Bladder wash as an alternative specimen for post-mortem toxicology: Feedback from 63 authentic samples

Abstract

Aim

In post-mortem toxicology, analysis of different biological matrices is crucial to confirm what has been identified in blood. Nevertheless, conventional matrices can be difficult to analyze with standard extraction protocol or missing. The concept of bladder washing has been introduced in 2022 at the TIAFT annual meeting [1]. The evaluation was carried out after emptying bladder and then rewashing it. But real-life conditions can be difficult with putrefied, polytraumatized or carbonized corpses. The first bladder wash was performed in Grenoble in November 2022 by forensic pathologist team and is now performed routinely. The authors aim to describe bladder wash sampling technique and present a critical evaluation in current post-mortem toxicology practice.

Method

Bladder wash was achieved when bladder was empty, with a 5 mL wash of 0.9% NaCl solution during the forensic autopsy.

This study included authentic bladder washes from November 2022 to November 2024. Toxicological analysis were performed on request of police officers or prosecutors. The analysis was carried out in LC-MS/MS, LC-HRMS for screenings of drugs of abuses and medications and GC-FID for volatiles.

Sample preparation for screening consisted in a phospholipid removal using Ostro® plates. Targeted LC-MS/MS screenings were carried out on an Acquity UPLC HSS C18 column (2.1 × 100 mm) and a Xevo TQ-XS mass spectrometer (Waters). Targeted LC-HRMS screenings were carried out on an Accucore Phenyl-Hexyl column (2.1 × 100 mm) and an Exploris 120 (Thermo Scientific) mass spectrometer.

To evaluate the efficiency of the wash, creatininuria and proteinuria assays were carried out on an Atellica system (Siemens Healthineers®) using automated enzymatic and colorimetric techniques.

Results

This study included 63 authentic bladder washes. Mean bladder wash volume was 2,3 mL. Subjects were aged from 1 month to 92 years with mean body mass index of 26.6. Mean post-mortem interval was 2.5 days.

No substance was identified in 8 washes. Among the substances identified, psychotropic drugs (lithium, cyamemaine, venlafaxine, diazepam, vortioxetine, valproic acid, pregabaline…), drugs of abuse (cocaine, MDMA, THC-COOH, oxycodone, methadone, morphine…), cardiotropic and antidiabetic medications (amlodipine, sotalol, irbesartan, amiodarone, ramiprilat, metformine, sitagliptine, gliclazide…), anesthesic drugs (sufentanil, midazolam, laudanosine, etomidate, propofol, ketamine) or metabolites (ethylglucuronide, cotinine, acetone) were correctly detected in bladder wash.

Between blood and bladder wash results, a concordance of 76.2% was observed in this study, including ethanol. In 15.9% cases, bladder wash was negative and only ethanol was identified in blood. Therefore, ethanol is difficult to detect in bladder washes, due to the dilution. Regarding other xenobiotics, blood was positive for acetaminophen, urapidil, THC-COOH, pantoprazole, and were not detected in bladder washes in several cases.

Regarding biochemical analysis, 86.2% of the washes had undetectable levels of creatinine (inferior to 0.18 mmol/L) and elevated proteinuria (mean 4.32 g/L).

Conclusion

The feedback from 63 authentic bladder washes compared to blood in forensic routine analysis over 2 years is satisfying. It is possible to identify a large panel of substances with standard procedure.

Bladder wash may not require highly sensitive technique regarding our simple sample preparation protocol. Given the sensitivity of recent mass spectrometers, bladder wash is more convenient to analyze that bile or gastric content. Indeed, less matrix effect was observed than bile on our MS/MS and HRMS screening techniques.

Bladder wash may not be resuspended urine regarding biochemical analysis but a mix of urine and necrotizing urothelium. Further analysis may be required to properly characterize this new matrix.

Bladder wash provided satisfactory results as biological matrix for blood confirmation purposes in routine activity. This study highlights the benefits of collaboration with forensic pathologist in the evolution of practices.