Bloodstain Analysis & DNA Fingerprinting

Biological stains — blood, semen, saliva, hair — are the workhorses of forensic identification. This topic moves stepwise from "Is it blood?" to "Is it human?" to "Whose is it?", culminating in DNA profiling, which has displaced classical serology as the gold standard for individualisation. It is a favourite NEET PG area because each test has a precise principle, a named eponym, and a characteristic colour or band pattern.

The forensic logic of identification

Identification of a stain follows a fixed, examinable sequence. Each step narrows the possibilities — you never jump straight to DNA without first establishing that the stain is biological.

Stain found → Presumptive (screening) test: is it blood? → Confirmatory test: is it really blood? → Species test: is it human? → Blood grouping → DNA profiling (individualisation)

High-yield: A presumptive (screening) test is sensitive but NOT specific — a negative result rules blood out, a positive result only suggests it. A confirmatory test is specific. DNA fingerprinting is the only test that achieves true individualisation (identifies a single person).

Classification of stain tests

Category	Purpose	Examples	Specificity
Presumptive / screening	Detect possible blood	Kastle–Meyer (phenolphthalein), benzidine, leucomalachite green, luminol, Hemastix	Low (sensitive, not specific)
Confirmatory (crystal)	Prove it is blood	Teichmann (haemin), Takayama (haemochromogen)	High
Species identification	Prove it is human	Precipitin (Uhlenhuth), gel diffusion, mixed agglutination, anti-human haemoglobin	High
Individualisation	Identify the person	DNA fingerprinting (RFLP, PCR-STR)	Highest

Presumptive (screening) tests for blood

These exploit the peroxidase-like activity of haem, which catalyses oxidation of a colourless reagent by hydrogen peroxide, producing colour or light.

• Kastle–Meyer test — reduced phenolphthalein (phenolphthalin) + H₂O₂ → deep pink colour. Most popular field screening test.
• Benzidine test (Adler test) — gives a blue/blue-green colour. Highly sensitive (detects up to 1 in 300,000 dilution) but benzidine is carcinogenic, so it is now largely abandoned.
• Leucomalachite green — produces a green colour.
• Orthotoluidine / Hemastix — blue colour; Hemastix is a urine dipstick adapted for field use.
• Luminol — emits bluish-white chemiluminescence in the dark; used to detect diluted, washed, or invisible bloodstains over large areas (e.g., a cleaned crime scene). It does not destroy DNA, so samples can still be profiled afterwards.

High-yield: Benzidine is the most sensitive presumptive test but is carcinogenic. Kastle–Meyer (phenolphthalein) gives a pink colour. Luminol detects washed/latent blood by chemiluminescence.

False positives of peroxidase-based tests: vegetable/plant peroxidases (potato, horseradish, turnip), certain metallic salts (copper, iron rust), and oxidising agents. This is exactly why a positive screen always demands confirmation.

Confirmatory crystal tests

These convert haemoglobin into characteristic microscopic crystals.

Test	Reagent	Crystal formed	Colour/shape
Teichmann	Glacial acetic acid + NaCl, heated	Haemin (haematin) chloride	Rhombic, brown crystals
Takayama	Pyridine + glucose + NaOH	Haemochromogen (pyridine ferroprotoporphyrin)	Pink, feathery/needle-shaped

High-yield: Teichmann test → haemin chloride crystals (rhombic, brown). Takayama test → haemochromogen crystals (pink, feathery). Mnemonic: "Teichmann = T = haematin/Tan-brown rhombic; Takayama = pink feathers."

Spectroscopic examination of haemoglobin and its derivatives (oxyhaemoglobin, methaemoglobin, sulph-met-Hb, carboxyhaemoglobin) also confirms blood and can indicate the age/condition of the stain.

Species identification — is it human?

Once confirmed as blood, the next question is whether it is human or animal. Tests rely on the antigen–antibody reaction using antiserum raised against human protein in a rabbit.

• Precipitin test (Uhlenhuth test) — the classic. Anti-human serum (from a rabbit immunised with human serum) is layered over the stain extract; a white precipitin ring at the interface indicates human protein. Can detect blood as old as 10–15 years and even in dilutions of 1:1000.
• Gel diffusion (Ouchterlony double diffusion) — antigen and antibody diffuse through agar gel; precipitin lines form where they meet in optimal proportion.
• Precipitin (rocket) immunoelectrophoresis — faster, current-driven version.
• Mixed agglutination — used for minute, old, or dried stains where the antigen survives even when serological activity is reduced. Group-specific antigens on the stained fibres bind added antibody, then indicator red cells of the same group agglutinate around the fibre.
• Anti-human haemoglobin / HemoTrace (ABAcard) — modern immunochromatographic strip specific for human haemoglobin; rapid bedside-style confirmation of human blood.

High-yield: The precipitin test (Uhlenhuth) is THE species-identification test — anti-human serum raised in a rabbit gives a white ring with human blood. Mixed agglutination is preferred for old, dried, minute stains.

High-yield: Precipitin test only tells you the blood is human (or which species). It cannot tell you which human — that needs grouping and DNA.

