Brain Protein Stains Reveal Hidden Damage in Fatal Drug Poisonings

Forensic pathologists in Finland have found that a routine brain stain can reveal signs of fatal drug poisoning that toxicology alone may miss. In a series of 14 medico-legal autopsies, beta-amyloid precursor protein immunoreactivity, a marker of axonal injury, appeared in seven of ten fatal polysubstance poisonings, most of them driven by buprenorphine. The findings, published in the International Journal of Legal Medicine, suggest that this staining technique could become a valuable supplementary tool in the investigation of suspected poisoning deaths.

The study addressed a persistent problem in forensic medicine: it is not always possible to definitively establish fatal poisoning by postmortem toxicology alone. Postmortem redistribution of drugs, polysubstance use, poor sample quality, and interindividual variability in metabolism and toxic effects all complicate interpretation. Among opioid users, tolerance adds another layer of difficulty, because therapeutic concentrations can be of similar magnitude to fatal ones. Buprenorphine, a partial mu-opioid receptor agonist, illustrates the challenge. It has a ceiling effect on respiratory depression, yet concomitant use of other psychoactive substances may attenuate that effect and raise the risk of fatal intoxication. In the Nordic countries, buprenorphine is often combined with other drugs, and it is the drug most frequently attributed to fatal poisonings in Finland.

Beta-amyloid precursor protein immunohistochemistry is widely used in forensic neuropathology. Originally developed to detect traumatic axonal injury, the method has since found applications in non-traumatic deaths. Staining patterns differ to some extent between causes of axonal injury: geographical and patchy patterns of axonal staining are considered indicative of ischaemic or metabolic origin, termed vascular axonal injury. This type of immunoreactivity has previously been reported in the brains of individuals with polysubstance use, and opioids in particular have been noted as a common substance relating to beta-APP positivity. A recent study of fentanyl-related deaths reported prominent positivity in the frontal white matter and hippocampus, proposing the marker as an ancillary diagnostic tool linking respiratory depression and hypoxia-ischaemia to early axonal injury before conventional neuronal changes appear.

The Finnish team analysed a consecutive series of neuropathologically examined medico-legal autopsy cases from 2023 to 2025 at the Helsinki office of the Finnish Institute for Health and Welfare, which conducts approximately 3000 medico-legal autopsies per year. Inclusion criteria required a history of polysubstance use, a suspected fatal acute poisoning as the cause of death, and a neuropathological examination of the formalin-fixed brain. Of 11 cases meeting these criteria, 10 were certified as fatal polysubstance poisonings and one was a natural cardiac death. The series was supplemented with three control cases: a sudden violent suicide in a person with previous drug intoxications, a fatal diabetic ketoacidosis, and a death from crush injuries and asphyxia showing characteristic traumatic and vascular axonal injury patterns.

The 14 cases were divided into three groups: poisonings with vascular axonal injury, poisonings without it, and controls. Most of the deceased were male, and ages ranged from 15 to 52 years, with nearly four in five cases among 20 to 30-year-olds. All ten poisoning deaths were classified as accidental. Toxicological analyses were based on postmortem femoral venous blood and urine, using validated and quality-controlled methods. Every poisoning involved multiple substances. In eight cases, the substance considered most significant was an opioid: buprenorphine in five, and tramadol, oxycodone and fentanyl in one case each. One remaining case was amphetamine-driven and the other ethanol-driven combined with pregabalin. All opioid- and amphetamine-driven deaths also involved benzodiazepines.

Neuropathological examination followed a standardized protocol. The cerebrum, cerebellum and brainstem underwent gross examination after at least two weeks in formalin, with tissue sampling from regions known to be vulnerable to axonal injury: the corpus callosum, internal capsule and pons. Sections were immunolabelled with antibodies targeting the N-terminal region of APP, and each staining round included a positive control and at least one immunonegative sample. In the seven positive poisoning cases, beta-APP positivity was localised in the corpus callosum and pons in all of them, and in the internal capsule in six. Positivity also appeared in other regions, including frontal white matter, lenticular nucleus and cerebellum. Oedema was frequently observed, whereas conventional haematoxylin and eosin staining revealed hypoxic-ischaemic neurons in only two cases.

