Main results of the IN CADAVER protocol: The value of using dried blood spots (DBS) in post-mortem forensic toxicology

Abstract

Aim

In recent years, post-mortem analytical toxicology has undergone significant technical advances (pre-analytical extraction stage and analytical instruments), improving analysis performance. However, two challenges remain unchanged: (1) biological samples, usually autopsy samples, of several milliliters in size, constituting time-consuming and costly pre- and post-analytical management seals, and (2) difficulties in interpretation due to in cadaver phenomena during the post-mortem period (degradation and PMR), in vitro instability, and imperfections of reference systems for interpretation. In addition, in our experience, toxicological analysis as part of the search for the cause of death can be carried out correctly 9 out of 10 cases using post-mortem blood analysis alone. To overcome the two challenges mentioned above, the IN CADAVER protocol explores the possibility of carrying out such tasks using 70 μL of capillary blood collected at the time of foreign body removal. With the agreement of the judicial authorities, this single-center protocol aims to compare the toxicological results obtained (1) in micro-samples versus conventional samples taken during autopsy (T2), and (2) in micro-samples taken during body removal (T1) versus autopsy ones (T2). Here are the main results.

Method

The blood micro-samples consist of 2 DBS of 10 μL (VAMS Mitra Neoteryx) and 50 μL on a fluorinated microtube dedicated to capillary blood sampling. These micro-samples are taken (capillary vein of the foot or femoral artery) at T1 and T2, and are only analyzed when toxicological analyses are required. All samples were analyzed using the same methods (LC-HRMS, LC-MS/MS and HS-CG-FID), with additional assays for alcohol biomarkers (PEth and EtG) in the micro-samples. Only cases with positive toxicological results are finally included in the IN CADAVER protocol.

Results

Seventy-two cases of death were sampled, 35 of which were requisitioned for toxicological analysis: 33 revealed the presence of xenobiotics (mean age 41 years-old; sex-ratio 3.4; 45 hours as mean time between T1 and T2).

At T2, all the molecules detected in the autopsy blood samples were also detected in the micro-samples, and the differences between the concentrations measured in the autopsy blood samples and the autopsy micro-samples do not affect the interpretation. There was no significant difference between the blood concentrations of ethanol, PEth and ETG measured at T1 and T2. With regard to opiates, the main finding was that in 2 out of 3 cases, 6-MAM was only detected (at T1 and T2) in DBS. Certain opioids such as tramadol (and N-desmethyltramadol) showed an increasing trend between T1 and T2 (suggesting PMR). THC and related metabolites were detected in all 3 blood samples, but the concentrations measured in DBS were lower (probably due to the extraction difficulty from this sampling system). Concentrations of cocaine and its metabolites are generally DBS T1 > DBS T2 > blood T2 due to both in cadaver degradation and in vitro stabilization on DBS. The same trend was observed for benzodiazepines (e.g. oxazepam, temazepam, alprazolam, bromazepam, 7-aminoclonazepam).

Conclusion

On an analytical point of view, quantitative results obtained in micro-samples versus conventional samples are similar (with the exception of cannabinoids). On a forensic point of view, use of DBS sample enhances interpretation (e.g. heroine, cocaine, benzodiazepines intakes) and provides an easier way to take blood samples during body removal. This approach favors the interpretation of results obtained from a blood sample taken as close as possible to the time of death, regardless of the sampling area [1]. In this way, as a first step, DBS samples will be systematically added to other autopsy samples for toxicological investigations at the Lille Institute of Forensic Medicine in 2025.