Blood as Biological Evidence

Whole blood is approximately 55 percent plasma and 45 percent formed elements. Plasma is a water-based solution of proteins, electrolytes, hormones, and metabolites. The formed elements are red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Each fraction has distinct forensic implications.

Mature human red blood cells lack a nucleus, which distinguishes them from the red cells of most other vertebrates. This is why a basic microscopic examination of a stain can suggest a mammalian (anucleate) versus non-mammalian (nucleate) source. Species-specific confirmatory tests, typically using anti-human precipitin antisera or ELISA-based immunoassays, are required to confirm human origin before DNA profiling proceeds.

Leukocytes are the DNA source of choice. A microlitre of blood contains roughly 7,000 leukocytes on average. A 1-centimetre dried bloodstain may have held 20 to 50 microlitres of liquid blood, meaning it contains on the order of 140,000 to 350,000 leukocytes. Even with significant degradation, enough intact nuclear DNA usually survives for PCR amplification of STR loci. The sensitivity of modern STR kits, capable of producing profiles from as little as 0.5 nanograms of DNA, means that trace bloodstains are routinely profiled.

Each leukocyte nucleus contains two copies of the human genome distributed across 23 pairs of chromosomes. The genome comprises approximately 3.2 billion base pairs, of which a small fraction consists of the highly polymorphic STR loci used in forensic typing. Current forensic STR multiplexes in the United States (CODIS 20-locus kit), the European Union (ESS 16-locus minimum), the United Kingdom (DNA-17 profile), and India (NDNAD India uses a subset consistent with Interpol standards) all target autosomal STRs that are unlinked, inherited independently, and achieve discrimination powers exceeding one in a trillion across unrelated individuals.

The cell also contains mitochondria, each carrying a copy of the 16,569 base pair mitochondrial genome. Because a single cell holds hundreds to thousands of mitochondria, mtDNA is present in far higher copy number than nuclear DNA per cell. This copy-number advantage makes mtDNA recoverable from samples where nuclear DNA is too fragmented or too dilute for STR profiling, particularly hair shafts without follicular tissue, teeth, and severely degraded dried bloodstains. The drawback is biological: mtDNA is maternally inherited and is identical among all maternal-line relatives, so it cannot distinguish siblings, cousins, or any two people sharing the same maternal ancestor.

For a detailed treatment of DNA structure, see DNA Double Helix and Base Pairing and Chromosomes, Genes, and the Human Genome.

Blood is the most frequently encountered biological evidence but not the only one. Forensic biologists routinely analyse a range of biological substrates, each with distinct DNA yield, typing strategy, and collection requirements. Understanding the range of biological evidence types prevents an investigator from overlooking a valuable sample because it does not look like a bloodstain.

• Semen and saliva: Semen contains spermatozoa (nuclear DNA) and seminal fluid proteins. Saliva contains epithelial cells shed from the oral mucosa. Both are addressed in the companion topic on semen, saliva, and other body fluids in this subject.
• Hair: Hair with an attached root follicle yields nuclear DNA. Hair shaft without a follicle yields mtDNA only. Microscopic morphology (cortex pigmentation, medulla pattern, cuticle scale) provides preliminary species and racial group indication, but morphology alone cannot individualize to a single source. DNA analysis is required for individualization.
• Bone and teeth: Bone is the most durable biological tissue and the primary DNA source in decomposed, burned, or ancient remains. Cortical bone and dense tooth root dentin protect DNA from environmental exposure longer than soft tissue. Modern protocols extract DNA from powdered bone or tooth root after demineralization. Skeletal biology and forensic anthropology are treated in the forensic anthropology subject.
• Tissue and organ fragments: Soft tissue from muscle, liver, and skin yields good-quality DNA when fresh. Formalin-fixed paraffin-embedded tissue from autopsy blocks contains heavily crosslinked DNA requiring specialised extraction protocols. Decomposed tissue may be limited to mtDNA recovery.
• Touch DNA: Epithelial cells deposited on surfaces through handling. Yields are often low (below 500 picograms), making mixture interpretation and low-template analysis necessary. Touch DNA is covered in the touch DNA and trace biological material topic in this subject.

The forensic biology laboratory must decide, on receipt of each item, which biological material is likely present and what the optimal analytical workflow is. A stained garment may carry blood, semen, and touch DNA from three different sources, each requiring a separate extraction and profiling strategy. Triage decisions at case intake directly affect how much DNA is consumed and how much sample remains for re-examination.