Three poisoning cases showed no beta-APP immunoreactivity at all. The researchers speculate that these deaths may have occurred rapidly, before sufficient time had passed for vascular axonal injury to develop; literature suggests the time window for immunoreactivity to form is approximately 30 minutes. One negative case had at least 50-fold higher blood buprenorphine concentration than the other buprenorphine-driven deaths, possibly reflecting a higher dose. Another was the only fentanyl-driven poisoning in the series, and its lack of positivity contradicts previous literature in which all 11 fentanyl-related deaths examined showed beta-APP positivity. The third negative case was the only stimulant-driven poisoning, an amphetamine death. Previous reports of fatal stimulant poisonings have described both lack and presence of beta-APP positivity, with survival times and sampling extent varying across studies.

The control cases provided noteworthy observations. A case initially suspected to be a fatal poisoning but certified as natural coronary artery disease showed no immunoreactivity. A control case with several drug intoxications during the previous year was also negative, which the authors interpreted as further evidence that beta-APP positivity reflects a relatively recent event rather than an episode occurring months earlier. A fatal diabetic ketoacidosis case was negative as well, suggesting that not all conditions associated with delayed death or coma produce similar beta-APP findings to those seen in fatal poisonings. In contrast, the control case of traumatic brain injury and asphyxia showed axonal positivity consistent with both traumatic and vascular axonal injury, in keeping with the circumstances of death.

The authors conclude that beta-APP immunohistochemistry can serve as an ancillary tool in the forensic cause-of-death investigation of suspected fatal poisonings, particularly in deaths that may involve a delayed nature, such as those caused by central nervous system depressants. They caution, however, that the method is far from definitive. Relying on negativity can be misleading, especially when the interval between substance use and death is short, as in stimulant-driven sudden deaths. Beta-APP positivity cannot be considered a confirmatory marker of poisoning, because vascular axonal injury has been reported in a range of conditions other than poisoning. At best, the marker supports diagnosis when interpreted together with toxicological and circumstantial information. Comprehensive and systematic sampling is also required, and the findings suggest that the corpus callosum, pons and internal capsule may represent suitable sampling sites, as these regions are established sites for traumatic axonal injury and can be identified even from a fresh brain.

The study is the first report describing beta-APP immunoreactivity in buprenorphine-related deaths, which is significant because buprenorphine’s partial agonist properties set it apart from other misused opioids. The authors acknowledge limitations: the sample was small, and because neuropathological examination is not routinely performed in all intoxication cases in Finland, the sample may not represent all fatal intoxications. They also note that additional immunohistochemical markers and more brain regions could provide further value, and that more detailed circumstantial information and precise timelines could have allowed more definitive conclusions. Further research is needed to characterise the exact mechanisms linking beta-APP positivity and vascular axonal injury to poisoning deaths, and future studies should aim to delineate how substance concentrations, drug combinations and the time interval between use and death are reflected in staining patterns and regional distribution of positivity in the brain.

Subject of Research: Beta-amyloid precursor protein immunoreactivity in fatal polysubstance poisoning deaths examined at medico-legal autopsy

Article Title: β-amyloid precursor protein immunoreactivity in fatal polysubstance poisonings: a neuropathologically examined autopsy case series

Article References: Kivelä, T., Vauhkonen, P. K., Koskela, R., Kriikku, P., & Oura, P. (2026). β-amyloid precursor protein immunoreactivity in fatal polysubstance poisonings: a neuropathologically examined autopsy case series. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-04049-9

Image Credits: AI Generated

DOI: 10.1007/s00414-026-04049-9

Keywords: beta-amyloid precursor protein, vascular axonal injury, forensic neuropathology, buprenorphine, polysubstance poisoning, medico-legal autopsy, postmortem toxicology, opioid-related death, axonal injury, corpus callosum, pons, internal capsule