relevant to testing: RBCs carry haemoglobin (haem iron gives all presumptive tests their peroxidase-like activity); WBCs carry nuclear DNA; plasma carries antibodies.
• Presumptive (catalytic) tests — haem catalyses oxidation of a colourless reagent by hydrogen peroxide:
  • Kastle-Meyer (phenolphthalin) → deep pink. Sensitivity about 1:10,000 to 1:100,000 dilution.
  • Leucomalachite green → blue-green; Tetramethylbenzidine (TMB)/Hemastix → blue-green; o-tolidine → blue.
  • Benzidine test is now obsolete and prohibited in practice — benzidine is a proven human carcinogen.
  • Luminol → blue chemiluminescence, detects dilutions to about 1:1,000,000 and reveals wiped-up patterns; fluorescein gives a longer-lasting glow and less dilution of the stain.
  • False positives: plant peroxidases (potato, horseradish, turnip), rust and metal salts (Cu, Ni), chemical oxidants (hypochlorite bleach), some vegetable juices. Sodium hypochlorite gives a colour before peroxide is added — hence the three-step sequence (reagent, observe, then peroxide).
• Confirmatory for blood:
  • Takayama (haemochromogen) test — pyridine + glucose + NaOH forms salmon-pink needle-shaped pyridine haemochromogen crystals; works on old stains.
  • Teichmann test — glacial acetic acid + NaCl, heated, gives brown rhombic haemin (ferriprotoporphyrin chloride) crystals; fails on very old or heated stains.
  • Microspectrophotometry/absorption spectra of oxyhaemoglobin, methaemoglobin, haematin; immunochromatographic human-haemoglobin strips (HemaTrace/ABAcard) and RSID-Blood (glycophorin A) are both confirmatory *and* human-specific.
• Species determination (medico-legal "is it human?"):
  • Precipitin/ring test — stain extract layered over anti-human serum; a white ring at the interface indicates human protein.
  • Ouchterlony double diffusion in agar gel, crossover (counter-current) electrophoresis, ELISA, and anti-human haemoglobin immunoassay.
  • Cross-reactivity with higher primates is the known limitation; for wildlife matter, species is confirmed by hair/bone morphology or mitochondrial markers at WII Dehradun.
• Blood grouping from dried stains (historic but still examined):
  • Absorption-elution — antisera absorbed by antigens in the stain, washed, then eluted at 56 °C and detected with indicator cells. Most sensitive and most used.
  • Absorption-inhibition — residual unbound antiserum is titrated against known cells.
  • Mixed agglutination — for fabrics and single fibres.
  • Lattes crust test — detects *antibodies* (anti-A/anti-B) in the blood crust; needs a fairly large crust.
  • Secretor status: about 80% of people are secretors (functional *FUT2*/Se gene; the separate Lewis gene is *FUT3*) and express ABH antigens in saliva, semen and sweat; non-secretors cannot be grouped from such stains.
  • Grouping has very low discriminating power and is now only a screening or exclusion tool.

Bloodstain pattern analysis (BPA)

• Passive stains: drip stains, drip trails, pools, flows, saturation and transfer/contact patterns (including swipe — moving bloodied object onto a clean surface — and wipe — a clean object through a wet stain).
• Spatter by energy: low-velocity/gravitational (drops > 4 mm, falling blood); medium-velocity/impact (1–4 mm, beating or stabbing); high-velocity/mist (< 1 mm, gunshot, high-speed machinery). Modern BPA prefers mechanism-based terms (impact spatter, gunshot spatter, cast-off, expirated, arterial spurt) over velocity labels.
• Other diagnostic patterns: cast-off (blood flung from a swung weapon — counts can indicate minimum number of blows), arterial gush/spurt (large-volume zig-zag), expirated blood (bubble rings, diluted, with saliva), void (absence showing an object or person was present), skeletonised stain (drying ring shows the stain was disturbed later).
• Surface effect: a smooth glazed tile gives round stains with few spines; rough concrete or cloth gives heavily scalloped, spiny stains — always test the same surface with known blood before interpreting.
• Directionality: the pointed/spiny end and satellite spatter point in the direction of travel. Area of convergence (2-D, by back-projecting long axes) and area of origin (3-D, adding the impact angle) locate where the blood source was.
• Limits: BPA is interpretative; drop volume, surface texture, drying and overlapping events all introduce error, and the stain tells you about the blood source, not about who struck whom.