Before DNA extraction, a stain must be confirmed as blood of human origin. The workflow has two steps: a presumptive (screening) test to detect catalytic activity or a blood-associated molecule, followed by a confirmatory (specific) test that identifies a human-specific protein.

Common presumptive tests exploit the pseudo-peroxidase activity of haemoglobin. Haemoglobin catalyses the oxidation of a chromogenic substrate by hydrogen peroxide, producing a colour change. The Kastle-Meyer test (phenolphthalein) gives a pink colour; leuco-malachite green gives a green colour. Luminol is a chemiluminescent version used to detect traces on surfaces, including stains that have been cleaned or are present in large areas. All presumptive tests can produce false positives from plant peroxidases, certain metals, and some household chemicals, so a positive result must be confirmed.

Confirmatory tests for human blood use antibodies specific to human haemoglobin, human serum albumin, or human immunoglobulin. Lateral flow immunochromatographic strips (for example, the Hexagon OBTI strip for haemoglobin and the ABAcard HemaTrace) are the current standard of practice: they are rapid (result in five minutes), specific to human or primate blood, and the result is documentable by photograph. A positive confirmatory test establishes human blood; species-level exclusion of non-human sources is built into the test design.

The integrity of biological evidence depends on decisions made within the first hour of collection. Three variables drive outcome: packaging material, moisture at packaging, and temperature during storage and transport. Errors here are irreversible; degraded DNA cannot be recovered.

• Liquid blood: Collect into EDTA anticoagulant vacutainer tubes (purple cap). EDTA chelates magnesium ions required by DNase enzymes, substantially slowing DNA degradation. Refrigerate at 4 degrees Celsius for short-term (up to 2 weeks); freeze at minus 20 degrees Celsius for longer storage. Do not freeze in a non-frost-free freezer without secondary containment: repeated freeze-thaw cycles shear DNA.
• Dried stains on portable items: Air-dry the item fully if damp. Package in paper bags or paper envelopes, not plastic. Plastic traps moisture, promoting mould growth and bacterial nuclease activity that destroys DNA within days. Paper allows vapour exchange. Label the exterior without contacting the stain.
• Dried stains on fixed surfaces: Use a sterile cotton swab moistened with a small volume of sterile distilled water. Swab the stain with firm circular pressure. Air-dry the swab fully before placing it in a paper swab box or cardboard tube. Dry swabbing of a companion area beside the stain provides a substrate control for environmental contamination.
• Reference samples: Collect buccal swabs or EDTA blood from all known individuals whose DNA is needed for comparison. A reference sample from the victim is mandatory for sexual assault cases to establish the victim's profile and separate it from any unknown contributor profile in the mixture.

Legal frameworks governing collection vary by jurisdiction. In India, the Bharatiya Nagarik Suraksha Sanhita 2023 (replacing the CrPC) governs search and seizure of physical evidence, while the Bharatiya Sakshya Adhiniyam 2023 (replacing the Indian Evidence Act 1872) governs admissibility. In the United States, the Fourth Amendment constrains warrantless collection; in England and Wales, the Police and Criminal Evidence Act 1984 (PACE) provides the framework. The European Union's General Data Protection Regulation (GDPR) applies to storage and cross-border transfer of DNA profiles from EU nationals. All frameworks share the requirement for documented chain of custody from collection through analysis.

DNA degradation reduces fragment length and creates chemical modifications that block polymerase extension during PCR. The result is allele dropout (failure to detect a true allele), reduced peak heights, and in severe cases, a completely uninformative profile. Understanding the mechanism of degradation helps the analyst interpret partial profiles and informs decisions about re-extraction or alternative analytical approaches.

Degraded samples can sometimes be profiled with low-copy-number (LCN) or probabilistic genotyping methods, which tolerate higher levels of stochastic variation and incomplete profiles than conventional threshold-based interpretation. However, these methods require stringent contamination controls and laboratory validation before use in casework. The Forensic Science Regulator (England and Wales), the Scientific Working Group for DNA Analysis Methods (SWGDAM) in the United States, and equivalent bodies in other jurisdictions publish mandatory validation requirements for LCN methods.

FTA (Flinders Technology Associates) cards are a dry-preservation medium impregnated with chemicals that lyse cells, denature proteins, and chelate metal ions. Blood spotted onto an FTA card and dried at room temperature in ambient air retains amplifiable DNA for decades. FTA cards are standard for reference sample collection in national DNA databases, including the UK National DNA Database (NDNAD), the US Combined DNA Index System (CODIS), and India's national database maintained under the DNA Technology (Use and Application) Regulation Act 2019. The Act governs consent, database use, and data protection for DNA profiles in India.