Detection of diatom in formalin-fixed tissue by proteinase K digestion

Abstract

The diatoms detection has been proposed to be useful in the diagnosis of drowning. Enzymatic digestion of unfixed lung tissues and other organs with proteinase K is widely employed to detect diatoms. Handling unfixed organs or blood from the bodies with some infectious diseases could prove to be dangerous. In this study, we examined the application of enzymatic digestion for diatom detection to formalin-fixed lung obtained at autopsy. Furthermore, we assessed the effect of hydrogen peroxide on the contamination of the lung specimen with foreign bodies inhaled in the course of drowning, smoking, or air pollution. Formalin-fixed lung was heated in 0.01 M Tris–HCl buffer (pH 7.5) containing sodium dodecyl sulfate (SDS) (tissue lysis-buffer), with or without glycine. Thereafter, the lung was subjected to enzymatic digestion with proteinase K. A part of formalin-fixed or unfixed samples digested with proteinase K were incubated with hydrogen peroxide at 80 °C for 6 h or 12 h, while the residues were processed without incubation. Formalin-fixed samples heated in tissue lysis-buffer with glycine could be digested with proteinase K; further, the number and proportion of diatoms detected in both formalin-fixed and unfixed samples were observed to be similar. The results suggest that enzymatic detection of diatoms can be applied to formalin-fixed organs by heating the samples in glycine-containing tissue lysis-buffer. As the use of formalin-fixed tissue for diatom detection can decrease risk of contamination by pathogenic organisms during the course of enzymatic digestion, the method presented in this study would be beneficial, to some extent, to individuals performing diatom analysis. Moreover, our results suggest that archival organs stored in formalin solution could be available in diatom detection over a long time-period following autopsies. Clearer image of diatoms was observed in the specimen incubated with hydrogen peroxide for 6 h, in which inhaled foreign bodies were discolored, than those not subjected to incubation. Therefore, incubation of sample digested with hydrogen peroxide in the limited time would be helpful for quantitative and qualitative diatom analysis.