RSID-Blood and ABAcard HemaTrace

A lateral-flow strip is a sandwich immunoassay on a membrane. In the ABAcard and RSID-Blood format, the key players are a detection antibody labelled with colloidal gold (conjugate pad), a capture antibody immobilised at the test line, and a second capture antibody at the control line. When the sample extract flows along the strip, it first rehydrates the labelled detection antibody. If human haemoglobin is present, it binds to the detection antibody. This complex travels forward and is captured at the test line, where the gold label accumulates and produces a visible pink or red band.

The control line captures any remaining labelled antibody, confirming the fluid migrated the full length of the strip. An invalid strip (no control band) must be discarded and the test repeated. This built-in quality check is one reason lateral-flow results are reliable even in field conditions: an analyst cannot misread an invalid strip as negative without noticing the absent control line.

ABAcard HemaTrace (Abacus Diagnostics) was one of the first lateral-flow assays to achieve widespread forensic validation for blood identification. The operating protocol is simple: the stain is extracted with a small volume of distilled water or buffer, the extract is added to the sample well, and the card is read after 10 minutes. A positive requires two bands (T and C). The test line can be very faint and still count as positive; analysts are taught to read any visible T line, however pale, as a positive result.

• Sensitivity: can detect haemoglobin at concentrations of approximately 1:100,000 to 1:1,000,000 depending on substrate and extraction efficiency. This far exceeds the sensitivity of classical crystal tests.
• Substrate range: validated on fabric (cotton, wool, nylon, denim), skin swabs, paper, wood, carpet, leather, and soil.
• Sample volume: requires approximately 300 microlitres of extract, a fraction of what classical tests consume from the stain.
• Shelf life: commercial cards have a printed expiry; storing at 2-8 degrees Celsius extends performance. Cards must not be used beyond expiry in casework.

Validation studies published across multiple jurisdictions have confirmed HemaTrace specificity against large panels of non-blood substances including plant peroxidases, rust, bleach residue, and a range of animal blood species. The test does not react to rust, coffee, ketchup, chocolate, or the peroxidase-containing plants (horseradish, spinach) that cause phenolphthalein to react.

RSID-Blood (Rapid Stain Identification of Blood, Independent Forensics) was developed later than HemaTrace but has accumulated a comparable validation record. It uses a different anti-human haemoglobin monoclonal antibody pair and a different detection label. The format and reading rules are the same: two bands positive, one band negative, no band invalid.

Laboratory choice between HemaTrace and RSID-Blood is typically determined by the laboratory's internal validation, procurement, and the jurisdiction's accreditation requirements. Some laboratories validate both and treat a concordant result from both assays as a stronger confirmatory package. Discordant results between the two tests, which are rare, prompt further investigation.

The anti-human haemoglobin antibodies in both assays are highly specific for the human haemoglobin sequence, but they are not perfectly exclusive. Ferret haemoglobin has sufficient structural similarity to human haemoglobin to cross-react with HemaTrace antibodies, giving a positive test result despite the blood being non-human. Higher primates, particularly great apes (gorillas, chimpanzees, orangutans, and bonobos), also cross-react because their haemoglobin is closely related to the human sequence.

• Cross-reactive: ferret, gorilla, chimpanzee, orangutan, bonobo, and most other great apes.
• Not cross-reactive (documented): dog, cat, cow, pig, horse, sheep, chicken, rat, mouse, rabbit, deer. Standard domestic and farm animals do not trigger a false positive.

In the overwhelming majority of forensic casework the cross-reactivity issue is irrelevant because the scene context makes ferret or primate exposure implausible. The analyst is trained to note the context and judge whether it warrants additional species testing. This is a scientific disclosure, not a test failure.

The outstanding sensitivity of lateral-flow assays, routinely detecting blood at dilutions of 1:100,000 or greater, means they perform reliably on stains that would be too small or too old for crystal tests. Validation studies have confirmed positive results from stains on fabric aged two to five years in ambient storage, from dried stains on wood and brick, and from samples subjected to moderate UV exposure.

Degradation does eventually reduce sensitivity. Haemoglobin denatures over time, and severely degraded or chemically treated samples may yield negative results. Bleach is the most damaging: sodium hypochlorite breaks the haemoglobin molecule in ways that destroy the relevant epitopes, so a bleach-treated bloodstain can produce a negative HemaTrace result even when prior treatment was substantial. This is a known limitation that appears in validation literature and must be acknowledged when reporting negative results from potentially bleach-treated scenes.

Both HemaTrace and RSID-Blood have extensive peer-reviewed validation records. Studies have systematically characterised sensitivity, specificity against panels of non-blood substances and animal blood species, performance across diverse substrates, and behaviour on aged or environmentally challenged samples. Accreditation standards in the United States (FBI Quality Assurance Standards, ASCLD/LAB), the United Kingdom (Forensic Science Regulator guidelines), and Australia (NATA) all require laboratories to validate immunochromatographic assays before casework use.

Reporting a positive result from either assay should include: the test used, the substrate, the extraction method, the result (T and C bands visible), any relevant caveats (cross-reactivity disclosure if context warrants, prozone if noted), and the analyst's conclusion. The conclusion states that the substance is confirmed as blood consistent with human origin, or, if species context was relevant, that the result is positive for human haemoglobin or a cross-reactive species.