Composition and Properties of Blood

Human blood is classified as a connective tissue: a matrix of plasma in which cellular elements are suspended. A haematocrit tube spun at 3,000 rpm will separate the four main fractions by density. Plasma floats at the top, forming a straw-yellow to clear layer. The buffy coat, a thin whitish band, holds the white blood cells and platelets. The red cell pellet, the densest layer, forms the bottom and accounts for about 45% of the total volume in a healthy adult male.

• Plasma: carries species-specific proteins (immunoglobulins, albumin), clotting factors, and small amounts of cell-free DNA. The main target of precipitin and lateral-flow species tests.
• Red blood cells (erythrocytes): anucleate (no nucleus) and enucleate in mature form. They carry haemoglobin and surface ABO, Rh, and other blood-group antigens. No nuclear DNA, but mitochondrial DNA is present in trace amounts from remnant mitochondria.
• White blood cells (leucocytes): nucleated cells that are the primary source of nuclear DNA for STR profiling. Present at about 4,000 to 11,000 per microlitre of blood in a healthy adult.
• Platelets (thrombocytes): small anucleate cell fragments involved in clotting. Forensically they contribute to clot formation that alters stain morphology, but carry no nuclear DNA.

Haemoglobin is a tetrameric protein with four haem groups, each containing a ferrous iron atom (Fe2+) coordinated to a porphyrin ring. In arterial blood, the haem iron binds oxygen reversibly. The iron also has a weak catalytic ability to decompose hydrogen peroxide, a pseudo-peroxidase activity that is unrelated to its oxygen-transport function but extremely useful in forensics.

The Kastle-Meyer (phenolphthalein) test and leucomalachite green (LMG) test both exploit this pseudo-peroxidase activity. Hydrogen peroxide is added to a stain extract along with a chromogen (phenolphthalein or LMG). If haemoglobin is present, it catalyses the oxidation of the chromogen, producing a pink or green colour respectively, within about 30 seconds. Both tests are highly sensitive (down to nanogram quantities) but not specific: plant peroxidases, bleach, and some metallic ions can give false positives, which is why they are classified as presumptive.

Luminol and Bluestar operate on the same principle but produce chemiluminescence (light emission) rather than colour change. They are sprayed onto surfaces in the dark and emit blue-white light wherever haem is present. Their advantage is sensitivity over large areas and some resistance to surface cleaning, their disadvantage is that positive areas must be photographed quickly before the reaction fades, and the chemicals can inhibit subsequent DNA extraction if not rinsed.

When blood leaves the body it begins a series of chemical changes driven by drying, oxidation, and microbial activity. The colour sequence is one of the most visible indicators: oxyhaemoglobin (bright red) converts first to deoxyhaemoglobin (dark red/purple) as oxygen dissociates, then to methaemoglobin (dark brown) as the iron oxidises from Fe2+ to Fe3+, and over weeks to haemichrome and other degradation products that shift the colour toward dark brown and eventually near-black.

Temperature, humidity, UV exposure, and substrate all modulate the rate of these changes. A bloodstain in a warm, sunny outdoor environment degrades faster than one in a cool, dark room. A stain on a porous substrate such as soil or unfinished wood absorbs and disperses differently from one on a smooth non-porous surface. These variables mean that colour alone cannot reliably date a stain, though it can give a rough relative sequence when multiple stains are present in the same environmental conditions.

DNA degradation does not follow the same timeline as haemoglobin degradation. White-cell nuclear DNA can survive for centuries under dry, cool, dark conditions (think ancient mummy specimens). Conversely, DNA can be destroyed within days by microbial nucleases in wet, warm environments. Forensic DNA extraction from a visually well-preserved stain may yield no profile if the stain was wet and warm for an extended period, while a visually degraded old dry stain may still yield a partial STR profile.

Plasma proteins are the targets of species-confirmation tests. Human immunoglobulin G, albumin, and transferrin are present at gram-per-litre concentrations in plasma, several orders of magnitude higher than the concentration of cell-free DNA. Because proteins are more thermostable than DNA under many degradation scenarios, a stain too degraded for DNA typing can still yield a positive species result. This is one reason the sequential testing protocol places species confirmation before DNA extraction.

The lateral-flow haemoglobin card (such as the Hexagon OBTI test or ABAcard HemaTrace) uses a monoclonal antibody raised against human haemoglobin. A visible test line appears within five minutes at a detection threshold of approximately 1 microgram per millilitre. The test is validated as specific to human and some higher primate haemoglobins. Ferret haemoglobin can give a weak cross-reaction, a limitation that has been flagged in the literature but is rarely relevant in practice.

The forensic DNA laboratory depends on nuclear DNA from leucocytes. A single microlitre of blood contains approximately 4,000 to 11,000 white cells, each with about 6 picograms of genomic DNA. A bloodstain the size of a small coin on fabric, containing perhaps 20 to 50 microlitres of blood, can yield 200 to 500 nanograms of DNA, comfortably above the threshold for full STR profiling.

The catch is that leucocytes are fragile. They lyse in hypotonic solutions, they are attacked by bacterial and fungal nucleases in wet conditions, and they are sensitive to freeze-thaw cycles in cold storage. Haemolysis, the rupture of red cells, releases free haemoglobin into the surrounding fluid and can inhibit PCR. Standard DNA extraction protocols (Chelex, differential extraction, solid-phase extraction) include steps to remove haemoglobin and other inhibitors, but heavily haemolysed or degraded samples can still fail to amplify.

This is why correct evidence packaging matters as much as the chemistry. Bloodstained items should be air-dried before packaging in paper (not plastic) bags. Plastic bags trap moisture, promoting bacterial growth and nuclease activity. Dried stains stored in cool, dark conditions can retain amplifiable DNA for decades, as demonstrated by successful STR profiling from stains retained as evidence in cases from the 1980s and earlier.

Outdoor bloodstains face UV radiation, rain, microbial colonisation, and temperature swings simultaneously. UV breaks protein structure and fragments DNA. Rain both dilutes and potentially preserves a stain depending on whether it washes the sample away or deposits it into a crack. Heat above 60 degrees Celsius denatures proteins and accelerates DNA fragmentation. Freezing can preserve samples very well if freeze-thaw cycles are avoided.

• UV radiation: accelerates protein denaturation and DNA strand-break accumulation; haemoglobin colour shifts faster; long exposure may bleach the stain to near-invisibility.
• Moisture: wet conditions promote bacterial nuclease activity and haemolysis; short wetting followed by drying at a boundary (e.g., a puddle edge) can concentrate and preserve stains.
• Heat: temperatures above 60 degrees Celsius rapidly denature proteins; fire scenes present charred, heat-denatured stains that can still yield PCR product if the carbonised matrix is processed correctly.
• Bleach and oxidising cleaning agents: destroy haemoglobin peroxidase activity; can produce false negatives with colorimetric presumptive tests; luminol is more resistant.
• Substrate porosity: porous substrates (concrete, wood, soil) absorb blood deep into the matrix, partially protecting it from surface cleaning and UV; non-porous surfaces retain stains accessibly but also make them easier to clean.