Blood grouping from stains

After species identification, ABO, Rh, and other systems (MN, secretor status, serum proteins, enzyme polymorphisms) were classically determined to narrow identity.

• Fresh blood — direct agglutination (slide/tube) for ABO and Rh.
• Dried stains — absorption–elution technique (antibody is absorbed onto stain antigen, then heat-eluted and detected with indicator cells); absorption–inhibition is an alternative.
• Lattes crust method — detects naturally occurring antibodies (anti-A, anti-B) in a dried blood crust, indicating the person's group.
• Secretors (≈80% of people) secrete ABO antigens in saliva, semen, sweat, and other body fluids — so grouping can be done from a saliva or semen stain in secretors.

Grouping gives exclusionary value only; it can exclude a suspect or assign a probability but cannot individualise. DNA profiling has largely superseded it for positive identification, though grouping remains useful as a quick, cheap screen.

DNA fingerprinting — principles

DNA profiling exploits the fact that 99.9% of human DNA is identical between individuals; identification rests on the 0.1% that is polymorphic. Except for monozygotic (identical) twins, every individual has a unique DNA profile.

• Discovered by Sir Alec Jeffreys (1984, University of Leicester) — the father of DNA fingerprinting. First forensic use: the Colin Pitchfork case (1986–88, UK) — also the first time DNA exonerated an innocent suspect.
• The polymorphic regions used are non-coding (junk) DNA containing tandem repeats:
  • VNTRs (Variable Number Tandem Repeats) = minisatellites (10–100 bp repeat units) — used in RFLP.
  • STRs (Short Tandem Repeats) = microsatellites (2–6 bp repeat units) — used in modern PCR profiling.
• DNA is the same in every nucleated cell of an individual, so blood, semen, saliva, hair root, bone, or tissue all give the same profile.

High-yield: Alec Jeffreys invented DNA fingerprinting (1984). It uses polymorphic non-coding repetitive DNA (VNTR/STR). Identical (monozygotic) twins have identical DNA profiles — the one situation where DNA cannot distinguish two people.

DNA profiling techniques

Feature	RFLP	PCR–STR (current standard)
Target	VNTR / minisatellites	STR / microsatellites
DNA needed	Large amount (~µg)	Tiny amount (~ng or less)
Sample quality	Needs fresh, undegraded DNA	Works on degraded/old samples
Enzyme	Restriction endonuclease	DNA polymerase (Taq)
Detection	Southern blot + radioactive probe	Capillary electrophoresis + fluorescent primers
Time	Slow (weeks)	Fast (hours–days)
Discrimination	Good	Excellent (multiplex, many loci)

RFLP (Restriction Fragment Length Polymorphism)

Steps: Extract DNA → cut with restriction endonuclease at specific sites → separate fragments by gel electrophoresis → Southern blotting onto a membrane → hybridise with a labelled (radioactive ³²P or chemiluminescent) probe → autoradiograph shows a "bar-code" band pattern unique to the individual.

Limitations: needs large amounts of high-quality, non-degraded DNA, is slow, and uses radioactivity — hence largely replaced by PCR methods.

PCR–STR (the workhorse today)

PCR (polymerase chain reaction) amplifies target STR loci a million-fold, so even trace, degraded, or aged samples (a single hair root, cigarette butt, touch DNA) can be profiled.

Steps of PCR: Denaturation (94–95 °C) → Annealing of primers (~50–60 °C) → Extension by Taq polymerase (72 °C) → repeat 25–35 cycles.

Modern kits multiplex 13–24 STR loci. The international standard set is the CODIS loci (originally 13, now expanded to 20 core loci since 2017) used by the FBI's Combined DNA Index System. Amelogenin locus is co-amplified for sex determination (X vs XY band pattern).

High-yield: Amelogenin locus on PCR profiling determines sex (X = female, X+Y = male). CODIS = 13 core STR loci (expanded to 20). PCR allows profiling from minute/degraded samples.

Mitochondrial DNA (mtDNA)

• Maternally inherited, present in high copy number per cell.
• Used when nuclear DNA is absent or destroyed — old bones, teeth, hair shafts (without root), badly degraded remains.
• Useful for maternal lineage tracing and mass-disaster victim identification.
• Y-STR profiling traces the paternal lineage (useful in mixed male–female stains and sexual assault cases).

High-yield: mtDNA → maternal lineage, used for hair shaft, old bones; Y-chromosome STR → paternal lineage. Hair with root gives nuclear DNA; hair shaft alone gives only mtDNA.

Forensic applications of DNA profiling

1. Criminal identification — matching crime-scene biological evidence to a suspect.
2. Disputed paternity / maternity — a child's STR alleles must each be present in a true parent; DNA has replaced HLA and blood-group testing here.
3. Identification of mutilated/decomposed/burnt bodies and mass disasters (air crashes, tsunami).
4. Exchange of newborns in hospitals.
5. Establishing zygosity of twins.
6. Immigration disputes (proving biological relationship).
7. Wildlife forensics / paternity in animals.

