Enzymatic Assays

Although ADH is reasonably specific for ethanol, interferences from isopropanol, methanol, and ethylene glycol have been reported. Acetone is not a ADH substrate. ADH does, however, show varying degrees of cross-reactivity with isopropanol (6%), methanol (3%), ethylene glycol (4%), and n-propanol (1%) (29). Disinfection of skin with isopropanol before blood collection does not affect ethanol concentrations if the isopropanol is allowed to completely dry before phlebotomy. Use of ethanol to disinfect the skin has been shown to result in increases in measured blood ethanol concentrations (34).

Falsely increased ethanol concentrations have also been found in patients with increased lactic acid and lactate dehydrogenase (LD) when ethanol is measured using enzymatic assays. This is because LD catalyzes the conversion of lactate to pyruvate and NAD+ to NADH, thus generating a false signal even in the absence of any blood alcohol. However, the concentrations of lactate and LD necessary for such falsely increased results need to be at least 10 times the upper reference interval limit before an effect is seen (35). The EMIT serum alcohol assay (Behring, San Jose, CA) has been reported to produce false-positive results in postmortem samples because of increased concentrations of lactate and LD. This phenomenon was also observed in living subjects with high concentrations of lactate and LD. A patient with lactic acidosis may have high serum LD concentration because of cellular breakdown. End-stage liver disease, liver transplant (biliary atresia), Duchenne muscular dystrophy, and chronic myelogenous leukemia may also lead to high LD and lactate in living patients that may cause false-positive ethanol results by immunoassays. Nine et al. (36) observed a correlation between increasing lactate and LD concentration and false-positive ethanol results. This interference was most noticeable with the EMIT assay for alcohol and less remarkable with Abbott and Roche assays. With EMIT assay, false-positive ethanol started at LD activity of 682 U/L and lactate concentration of 14 mmol/L. The threshold was much higher for Abbott and Roche assays. Interestingly, authors observed apparent disappearance of this interference in the EMIT assay with high levels of lactate and LDH. This may be related to depletion of the NAD coenzyme (36).

Lactate concentrations tend to increase in trauma patients and Dunne et al. reported that 27% of 15,179 patients they studied had positive alcohol screen (mean alcohol: 141 mg/dL, 1 SD: 95 mg/dL, range: 10-508 mg/dL) and lactate correlated with magnitude of injury (37). Therefore, measurement of alcohol using immunoassays in hospital laboratories may be of concern. However, Winek et al. (38) compared alcohol concentration obtained by using an immunoassay (Dimension, Dade Behring) and GC in trauma patients and observed no false positive using immunoassays. Alcohol concentrations obtained by using immunoassays correlated well with GC values, and only in six specimens (out of 27) the differences between GC and immunoassay values exceeded 10%, and the highest difference was 22%. Authors concluded that immunoassay method can be used in hospital laboratory for determination of alcohol concentrations in trauma patients (38).

The interference of LD and lactate in enzyme assays for alcohol can be eliminated by taking advantage of the high molecular weight of LD, and low molecular weight of ethanol. LD is absent in the protein-free ultrafiltrate, but alcohol is not bound to serum proteins and is present in the protein-free ultrafiltrate. Although lactate is present in the ultrafiltrate, lactate alone cannot cause this interference in the absence of LD. Measuring alcohol concentrations in protein-free ultrafiltrate (prepared by centrifugation of sera for 20min at 1500 x g using Centrifree Micropartition System, MW cutoff of filter: 30,000 Da) can completely eliminate interference of lactate and LD in alcohol determination using immunoassays (39).

Serum or plasma is the most common specimen for ethanol analysis using ADH-based methods. Although whole blood may be used directly with some methods, others require a precipitation step before analysis to avoid interference from hemoglobin (40).

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