Semen, saliva and other body fluids

• Semen — most examined evidence in sexual-assault cases:
  • *Presumptive:* ALS fluorescence; acid phosphatase (AP) test — Brentamine Fast Blue B/Walker's test gives purple in seconds (semen AP is far higher than vaginal, so a rapid strong colour is significant); Florence iodine test — dark brown choline periodide crystals; Barberio's test — yellow needle-shaped spermine picrate crystals with picric acid.
  • *Confirmatory:* microscopic identification of spermatozoa (head, neck, tail; heads alone persist longest), aided by Christmas tree stain (nuclear fast red stains heads red, picroindigocarmine stains tails green); PSA/p30 immunoassay (ABAcard p30) and RSID-Semen (semenogelin) are the accepted confirmatory tests for azoospermic, oligospermic or vasectomised donors.
  • Sperm persistence: roughly up to 3 days in the vagina, shorter in the mouth, days longer on dried clothing; figures vary widely with case circumstances.
• Saliva: α-amylase — starch-iodine (amylase digests starch, so the blue colour disappears), Phadebas press test (blue zone on a reagent paper) for mapping stains on cloth; RSID-Saliva is specific for human salivary amylase. Useful on bite marks, cigarette butts, envelopes, gags, bottles.
• Urine: urea by urease/DMAC reagent, creatinine by Jaffe's test (picric acid in alkali → orange-red); characteristic odour and UV fluorescence are indicative only.
• Faeces: urobilinogen (Edelmann's test), microscopic plant and muscle debris.
• Vaginal secretion: glycogenated squamous epithelial cells stained with Lugol's iodine or PAS; not conclusive. Menstrual blood is indicated by fibrin degradation products/D-dimer and endometrial cells.
• Sweat: no reliable confirmatory test; relevant mainly as a DNA source on worn clothing.
• mRNA body-fluid profiling (tissue-specific transcripts) is the research-grade route to true fluid specificity and is being adopted slowly in Indian labs.

Hair, fibre-adjacent biology, and wildlife material

• Structure: cuticle (overlapping scales), cortex (pigment granules, ovoid bodies, cortical fusi) and medulla (continuous, interrupted, fragmented or absent).
• Medullary index distinguishes human from animal; scale pattern — coronal (crown-like; rodents, bats), spinous/petal (cat, seal, mink), imbricate/flattened (human and most large mammals).
• Growth phases: anagen (active, sheath present — best for nuclear DNA), catagen (transition), telogen (resting, club-shaped root — usually mitochondrial DNA only).
• Determinable from a hair: species, somatic origin (head, pubic, beard, body), whether cut/pulled/burnt/crushed, artificial colouring, disease and some drug/heavy-metal exposure. Not determinable with certainty: age, sex (without DNA), or individual identity — microscopy gives class characteristics only.
• Wildlife and plant forensics: species identification from hair, quills, claws, horn, bone and skin under the Wild Life (Protection) Act, 1972 (as amended, 2022) — for example shahtoosh (Tibetan antelope) versus pashmina goat hair; pollen and diatom assemblages (forensic palynology) link a body or vehicle to a place.
• Diatom test for drowning: siliceous diatoms carried by inhaled water into the circulation lodge in femoral bone marrow; recovery of diatoms matching the water body supports ante-mortem drowning. Needs an acid-digestion control, because diatoms are environmentally ubiquitous.
• Forensic entomology: blowfly (Calliphoridae) colonisation and faunal succession estimate the minimum post-mortem interval through accumulated degree-days/hours and species development data; Indian species data are region-specific, so local temperature records matter.

