The review, conducted by an international team led by researchers at Universiti Malaya in Kuala Lumpur, followed PRISMA 2020 guidelines and was registered on PROSPERO. The team searched PubMed, Scopus and Web of Science for peer-reviewed studies published between January 2015 and December 2025 that used spectroscopic methods to estimate the post-mortem interval, or PMI, of skeletal remains. From 167 initial records, 88 duplicates were removed and, after screening, 19 studies made the final cut. Risk of bias was assessed with the Joanna Briggs Institute critical appraisal tool, and every included study scored 6.0 or above, indicating moderate to high methodological quality across the board.

The technologies at the heart of this field are Fourier-transform infrared spectroscopy, known as FTIR, and Raman spectroscopy. FTIR was the most frequently used modality, appearing in ten of the 19 studies, most commonly in its attenuated total reflection configuration, while Raman spectroscopy featured in seven. Both techniques probe the molecular structure of skeletal tissue, but in different ways. Infrared spectroscopy measures how chemical bonds absorb mid-infrared light, typically across the 400 to 4000 per centimetre range, revealing the composition of the mineral phase and the organic matrix. Raman spectroscopy, by contrast, detects the tiny shifts in wavelength produced when laser light scatters inelastically off molecular vibrations, offering the practical advantage of being largely unaffected by water, which makes it well suited to hydrated or minimally prepared specimens.

The molecular targets of these analyses are the building blocks of bone itself. Bone consists of poorly crystalline carbonate-substituted hydroxyapatite embedded in a matrix dominated by type I collagen, while tooth enamel is far more heavily mineralised and crystalline. As bone degrades after death, measurable changes accumulate: the crystallinity index shifts, carbonate-to-phosphate ratios drift, collagen-related amide bands weaken, and the balance between mineral and organic content changes. Studies in the review typically acquired around 120 scans per spectrum at resolutions of 2 or 4 per centimetre, then extracted peak intensities, integrated band areas and derived ratios. Energy-dispersive X-ray spectroscopy, the second most common technique, added an elemental dimension, tracking calcium, phosphorus and trace elements such as barium, zinc and iron whose distributions shift with depositional environment and time.

The evidence base is strikingly diverse. Sample sizes ranged from 6 to 145 specimens, drawn from human remains in most studies and from domestic pigs, Wistar rats and Eastern grey kangaroos in four. Nine studies combined forensic and archaeological material, with archaeological specimens originating from burial sites across Italy, Spain, Slovenia, Portugal and Austria, including remains recovered from World War II mass graves. The femur was the most analysed bone, followed by ribs, skull bones and tibiae, while dental studies focused on incisors and molars. Depositional environments spanned surface exposure, burial, indoor storage and even freshwater and saltwater immersion. Notably, 58 percent of the included studies built directly on the foundational work of Nagy and colleagues and Patonai and colleagues, published before 2015, underscoring how heavily the field leans on a small number of seminal papers.

Where spectroscopy shines brightest is in separating the old from the very old. Several studies reported consistent discrimination between forensic and archaeological remains, reflecting cumulative diagenetic changes that unfold over decades and centuries. Machine-learning models integrating spectral data achieved classification accuracies ranging from 58 to 100 percent. Among the standout results, an orthogonal partial least squares model built on Raman data achieved a root mean square error of cross-validation of 1.25 years with a cross-validated coefficient of determination of 0.89. A genetic algorithm-assisted PLS model trained on second-derivative FTIR spectra predicted PMI with an error of 54.36 days for buried samples and 68.48 days for unburied ones. A logistic regression model combined with receiver operating characteristic analysis correctly classified 87 percent of samples with a ten-year PMI, 76 percent at 25 years and 80 percent at 50 years.

Yet the review also exposes a critical weakness: the first 90 days after death remain stubbornly opaque. Studies evaluating shorter post-mortem intervals frequently reported weaker or statistically insignificant associations, with differences between sample groups sometimes failing to reach significance at p greater than 0.05. The authors attribute this to the interplay of intrinsic biological factors with extrinsic environmental conditions, which produce different kinetics of bone degradation in the earliest stages. In that window, decomposition-related biochemical changes are simply too subtle to rise above biological and environmental noise. This matters because the early post-mortem period is precisely where conventional methods still work, meaning spectroscopy currently struggles most where it is least needed, and performs best where no alternative exists.

Environmental context emerged as one of the principal sources of variation. Ultraviolet radiation, burial conditions, aquatic immersion and storage conditions all altered the rate and extent of physicochemical change in skeletal tissue. One study showed that environmental conditions changed the expression of commonly used FTIR markers, while another reported distinct elemental profiles depending on depositional environment, and a third linked changes in elemental composition to soil interactions and fracture-related alterations in bone hydration. These findings align with established taphonomic principles, in which temperature, moisture, microbial activity, oxygen availability and soil chemistry collectively shape skeletal diagenesis. The implication is clear: spectral markers cannot be interpreted in isolation from the environment in which remains were deposited, and future predictive models must incorporate environmental variables alongside spectral parameters.

