Study of forensic and clinical source hemoglobin interference with the duPont aca ethanol method

The enzymatic alcohol (ethanol, ALC) analytical pack for the duPont aca is intended primarily for the analysis of serum or plasma samples containing no visibly detectable free hemoglobin (non-hemolyzed samples). It has become apparent, through clinical and medicolegal consultation, that this methodology has been applied for assay of ethanol content in hemolyzed samples (sample sources: clinical and forensic cases). The influence of sample free hemoglobin on the ace ethanol method was investigated. With 'hemolyzed' clinical samples (containing primarily oxyhemoglobin) a significant decrease was found in known ethanol concentration at 0.5 g per dl and greater of hemoglobin with the influence being greatest at lower ethanol concentration. With forensic samples (containing varying amounts of oxy-, reduced, and methemoglobins), the same observation was made with measured decrease in ethanol concentration beginning at 0.1 g per dL hemoglobin and greater. In essence, the higher the sample hemoglobin concentration the greater would be the effect in decreasing the apparent ethanol concentration. Preparation of protein-free trichloroacetic acid supernatants and their assay on the aca and by head-space gas chromatography resulted in excellent recovery of the sample ethanol. It is concluded that the effect of sample free hemoglobin is spectral in nature, and its presence results in potential and real overblanking for the aca ethanol enzymatic reaction sequence.