Key points

• Presumptive test - sensitive, non-specific screening (Kastle-Meyer, luminol); positive only means "proceed further".
• Confirmatory test - specific identification of the fluid or species (Takayama, Teichmann, sperm microscopy, p30).
• Catalytic principle - haem iron mimics peroxidase and oxidises the reagent in presence of H₂O₂.
• Luminol - detects blood diluted about 1:1,000,000 and reveals cleaned-up stains; does not destroy DNA at working concentrations but must be used after documentation.
• Takayama gives pink haemochromogen needles; Teichmann gives brown haemin rhombs.
• Precipitin/Ouchterlony - antigen-antibody precipitation proves human (or named animal) origin.
• Absorption-elution - the standard ABO grouping method for dried stains; Lattes detects antibodies in the crust.
• Secretors (~80%) express ABH antigens in saliva and semen; non-secretors cannot be grouped from those fluids.
• Acid phosphatase - fast presumptive for semen; spermatozoa or PSA/semenogelin is confirmatory, essential in azoospermic cases.
• Christmas tree stain - nuclear fast red (heads) + picroindigocarmine (tails).
• α-amylase - marker for saliva (starch-iodine, Phadebas); creatinine/urea for urine; D-dimer for menstrual blood.
• Angle of impact sin θ = width/length; stains become more elongated as the angle falls.
• Area of convergence is 2-D; area of origin adds height using tan θ.
• Cast-off, expirated, arterial and void patterns each carry a distinct mechanistic meaning.
• Medullary index < 0.3 = human; > 0.5 = animal; scale patterns are coronal, spinous or imbricate.
• Anagen roots give nuclear DNA; telogen usually only mitochondrial DNA.
• Diatoms in femoral marrow support ante-mortem drowning; controls are mandatory.
• BNSS, 2023 s.176(3) mandates forensic investigation for offences carrying seven years or more; expert opinion is admitted under BSA, 2023 s.39.

Formulas and examples

Example 1 — Angle of impact and height of origin. A spatter stain on a wall measures L = 12.0 mm, W = 6.0 mm; the area of convergence is 80 cm horizontally from it.

• sin θ = 6.0 / 12.0 = 0.500 → θ = 30.0°.
• h = 80 × tan 30° = 80 × 0.5774 ≈ 46.2 cm above the convergence plane.
• Interpretation: the blood source was roughly 46 cm above that line — consistent with a victim seated or crouching, not standing.

Example 2 — Dilution and presumptive sensitivity. 0.1 mL of blood is washed into a bucket holding 10 L of water.

• DF = 0.1 mL / 10,000 mL = 1 : 100,000.
• Kastle-Meyer (limit about 1:100,000) is at the edge of detection and may read weak or negative; luminol (about 1:1,000,000) should still chemiluminesce. Hence luminol is the reagent of choice for washed floors.

Example 3 — Blood loss estimate. A 68 kg adult male; the scene shows pooled blood estimated at 1.9 L.

• TBV ≈ 0.07 × 68 = 4.76 L.
• Fraction lost = 1.9 / 4.76 = 0.399 ≈ 40% — Class III haemorrhage (30–40%), right at the Class IV threshold (>40%), consistent with a potentially fatal exsanguinating injury. (Pool-volume estimates from area are crude; state them as approximations.)

Example 4 — Discriminating power of serological grouping. A vaginal swab stain types as group B, and the donor is a secretor. Taking approximate Indian frequencies group B ≈ 0.33 and secretors ≈ 0.80 (frequencies vary considerably between regions and communities):

• P = 0.33 × 0.80 = 0.264, i.e. about 1 in 3.8 unrelated men would match.
• Conclusion to be written as "cannot be excluded" — never as identification; DNA typing is required for individualisation.

Example 5 — Minimum PMI from accumulated degree-hours. A blowfly species needs 250 ADH above a base of 10 °C to reach the third instar; the recorded mean ambient temperature is 30 °C.

• Accumulation per hour = 30 − 10 = 20 degree-hours.
• Time = 250 / 20 = 12.5 h, so the minimum PMI is about 12–13 hours.
• If the mean temperature were 20 °C: 250 / (20 − 10) = 25 h — showing how strongly temperature drives the estimate.

Example 6 — Medullary index. Hair A: medulla 16 µm, shaft 80 µm → MI = 16/80 = 0.20 → human. Hair B: medulla 54 µm, shaft 90 µm → MI = 54/90 = 0.60 → animal (non-human).

