Time of Death Clinical Trial
Official title:
Estimate of the Time Since Death Based on the Post Mortem Quantification of the Tau Protein in the Cerebrospinal Fluid
Background: The accurate estimation of the time of death is a challenge in forensic medicine,
as the methods routinely used to assess the post-mortem interval (PMI) are far from being
precise. Recent developments in biochemical techniques may provide the opportunity to assist
in more precise estimation of the time of death. The focus has been placed on the study of
the biochemical profiles of closed compartment body fluids, as they are longer preserved than
blood after death and subject to confined post-mortem chemical changes. Cerebrospinal fluid
(CSF) has been considered as a suitable fluid to investigate these changes, as it is easy to
sample and found in large amount.
Due to its closeness to the central nervous system (CNS), CSF is used in clinical settings
for the diagnosis of various CNS disorders such as Alzheimer's disease, whose diagnosis is
mainly based on the increase of the concentrations of Tau protein and its phosphorylated
isoform (p-Tau) in CSF. A post mortem leakage of Tau into the CSF has also been shown,
reflecting progressive neuronal death as in Alzheimer's disease. In this exploratory,
cross-sectional study, we investigated Tau in post mortem CSF as a potential biomarker of the
time of death.
Objectives: The main objective was to assess the correlation between the concentration of Tau
in CSF and the PMI. The secondary objectives were (1) to determine the inter-individual
variability of the concentration of Tau for a same PMI; (2) to determine the kinetics of this
concentration over time in the same individual; (3) to determine the variability of this
concentration according to the site of collection (lumbar vs. sub-occipital).
Background: The accurate estimation of the time of death is a challenge in forensic medicine,
as the methods routinely used to assess the post-mortem interval (PMI) are far from being
precise. Recent developments in biochemical techniques may provide the opportunity to assist
in more precise estimation of the time of death. The focus has been placed on the study of
the biochemical profiles of closed compartment body fluids, as they are longer preserved than
blood after death and subject to confined post-mortem chemical changes. Cerebrospinal fluid
(CSF) has been considered as a suitable fluid to investigate these changes, as it is easy to
sample and found in large amount.
Due to its closeness to the central nervous system (CNS), CSF is used in clinical settings
for the diagnosis of various CNS disorders such as Alzheimer's disease, whose diagnosis is
mainly based on the increase of the concentrations of Tau protein and its phosphorylated
isoform (p-Tau) in CSF. A post mortem leakage of Tau into the CSF has also been shown,
reflecting progressive neuronal death as in Alzheimer's disease. In this exploratory,
cross-sectional study, we investigated Tau in post mortem CSF as a potential biomarker of the
time of death.
Objectives: The main objective was to assess the correlation between the concentration of Tau
in CSF and the PMI. The secondary objectives were (1) to determine the inter-individual
variability of the concentration of Tau for a same PMI; (2) to determine the kinetics of this
concentration over time in the same individual; (3) to determine the variability of this
concentration according to the site of collection (lumbar vs. sub-occipital).
Materials and methods: The study was reviewed and approved by the Ethics Committee of the
University Hospital of Montpellier. Post mortem CSF samples were collected from 80 adult
cadavers whose time of death was precisely known, at the mortuary of the University Hospital
of Montpellier. Individuals with neurological disorders and head trauma were excluded from
the study, as well as subjects with unknown past medical history or cause of death. CSF
samples were removed by cisternal and lumbar punctures at the time of arrival at the
mortuary, before refrigeration. A few mL of CSF were obtained at each tap in clean, sterile
polypropylene tubes, using a 18G lumbar puncture needle. The cadavers were divided into four
groups according to the PMI (n=25 in each group). The samples were taken 0-6 h (group A),
6-12 h (group B), 12-18 h (group C) and 18-24 h (group D) after death. Additionally, CSF
samples were collected every 3h from 10 cadavers during the first 15 h post mortem. All
cadavers were kept at room temperature (+20 ± 2°C) during sample collection. CSF samples were
transferred at +4°C to the laboratory where they were centrifuged for 10 min (+4°C, 1000 g).
The clear supernatant was divided into aliquots then stored at -80°C until analysis. The
rectal and tympanic temperatures were measured at the time of CSF collection using a probe
thermometer.
Concentrations of Tau and and its phosphorylated isoform (p-Tau) were measured by
conventional immunoassays, while total protein concentration was determined using a
bicinchoninic acid protein assay.
The correlation coefficient between the concentration of Tau in CSF and the PMI was
calculated in each case. The inter-individual variability was assessed by measuring the
standard deviation (SD) of the mean concentration of Tau in each group. Linear regression
analysis (adjusted for confounders) was used in assessing whether Tau concentration was
dependent on the PMI. Paired Student's t-test was used to assess the variability of Tau
concentration depending on the site of CSF collection
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