Comparing clinical laboratory beta‐hydroxybutyrate testing of vitreous humor against toxicological blood analyses in ketoacidosis deaths

Abstract

Beta‐hydroxybutyrate (BHB) is used in investigations to detect ketoacidotic states. Standard chromatographic methods of analysis of blood have traditionally been used to assess BHB levels. More recently, vitreous humor has been an alternative matrix/body fluid in clinical laboratory testing. A retrospective case–control study was conducted on 406 adult autopsies performed between 2016 and 2024 where toxicologic testing of blood BHB had been performed. Quantitated postmortem blood toxicology BHB concentrations and clinical laboratory values of vitreous humor were compared. Blood BHB concentration was measured either by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–tandem mass spectrometry (LC–MS/MS), while vitreous BHB concentration was quantified enzymatically. Cases were classified as no ketoacidosis, diabetic ketoacidosis (DKA), alcoholic ketoacidosis (AKA), hypothermia, or ketoacidosis not otherwise specified (NOS) based on BHB levels, postmortem glycated hemoglobin (HbA1c) concentration, case history, and autopsy findings. Correlation and regression analyses compared blood to vitreous BHB, and receiver‐operating characteristic (ROC) curves were used to establish separate thresholds for the diagnosis of DKA and AKA by vitreous biochemistry. Vitreous BHB correlated strongly with blood BHB across both toxicologic methods (Pearson r > 0.94). ROC curves identified vitreous BHB thresholds of 478 mg/L (4.60 mmol/L) for fatal DKA ( n  = 39; sensitivity 94.9%, specificity 100%) and 283 mg/L (2.72 mmol/L) for AKA ( n  = 33; sensitivity 97.0%, specificity 93.4%); both with areas under the curve exceeding 0.97. These findings support biochemical testing of vitreous humor as a reliable modality for BHB analysis and outline diagnostic thresholds for differentiating fatal DKA and AKA.