Order of Testing and DNA Preservation

The central constraint of biological evidence examination is that the original stain is finite and, once tested, partially consumed. A fabric cutting taken for a presumptive test, a swab used for a lateral-flow card, and an extraction plug removed for DNA profiling are all permanent reductions of the original evidence. If the full stain is consumed before a positive result is obtained at the confirmatory level, there is nothing left for DNA, nothing left for the defence expert to re-test, and nothing left if a method challenge requires reanalysis.

Sequential testing manages this constraint by applying the most informative and least consumptive tests first. Alternate light source (ALS) examination consumes nothing. A visual inspection and photography consume nothing. A KM test applied to the periphery of a stain on fabric, or to a small scraping from a hard surface, consumes a fraction of a microlitre. Only after those steps confirm that the stain warrants further analysis does the protocol move to the confirmatory test, and only after that to DNA extraction of the most promising sample area.

Presumptive tests are designed for high sensitivity and rapid screening, not definitive identification. For blood, the Kastle-Meyer phenolphthalein test and leucomalachite green test are the workhorses: both can be applied with a cotton swab to the periphery of a stain, leaving the central area intact. A positive result within 30 seconds warrants further examination. A negative result, in a properly conducted test with appropriate controls, is reliable enough to dismiss blood from the stain composition and redirect attention elsewhere.

Luminol and Bluestar occupy a special position in the presumptive stage. They are used for area-wide screening at scenes where the distribution of blood is unknown, after visible stains have been photographed and sampled. Applying luminol before sampling any stain reverses the correct order and should be avoided whenever possible. If luminol must be used first (because the scene is about to be disturbed or the exhibit will be handed over), any subsequently sampled areas should be noted as having received luminol exposure, and the DNA analyst should be informed so that appropriate inhibitor-removal steps can be included in the extraction protocol.

Two controls should accompany every presumptive test: a positive control (a known blood standard run in parallel) to confirm the reagent is working, and a negative control (the reagent applied to an unstained portion of the same substrate type) to detect substrate-related false positives. These controls must be recorded in the case notes alongside the test result.

Confirmatory tests are those with sufficient specificity to support a conclusion in a forensic report. For blood, the lateral-flow haemoglobin card (for example the ABAcard HemaTrace or Hexagon OBTI) is the most widely used confirmatory test. It detects human haemoglobin specifically and is validated to a defined lower limit of detection. A positive result on a validated card, supported by positive and negative controls run in the same batch, constitutes confirmatory identification of human blood in most laboratory protocols.

For semen, the Christmas tree stain applied to a slide made from a swab extract identifies spermatozoa by their characteristic morphology: the round to oval head with its acrosomal cap takes up red stain, while the midpiece and tail take up green. This is both confirmatory for semen and simultaneously identifies the cellular source of the sperm fraction for differential extraction. Where the depositor may be azoospermic (absent sperm due to vasectomy or medical condition), a PSA lateral-flow card detects prostate-specific antigen in the stain extract as the confirmatory alternative.

In most jurisdictions, the lateral-flow haemoglobin card already performs double duty: it both confirms blood and confirms human origin, because the antibodies used in validated commercial cards are specific to human haemoglobin. Where the card used is not species-specific, or where a non-blood biological fluid is being tested, a separate species test may be required.

The classic species test for blood is the tube precipitin test or its gel-based equivalent. It is now largely replaced by the lateral-flow format. For semen and saliva, species specificity is addressed by the test design: the RSID-Saliva card detects human salivary alpha-amylase (which has lower cross-reactivity with rodent amylase than earlier enzymatic assays), and the PSA card targets human prostate-specific antigen. Where a stain is of uncertain origin, mitochondrial DNA cytochrome b sequencing can identify species from a tiny amount of template, independent of any immunological reagent.

DNA preservation begins at the scene. Biological stains should be photographed and sampled before any chemical presumptive tests are applied. Swabs should be air-dried immediately after collection and placed in labelled paper envelopes, not plastic bags. If the substrate is a portable item (clothing, a weapon, a tile fragment), the entire item should be packaged for laboratory examination rather than performing scene-side swabbing that may introduce contamination or miss the best sample area.

