Identifying Species in Bloodstains Found on Sand and Soil Using Vibrational Spectroscopy

Two non-destructive spectroscopic techniques with statistical modeling can distinguish different living organisms’ blood.

According to a recent study published in Forensic Science International, attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) and Raman microspectroscopy, along with a chemometric technique called orthogonal partial least squares-discriminant analysis (OPLS-DA), can differentiate between human, dog, and cat bloodstains deposited on sand and soil.¹ The results speak to addressing a gap in crime scene analysis involving outdoor or buried evidence.

What did the researchers test in their study?

In their study, the research team investigated whether ATR-FTIR and Raman microspectroscopy, when paired with OPLS-DA, could identify the species source of bloodstains on environmental substrates without destroying the sample. ATR-FTIR consistently outperformed Raman microspectroscopy at distinguishing the three species, particularly on soil, where Raman failed to reliably detect blood signatures at all.¹

Why should researchers care about this study and its results?

In forensic science, one of the main concerns with current equipment is the susceptibility of false positives emerging when conducting analyses.² False positives are an important problem in forensic science because it could lead to investigations being extended or delayed before samples even reach a laboratory for DNA analysis.^1,2 False positives are also common at crime scenes when both human and animal blood is present.¹ And finally, bloodstains recovered from sand or soil are especially difficult to work with because the substrate itself interferes with chemical analysis, and investigators are often reluctant to consume limited stain material on tests that may not yield a definitive answer.¹

Vibrational spectroscopy addresses all these concerns. Vibrational spectroscopic techniques require no reagents or sample destruction, and portable versions of the instruments already exist, raising the possibility of on-site screening tools for investigators working active scenes.¹

How did ATR-FTIR and Raman microspectroscopy perform on sand and soil?

The two techniques performed differently depending on the substrate. On sand, both instruments picked up spectral signals from the blood and the substrate, but the chemometric models found species separation more difficult than expected.¹ On soil, ATR-FTIR still detected clear blood-related spectral bands alongside signals from the substrate, but Raman microspectroscopy could not consistently register blood at all.¹

The team also tested whether the techniques could distinguish the sex of the blood donor, which is a capability with obvious value in narrowing suspect or victim pools. Using a single donor per sex for each species, the researchers found only limited separation on neutral substrates.¹ On sand and soil, ATR-FTIR combined with chemometric analysis achieved some degree of sex differentiation, while Raman microspectroscopy did not produce distinguishable patterns.¹

What are the key takeaways from this study?

Although this study showed encouraging results for how vibrational spectroscopy can be used in forensic analysis, there are also a few caveats that the authors made clear in the conclusion of their paper. For one, the sample size only included a small number of donors, and the sex-discrimination testing in particular relied on just one donor per sex per species.¹ Because of the small sample size, it is not enough to establish population-level reliability. Second, the researchers acknowledged that they only tested one type of sand and one type of soil, and these natural substrates could vary in their mineral composition. This is important because it is a factor that can alter spectral readings and background interference.¹ The researchers called for follow-up studies with larger and more diverse donor cohorts, a wider range of soil and sand types, and refined Raman acquisition settings, including a longer-wavelength 1064 nm laser, to reduce the fluorescence that limited performance on soil.¹

However, the main takeaway from this study is that it demonstrated a proof-of-concept that ATR-FTIR, in particular, can retrieve usable biological information from small, degraded bloodstains on outdoor substrates without consuming the sample, which is a capability current presumptive tests and destructive assays cannot offer.¹ Whether the approach becomes a validated field or laboratory tool will depend on the larger-scale studies the authors say are still needed before it can support casework.

References

1. Cano-Trujillo, C.; Montalvo, G.; Garcia-Ruiz, C.; Toledo-Gonzalez, V. Forensic Approach For Distinguishing Species and Sex of Multispecies Bloodstains in Soil and Sand by Vibrational Spectroscopy. For. Sci. Int. 2026, 388, 113082. DOI: 10.1016/j.forsciint.2026.113082
2. Jackson, G. P. Error Terror in Forensic Science: When Spectroscopy Meets the Courts. Spectroscopy 2016, 31 (11). Available at: https://www.spectroscopyonline.com/view/error-terror-forensic-science-when-spectroscopy-meets-courts