Biological Sample Screening and Presumptive Tests

Blood presumptive tests exploit the peroxidase-like activity of haemoglobin to catalyse the oxidation of a chromogenic or chemiluminescent substrate. The result is a colour change or light emission that is detectable at very low concentrations. No presumptive test is specific for blood: the same catalytic activity is found in plant peroxidases, certain metals, and some household chemicals.

Luminol is the standard scene-level presumptive test. Applied as an aqueous spray in the dark, it produces blue-white chemiluminescence on contact with haemoglobin. Its detection threshold of approximately one part per ten million makes it suitable for detecting blood that has been wiped, washed, or heavily diluted. The test does not permanently alter the underlying stain and studies consistently show that subsequent STR profiling from luminol-treated samples remains feasible, though multiple applications or prolonged contact can cause DNA degradation. False positives include bleach, copper compounds, horseradish, and certain rust-coloured soils.

Tetramethylbenzidine (TMB) and phenolphthalein (Kastle-Meyer) are the two most widely used bench-level presumptive tests for blood. TMB produces a blue-green colour change; phenolphthalein produces a pink colour in the presence of hydrogen peroxide. Both are applied to a cotton swab moistened with distilled water and rubbed against the stain. A positive result in under 30 seconds, with no colour change in the reagent blank, is considered reactive. Leucomalachite green (LMG) is another catalytic test used in some laboratories. All three share the limitation of false positives from plant material and some metals.

Confirmatory tests for blood rely on the detection of haemoglobin derivatives rather than peroxidase-like catalytic activity. The two classic crystal tests, Takayama and Teichmann, produce characteristic crystals from haemoglobin reaction products that are identified by microscopy. The Takayama (haemochromogen) test reacts haemoglobin with pyridine in an alkaline reducing environment to produce salmon-pink feathery crystals of pyridine ferroprotoporphyrin. The Teichmann (haemin) test reacts haemoglobin with glacial acetic acid and potassium chloride to produce dark brown rhomboid crystals of ferriprotoporphyrin chloride.

Both crystal tests are specific for blood and are performed under microscopy. They require a visible amount of stain material and can be conducted on aged or dry stains. Crystal formation confirms the presence of haemoglobin but does not distinguish human from animal blood. Species identification requires an additional test, typically an immunological method such as the Ouchterlony gel diffusion assay or a species-specific antiserum-based lateral flow strip.

Immunochromatographic lateral flow strips that detect human haemoglobin are now available and widely used as a combined presumptive-confirmatory approach. Products such as the HemaTrace strip (Abacus Diagnostics) give a positive result within minutes and are specific for human haemoglobin, distinguishing human from most animal blood at the analytical threshold. However, these strips are designed as screening devices for human haemoglobin, not as forensic confirmation per se, and their status as a confirmatory test varies by laboratory accreditation protocol.

Semen identification follows the same two-tier logic as blood. The primary presumptive test is acid phosphatase (AP) activity, detected by the Brentamine Fast Violet B spray or the SERI AP spot test. Seminal plasma contains AP at concentrations roughly 400 times higher than other body fluids. A colour change to purple within 30 seconds is considered a strong positive; a slower reaction or a faint result is recorded as weakly positive and triggers caution in interpretation. False positives occur with vaginal secretions, which also contain AP at lower concentrations, so timing and intensity of the colour reaction matter.

Microscopic examination for spermatozoa is both a presumptive indicator and, when mature spermatozoa with visible head and tail are identified, a strong confirmatory result. A Christmas tree stain (nuclear fast red with picroindigocarmine) differentially colours sperm heads red and tails green, making identification straightforward under light microscopy. The limitation is that azoospermic donors, vasectomised individuals, and aged or degraded samples may yield no spermatozoa. In these cases, a semen identification depends entirely on PSA.

The prostate-specific antigen (PSA, also called p30 in older literature) immunochromatographic strip test is the standard confirmatory test for semen in most laboratories. PSA is present in seminal plasma at 0.5 to 2 mg/mL; vaginal secretions, urine, and blood contain PSA at levels below the strip's detection threshold in the vast majority of cases. A positive strip result, in the context of an appropriate investigative scenario, confirms seminal material. PSA is stable in dried stains for extended periods and has been detected in stains several years old, though sensitivity declines with age, humidity, and UV exposure.