• Scene collection: photograph first, then sample. Air-dry swabs before sealing. Use paper packaging. Avoid plastic, which traps moisture and accelerates DNA degradation.
• Before presumptive testing: apply KM or LMG to the stain periphery or to a small cutting that does not compromise the main body of the stain. Leave the central area untouched for DNA.
• Before confirmatory testing: the extract used for a lateral-flow card test can, in some protocols, also serve as the extract for DNA extraction if the card test is performed first from the same extraction and the remaining volume is routed to DNA. Verify the laboratory's validated protocol before combining steps.
• Luminol and PCR: if luminol was applied before DNA sampling, inform the DNA analyst. Additional clean-up steps (e.g., Chelex with extended wash cycles) can reduce but not eliminate the inhibitory effect. Short-amplicon STR kits are more tolerant of degraded or inhibited samples.
• Storage: dried biological exhibits should be stored at room temperature or 4 degrees Celsius in a cool, dark, dry location. Refrigerated storage extends DNA stability. Frozen storage is appropriate for liquid blood but can damage dried stains through freeze-thaw cycling.

Chain of custody in biological evidence is not merely a procedural formality. A defence challenge to DNA evidence will routinely question whether the sample was handled consistently, whether contamination was possible, and whether the person who extracted DNA was the same person who performed the preceding serological tests. Each step in the sequential protocol must generate a contemporaneous record that answers these questions before they are asked.

At minimum, the case notes should record for each serological test: the exhibit identifier and the description of the stain examined; the exact test applied and the lot number or batch reference of the reagent; the result (positive, negative, or equivocal) with a description of the reaction observed; the positive and negative control results; the analyst's name and signature; the date, time, and laboratory location; and the amount of substrate consumed or the area of the stain tested. For the DNA submission record, the serological findings and the rationale for the priority of sampling should be transferred to the DNA request form.

ISO/IEC 17025 accreditation requires that each test performed as part of a case is covered by a validated, documented method. The analyst should be able to point to the validation study that supports the sensitivity, specificity, and interference-substance profile of every test applied. Courts in the United Kingdom, the United States, Australia, and increasingly in India under the Bharatiya Sakshya Adhiniyam 2023 and allied criminal law reforms, expect that this documentation exists and is available to both prosecution and defence.

Sexual assault evidence introduces a specific challenge: the stain may contain a mixture of semen and vaginal epithelial cells, and the goal is to obtain a DNA profile from the semen donor and a separate profile from the complainant. This is the rationale for differential extraction, which exploits the greater resistance of intact sperm cells to lysis compared with epithelial cells. The protocol uses SDS and proteinase K to lyse the epithelial cells first, collecting the supernatant as the epithelial fraction, and then a stronger lysis step (SDS plus proteinase K plus dithiothreitol to break disulfide bonds in the sperm nucleus) to release the sperm fraction.

The serology stage before differential extraction is critical: the microscopic examination of a smear from the swab to identify and document the presence of spermatozoa, confirms the substrate is semen and sets up the two-fraction extraction. If no sperm are seen (either an azoospermic donor or an old stain where sperm heads have degraded), the analyst should note this explicitly and consider whether the PSA test confirms semen origin before proceeding to a standard single-fraction extraction.

1. Presumptive acid phosphatase (AP) test on the swab: rapid colorimetric screen for semen-level AP activity.
2. Prepare a smear slide from the swab and apply Christmas tree stain: confirm spermatozoa are present and document their morphology and density.
3. If spermatozoa are absent: perform PSA lateral-flow card to confirm semen origin despite azoospermia.
4. Proceed to differential extraction: epithelial fraction first (mild lysis), sperm fraction second (full lysis with DTT).
5. Profile each fraction separately: the epithelial profile is compared to the complainant's reference; the sperm profile is compared to the suspect's reference.