Biological variability adds a second layer of complexity. Animal models, particularly pigs, are widely used as analogues for human decomposition, but the review found that spectral patterns in animal remains were not always concordant with those in human samples, especially for phosphate-related parameters and mineral-to-organic matrix indices. Differences between skeletal tissues also matter: one study observed a decrease in the carbonate-to-phosphate ratio in teeth that ran counter to trends commonly reported in bone, likely reflecting the differences in mineral content, crystallinity and hydroxyapatite structure between the two tissues. Limited evidence even hints at sex-related differences in the magnitude of spectral change, although the overall direction of decomposition trends appears consistent between males and females.

The path forward, the authors argue, lies less in discovering new biomarkers than in standardising and validating the ones already known. Carbonate-to-phosphate ratios, crystallinity indices, collagen-related bands and mineral-to-organic matrix ratios have shown reproducible temporal trends across independent studies and now deserve large-scale, multicentre validation on human remains under realistic depositional scenarios. Machine learning will be central to that effort, but the review cautions that most existing models were built on small datasets and lack external validation, and that the limited interpretability of some algorithms may hinder forensic admissibility under legal standards such as Daubert, which demands tested, peer-reviewed methods with known error rates. The reviewers also call for multimodal integration, combining spectroscopy with micro-computed tomography or X-ray diffraction, and for greater attention to dental tissues, which remain underexplored. Spectroscopy, in short, has proven it can read the chemical autobiography written into bone. What remains is to teach it to testify.

Subject of Research: Spectroscopic estimation of the post-mortem interval of skeletal remains

Article Title: Post-mortem interval estimation of skeletal remains using spectroscopic analysis: a systematic review

Article References: Thrukkumar, S., Rahmat, R. A.-A., Soon, L. P., Hamdan, S. N. S. M., Rizky, B. N., Kurniawan, A., Humphries, M., Sani, S. F. A., Ibrahim, N., Rahim, A. H. A., Suzuki, T., & Linacre, A. (2026). Post-mortem interval estimation of skeletal remains using spectroscopic analysis: a systematic review. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-04010-w

Image Credits: AI Generated

DOI: 10.1007/s00414-026-04010-w

Keywords: forensic science, post-mortem interval, spectroscopy, FTIR, Raman spectroscopy, skeletal remains, bone diagenesis, machine learning, chemometrics, taphonomy, forensic taphonomy, systematic review

The research team, led by Catarina Ermida of the Laboratory of Forensic Anthropology, together with Eugénia Cunha, Francisco Gil and Maria Teresa Ferreira, drew on a uniquely well-documented resource: identified skeletal collections in Portugal, including the 21st Century Identified Skeletal Collection at Coimbra and clavicles recovered from autopsies at the Portuguese National Institute of Legal Medicine and Forensic Sciences. Because the identities and death dates of these individuals are known with documented precision, the researchers could work with genuinely known postmortem intervals, something that is remarkably rare in this field. In total, 38 unburned human clavicles were analyzed, spanning PMIs from 4 to approximately 500 years. To probe the effects of heat, the team added a subsample of 12 clavicles that had been burned and carried a PMI of only four years, providing a natural experiment in how thermal alteration reshapes bone mineral independently of time.

The technique at the heart of the study, X-ray diffraction, is one of the workhorses of materials science. When a beam of X-rays strikes a crystalline substance, the atoms scatter the radiation in patterns dictated by the regular arrangement of the crystal lattice. Bone mineral, a poorly crystalline form of calcium phosphate closely related to the mineral hydroxyapatite, produces characteristic diffraction peaks. Freshly formed bone contains many small, imperfect, strain-laden crystals, and their disorder broadens and smears the diffraction peaks. As bone ages, and especially as it is altered by the burial environment, crystals tend to recrystallize and coarsen: small crystals dissolve and redeposit onto larger ones, lattice defects are repaired, and the peaks become taller, narrower and sharper. Two parameters capture this maturation. The crystallinity index, or CI, is calculated from the relative sharpness of the diffraction pattern, essentially a ratio comparing the height of key peaks with the depth of the trough between them. Crystallite size, meanwhile, is estimated from the broadening of the diffraction peaks themselves, using the physical principle that smaller crystals produce wider peaks. If bone mineral truly aged like a clockwork, both numbers should climb steadily with time since death.

That is roughly what the study found for the crystallinity index, but not for crystallite size. Across the 38 unburned clavicles, crystallite size showed no consistent relationship with PMI at all; values fluctuated widely from sample to sample, swamped by variability that had nothing to do with elapsed time. The crystallinity index told a different story. It displayed a moderate positive correlation with postmortem interval, with a Spearman rank correlation coefficient of 0.587, a statistically significant result with a p value below 0.001 and a 95 percent confidence interval stretching from 0.194 to 0.810. In plain terms, older bones did tend, on average, to show sharper, more crystalline diffraction patterns, consistent with the long-standing picture of slow diagenetic recrystallization. For archaeologists sorting ancient from modern material, that trend is real and useful. But the confidence interval also reveals how noisy the relationship is, and the devil, as so often in forensic science, was hiding in the scatter.