Saliva is identified primarily by the detection of alpha-amylase activity. The Phadebas press test applies a Phadebas tablet (a cross-linked starch polymer with a blue dye) to a moistened membrane pressed against the stain. High amylase activity in the stain breaks down the starch, releasing the blue dye and creating a blue circle on the membrane. The intensity and speed of the reaction indicate the relative amylase concentration. Starch-iodine methods work on the same principle. These tests are presumptive: amylase is also present in sweat and pancreatic secretions, though at lower concentrations than in saliva.

A confirmatory approach for saliva uses immunological detection of salivary amylase isoforms or of other saliva-specific proteins. In some laboratories, mRNA profiling of body fluid-specific transcripts has replaced or supplemented conventional confirmatory tests, particularly for saliva where classical confirmatory tests remain limited. The mRNA approach detects transcripts such as statherin and histatin-3, which are expressed at high levels in salivary glands. This method is discussed further under forensic serology and molecular techniques.

Urine is one of the most difficult body fluids to confirm using classical methods. Creatinine concentration assays and urea detection have been used as presumptive indicators, but neither is specific. Some laboratories use immunological strips for urine-specific proteins. Vaginal secretions are identified by microscopic examination for vaginal epithelial cells and, where available, by detection of vaginal fluid-specific miRNA or protein markers. The identification of vaginal secretions is clinically important in sexual assault investigations but remains more challenging than blood or semen identification in terms of analytical specificity.

Messenger RNA (mRNA) profiling detects body fluid-specific gene transcripts in forensic stains. Unlike proteins, which are the targets of classical tests, mRNA transcripts are produced by specific tissues and are therefore potentially more specific as fluid identifiers. Saliva, semen, menstrual blood, peripheral blood, vaginal secretions, and skin cells each have characteristic transcript profiles. The method uses reverse transcription followed by quantitative PCR (RT-qPCR) or, increasingly, massively parallel sequencing of the transcript pool.

mRNA profiling offers several advantages over classical tests. It can identify multiple body fluids from a single extract, it is applicable to mixtures, and it can be performed on the same extract used for DNA profiling. Its limitation is that mRNA degrades more rapidly than DNA, particularly under humid or warm conditions, so old or environmentally compromised samples may yield insufficient transcript quantity for reliable identification. Validation studies have been published by research groups in the Netherlands, the United Kingdom, and the United States, and the method is entering routine use in some national laboratories.

Forensic serology, the classical discipline that developed most of the presumptive and confirmatory tests described in earlier sections, now interfaces closely with molecular biology. A laboratory receiving a complex case may apply classical screening to map stain locations on an exhibit, apply mRNA profiling to characterise the fluids present, and then extract DNA from the same stain. This integration is the standard workflow in high-volume sexual assault examination centres in England and Wales (operating under the Faculty of Forensic and Legal Medicine guidelines) and in comparable units in the United States, Australia, and India's Central Forensic Science Laboratories.

Screening results are only useful if the laboratory has a structured process for converting them into action. Most accredited forensic biology laboratories use a tiered prioritisation system. After initial documentation and photography of each exhibit, analysts apply rapid presumptive tests to map biological material. Items that give strong positive results in areas consistent with the alleged offence are designated high priority. Items with weak or ambiguous results, or results from areas inconsistent with the alleged act, are designated lower priority.

High-priority items proceed to confirmatory testing and then to DNA extraction. Lower-priority items are held pending the outcome of high-priority analysis. If the high-priority set yields a full DNA profile matching the suspect or providing an investigative lead, many cases do not require further analysis of lower-priority items. If the high-priority analysis fails, the lower-priority items are processed in order. This staged approach can significantly reduce analytical cost and turnaround time without compromising evidential completeness.

Documentation at every step is mandatory under accreditation standards including ISO 17025, which is the international standard for testing and calibration laboratories and is required for laboratories providing evidence in courts across India (under the Bharatiya Sakshya Adhiniyam 2023 and its predecessor), the United Kingdom (Forensic Science Regulator standards), the United States (FBI Quality Assurance Standards), and most other jurisdictions. Each screening test result, including negative results and equivocal results, must be recorded in the case notes. The reason for selecting or excluding each item from further analysis must be documented and defensible under cross-examination.