Simple protocol for extracting diatoms from lung tissues of suspected drowning cases within 3 h: First practical application

Abstract

Conventional acid digestion of tissues for analyzing diatoms obtained from suspected drowning cases is time-consuming, laborious and potentially dangerous. We propose a new protocol for solubilizing lung tissue using only Qiagen Proteinase K, Qiagen Buffer ATL, and 5N hydrochloric acid that can accelerate and simplify diatom extraction from suspected drowning cases. The lower lobe of the right lung (1g, inner region) and the upper lobe of the left lung (1g, peripheral region) of ten immersed victims were digested in 15-mL conical centrifuge tubes containing 9mL of Buffer ATL and 1mL of 20mg/mL Proteinase K solution at 56°C for 15–60min. The digest was washed with ultrapure water and then heated at 70°C for 15min in 5N hydrochloric acid. The acid residue was washed with ultrapure water followed by ethanol. The identification of 60–23,000 valves in 20 lung samples from the ten victims suggested that they had aspirated water before death. The proposed digestive protocol required only a low-speed centrifuge and a block incubator (or water bath) and diatoms could usually be extracted from lung samples within about 3h. Informative results of lung diatom tests were very helpful to confirm drowning as a cause of death. Therefore, the proposed protocol can be useful as a simple minimal test to support or challenge a diagnosis of death by drowning when characteristic autopsy findings of drowning are obvious and the probability of drowning is very high, when characteristic autopsy findings of drowning are absent and the probability of drowning is very low, and when conventional diatom testing is not performed due to logistic, personnel or budgetary limitations.