That scatter is precisely what limits the forensic dream. The authors observed substantial overlap in crystallinity values within the forensic timescale, meaning that a bone dead for five years might produce a CI indistinguishable from one dead for twenty or thirty years. Samples with identical known PMIs returned markedly different crystallinity values, a pattern the researchers attribute to taphonomic factors, the full suite of environmental insults that a body experiences after death: soil chemistry, moisture, pH, temperature swings, microbial attack, roots, and drainage. Two skeletons buried for the same number of years in different microenvironments can follow very different diagenetic trajectories, so the mineral fabric they present to an X-ray beam reflects burial history as much as burial duration. In forensic casework, where the difference between a recent death and a decades-old one can determine whether a crime is prosecuted, that ambiguity is decisive. A method that produces overlapping values across the critical range cannot pin a date, only gesture at one.

The burned-bone subsample added a further warning. Heat is known to drive dramatic recrystallization in bone mineral: as temperatures rise during burning, the small bioapatite crystals grow, merge and reorganize, sharpening diffraction patterns dramatically. The 12 burned clavicles in the study, all with a PMI of just four years, showed thermal alteration of crystallinity that could, in effect, mimic the signature of much older remains. A forensic investigator who analyzed a burned recent bone and interpreted its high crystallinity as a sign of antiquity would be badly misled. Conversely, cremated archaeological material could masquerade as fresh if the thermal signature were misread. The study’s authors stress that thermal history must be recognized and accounted for before any crystallinity measurement is interpreted as a proxy for time, and their data demonstrate concretely how fire can scramble the clock that X-ray diffraction is meant to read.

None of this means X-ray diffraction is useless in forensic contexts. The technique is fast, relatively inexpensive, minimally destructive, and its parameters do track genuine postmortem change in bone mineral. The crystallinity index’s moderate correlation with PMI confirms that mineral maturation is a real, measurable phenomenon, and the study’s use of documented skeletal collections shows how such measurements can be calibrated against known truth rather than assumed ages. What the findings deflate is the more ambitious claim: that CI or crystallite size alone can deliver a precise postmortem interval. The results align with a growing body of literature that has tested physicochemical dating proxies, from earlier X-ray diffraction studies to spectroscopic approaches, and found the same recurring pattern, a broad correlation with time that dissolves into scatter exactly where forensic questions need resolution. The authors conclude that, although XRD-derived crystallinity parameters provide useful insights into postmortem changes in bone mineral structure, their application for PMI estimation remains limited.

There is a broader lesson here about how forensic science progresses, and it is one worth savoring. The temptation in a young and under-resourced field is to chase a single magic number, a lone chemical or crystallographic readout that solves the dating problem once and for all. This study, grounded in 50 human clavicles with known histories, shows why that chase so often disappoints: death does not happen in a laboratory, it happens in soil, in water, in fire, and in a thousand uncontrolled environments that each rewrite the bone record in their own way. The measured effect of time is real but entangled with the effects of everything else. Progress will likely come not from a solitary indicator but from combinations of methods, larger and better-documented reference collections, and models that explicitly incorporate taphonomic variables rather than wishing them away.

For now, the crystals in our bones keep their secrets stubbornly. They do grow and ripen after death, and a diffractometer can watch it happen. But between a fresh forensic case and a centuries-old skeleton, the crystalline clock runs at different speeds in different graves, and fire can knock its hands sideways entirely. The Coimbra team’s careful negative result is, in its way, as valuable as a breakthrough: it tells every future investigator exactly where the limits lie, and redirects the hunt for the forensic clock of death toward approaches humble enough to respect the chaos of the burial world. Until then, the question of how long ago someone died will continue to demand the oldest tools of all, careful context, cautious inference, and the accumulated judgment of forensic anthropologists who know that every bone tells more than one story.

Subject of Research: X-ray diffraction analysis of bone mineral crystallinity as a potential marker of the postmortem interval in human skeletal remains

Article Title: X-ray diffraction analysis of bone crystallinity and its association with postmortem interval in human skeletal remains

Article References: Ermida, C., Cunha, E., Gil, F., & Ferreira, M. T. (2026). X-ray diffraction analysis of bone crystallinity and its association with postmortem interval in human skeletal remains. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-04020-8

Image Credits: AI Generated

DOI: 10.1007/s00414-026-04020-8

Keywords: X-ray diffraction, bone crystallinity, postmortem interval, forensic anthropology, human skeletal remains, crystallinity index, crystallite size, taphonomy, bone diagenesis, burned bone, time since death, International Journal of Legal Medicine