Chain of custody

The chain of custody (chain of evidence) is the chronological documentation of the seizure, custody, transfer, analysis, and disposition of physical/biological evidence. It ensures the sample produced in court is the same one collected, untampered.

Collect → label & seal → document handler at each transfer → secure storage → laboratory analysis → court production.

High-yield: A break in the chain of custody renders the evidence inadmissible even if the DNA match is perfect. Every person who handles the sample must be documented.

Biological evidence is air-dried before packaging (never sealed wet — moisture promotes bacterial growth and DNA degradation), packaged in breathable paper bags/envelopes (NOT airtight plastic, which traps moisture), and stored cold/frozen.

Bloodstain pattern analysis (BPA)

The physical pattern of bloodstains reconstructs events, independent of whose blood it is.

• Passive/drip stains — gravity; round when fallen vertically, with increasing spines/satellite spatter as drop height increases.
• Spatter (impact) stains — small drops from force (gunshot, beating); high-velocity spatter = fine mist (gunshot), medium-velocity = beating/stabbing, low-velocity = dripping.
• Cast-off stains — blood flung from a moving weapon.
• Arterial spurts — large volume, rhythmic, from arterial breach.
• Contact/transfer/wipe/swipe patterns.
• Angle of impact = arcsin (width ÷ length) of the elliptical stain; multiple stains let you triangulate the point of origin/convergence.

Complications, pitfalls & limitations

• Contamination — the biggest threat; foreign DNA from investigators or other samples. Mitigated by gloves, masks, and negative controls.
• Mixed samples — blood from multiple persons (e.g., assault) complicate interpretation; ratios and Y-STR help resolve.
• Degradation — heat, humidity, UV, bacteria, and time degrade DNA; PCR and mtDNA partly overcome this.
• PCR inhibitors — haematin, denim dye, humic acid (soil) can inhibit amplification.
• Identical twins — indistinguishable by standard STR profiling.
• Statistical interpretation — a "match" is reported as a random match probability; not absolute, but often 1 in billions.
• Allelic dropout / stutter peaks in low-template (touch) DNA.

Key differentials / contrasts to remember

Confusion point	Distinguishing fact
Screening vs confirmatory	Screening = sensitive not specific (Kastle–Meyer, benzidine, luminol); confirmatory = specific (Teichmann, Takayama)
Teichmann vs Takayama	Teichmann = haemin (brown rhombic); Takayama = haemochromogen (pink feathery)
Precipitin vs grouping	Precipitin = species; grouping/DNA = individual
VNTR vs STR	VNTR (minisatellite) = RFLP; STR (microsatellite) = PCR
RFLP vs PCR	RFLP needs much intact DNA, slow; PCR needs trace DNA, fast
mtDNA vs Y-STR	mtDNA = maternal (hair shaft, bones); Y-STR = paternal

Recently asked / exam angle

• "Father of DNA fingerprinting" → Alec Jeffreys (1984). First case → Colin Pitchfork.
• Teichmann test detects → haemin/haematin crystals (confirmatory test for blood).
• Takayama test forms → haemochromogen crystals.
• Most sensitive presumptive blood test → benzidine (but carcinogenic).
• Kastle–Meyer test colour → pink.
• Test for species identification → precipitin (Uhlenhuth) test.
• Test for old, dried, minute stains (species) → mixed agglutination.
• Luminol → detects washed/latent blood by chemiluminescence; does not destroy DNA.
• Amelogenin gene → sex determination in DNA profiling.
• mtDNA → maternally inherited, used for hair shaft/old bone, high copy number.
• Number of CODIS core loci → originally 13, now 20.
• Twins indistinguishable → monozygotic.
• DNA same in → all nucleated cells.
• Absorption–elution → grouping of dried bloodstains.
• Chain of custody break → evidence becomes inadmissible.

Rapid revision

1. Alec Jeffreys discovered DNA fingerprinting (1984); first case = Colin Pitchfork.
2. Screening tests are sensitive but not specific; confirmatory tests are specific.
3. Kastle–Meyer (phenolphthalein) → pink; benzidine → blue (most sensitive, carcinogenic); luminol → chemiluminescence for washed blood.
4. Teichmann = haemin chloride (brown rhombic); Takayama = haemochromogen (pink feathery).
5. Precipitin (Uhlenhuth) test = species identification using rabbit anti-human serum.
6. Mixed agglutination is best for old, dried, minute stains.
7. RFLP uses VNTR/minisatellites + restriction enzyme + Southern blot; needs lots of intact DNA.
8. PCR–STR uses microsatellites; amplifies trace/degraded DNA; capillary electrophoresis; current gold standard.
9. Amelogenin locus determines sex; CODIS = 13 (now 20) core STR loci.
10. mtDNA = maternal lineage (hair shaft, old bones, high copy number); Y-STR = paternal lineage.
11. Identical twins cannot be distinguished by standard DNA profiling; DNA is identical in all nucleated cells.
12. Dry the sample, use paper packaging, maintain chain of custody — a break makes evidence inadmissible.