Where it is used

• CFSLs under DFSS, MHA (Delhi, Kolkata, Chandigarh, Hyderabad, Pune, Bhopal, Guwahati) and State FSLs/RFSLs — Serology and Biology divisions handling murder, assault and sexual-offence exhibits.
• Sexual-assault casework in hospitals and police: SAFE/Nirbhaya kit collection by medical officers, examination under BNSS s.184, and FSL reporting of semen, saliva and blood on swabs and garments.
• Crime-scene units and mobile forensic vans (now scaled up in several States under BNSS s.176(3)) — luminol searches, bloodstain pattern documentation, videographed collection.
• Wildlife and customs enforcement: Wildlife Institute of India, Dehradun and TRAFFIC India — species identification of hair, skin, horn, bone and shahtoosh wool under the Wild Life (Protection) Act, 1972.
• Job roles: Scientific Assistant/Scientific Officer (Biology & Serology), crime-scene manager, medico-legal technician, research associate in NFSU/university labs, and QA roles maintaining NABL ISO/IEC 17025:2017 accreditation.
• Allied applications: paternity and relationship testing units, disaster-victim identification teams, insurance and veterinary fraud investigation, forensic entomology and palynology consultancies.

Interview questions

• Q: Why is the Kastle-Meyer test called presumptive and not confirmatory? A: It detects any peroxidase-like activity, so plant peroxidases, metal salts and oxidants also give a pink colour; it proves the possibility of blood, not its presence.
• Q: What is the sequence of adding reagents in the Kastle-Meyer test and why does it matter? A: Phenolphthalin first, observe, then hydrogen peroxide — a colour appearing before the peroxide indicates a chemical oxidant such as bleach, not blood.
• Q: Differentiate the Takayama and Teichmann tests. A: Takayama forms salmon-pink pyridine haemochromogen needles and works well on old stains; Teichmann forms brown rhombic haemin crystals and fails on heated or very aged stains.
• Q: A vasectomised man is the accused and no spermatozoa are seen. How do you prove semen? A: By immunoassay for prostate-specific antigen (p30) or for semenogelin (RSID-Semen), both of which are seminal-plasma markers independent of sperm.
• Q: How do you establish that a bloodstain is of human origin? A: By antigen-antibody methods — precipitin ring test, Ouchterlony double diffusion, crossover electrophoresis or a human-haemoglobin immunochromatographic strip.
• Q: What is a secretor and why does it matter in serology? A: A person (about 80% of the population) who secretes ABH antigens into body fluids; only their saliva or semen stains can be ABO grouped by absorption-elution.
• Q: How is the angle of impact calculated and what does a 90° stain look like? A: sin θ = width/length; a drop striking at 90° gives a circular stain with no directional tail.
• Q: Distinguish a swipe from a wipe. A: A swipe transfers blood from a bloodied object onto a clean surface; a wipe is a clean object passing through an existing wet stain and altering it.
• Q: What can and cannot be determined from a single hair under a microscope? A: Species, somatic origin, growth phase, damage and treatment can be determined; age, sex and individual identity cannot — those need DNA.
• Q: Why are diatoms sought in bone marrow rather than lung tissue in a suspected drowning? A: Lungs can be contaminated passively after death; diatoms reaching closed femoral marrow require circulation, implying the person was breathing and alive in the water.
• Q: Why must biological exhibits be packed in paper and not plastic? A: Plastic traps moisture, promoting bacterial and fungal growth that degrades proteins and DNA; paper allows the stain to stay dry.
• Q: Under which law is the FSL expert's report admitted in an Indian court? A: As expert opinion under Section 39 of the Bharatiya Sakshya Adhiniyam, 2023 (corresponding to the former Section 45 of the Indian Evidence Act, 1872), with the examination itself governed by the BNSS, 2023.

Common mistakes

• Reporting a positive Kastle-Meyer, acid phosphatase or luminol result as "blood found" or "semen found" instead of "indicated".
• Writing benzidine as a current reagent — it is carcinogenic and replaced by TMB, LMG or phenolphthalin.
• Confusing Florence (brown choline periodide crystals) with Barberio (yellow spermine picrate crystals), or reversing the Takayama and Teichmann crystal descriptions.
• Using the wrong trigonometric function in BPA — sine for the angle but tangent for the height — or measuring stain length including the tail, which inflates L and under-estimates θ.
• Treating ABO grouping or hair microscopy as individualising evidence; both give class characteristics only.
• Claiming sex or age can be read from hair morphology, or that the mere presence of diatoms proves drowning without controls from the water body and the body itself.
• Doing destructive or DNA-consuming tests before documentation, species testing and swab sub-sampling, and skipping the substrate control and reagent blank.

Related topics

DNA profiling · Forensic medicine basics · Forensic toxicology · Forensic chemistry · Crime scene management and chain of custody · Questioned documents and trace evidence