Chemistry. The Vitros and Axsym analyzers both run an "enzymatic" alcohol determination. The Vitros measurement relies upon oxidation of ethanol by the coenzyme nicotinamide adenine dinucleotide (NAD) in the presence of alcohol dehydrogenase (ADH). The reaction products are acetaldehyde and the reduced NADH. The acetaldehyde is trapped by a buffer, tris(hydroxymethyl)aminomethane (TRIS). The alcohol concentration is determined spectrophotometrically by measuring the increase in NADH concentration. The Axsym analyzer utilizes the same first step but then reacts the NADH with a monotetrazolium dye in the presence of diaphorase. The assay is based on Radiative Energy Attenuation (REA). With the Axsym,  whole blood can be run if different reagents are used.

Procedure. The Vitros analyzer pipets a 10-microliter drop of serum onto a test slide, which is a multilayered element coated on a polyester support.  The slide contains the NAD, TRIS, and ADH. The slide is incubated at 37 degrees for 5 minutes and then read spectrophotometrically. Appropriate controls and known values are run at specified intervals. The Axsym instrument autopipets an aliquot of sample-reagent mixture to a cuvette, and fluorescence changes are measured and compared against a calibration curve.

Reporting. The results are printed at the instrument and reviewed by the operator. Typically, the results are sent by computer to the patient floors where they are printed out in their final form. The printed results generated at the instrument may not be maintained.

Range of Tested Values. Values between 10 and 300 milligrams per deciliter (0.010 to 0.300 g/dL) are reportable on the Vitros analyzer and between 13 and 300 mg/dL on the Axsym. Values less than 10 (13) mg/dL are considered insignificant. Values greater than 300 mg/dL may be diluted and rerun.

Specificity. Some alcohols may cross-react with the reagents. Acetaldehyde, acetone, and methanol do not significantly interfere. On the Vitros, isopropyl alcohol at a concentration of 0.200 g/dL shows an apparent alcohol concentration of 0.020 g/dL. The Axsym gives a result of less than 0.005 g/dL with isopropyl alcohol at a concentration of 1.000 g/dL. Postmortem and immediately antemortem specimens might show erroneously high ethanol results because of extremely high levels of lactate dehydrogenase and lactate. On the Axsym analyzer, a lactic acid concentration of 270 mg/dL gave errors of less than 10%. No comparable data were reported on the Vitros analyzer.

Precision. When multiple replications were performed, a standard deviation of 0.0017 g/dL was obtained with the Vitros analyzer on specimens with mean alcohol concentration of 0.086 g/dL, and a standard deviation of 0.0043 g/dL was obtained with specimens with mean alcohol concentration of 0.195 g/dL. Values with the Axsym reflect a standard deviation of 0.00168 g/dL for 0.050 g/dL solutions and 0.00960 g/dL for 0.250 g/dL solutions.

Accuracy. When the results were compared with results obtained by head-space gas chromatography, a correlation coefficient of 0.990 was obtained with the Vitros. The correlation value for the Axsym analyzer was 0.994.

Case 1.  After an accident at 0121 hours, a blood specimen was drawn at about 0200 hours and analyzed by enzymatic analysis of serum. The result was 227 mg/dL (autoanalyzer unspecified). The hematocrit was calculated to be 41.5%. Prior to arrival, the suspect received a Lactated Ringer's infusion in the left arm at 0145 hours (slow rate) and a second Ringer's in the right arm at 0150 hours (wide open). A sample was drawn at the request of a peace officer at 0325 hours. Subsequent analysis by gas chromatographic analysis revealed an alcohol concentration of 0.16 g/dL. A search of the suspect's truck was conducted several days later; a 12-pack cardboard beer package and an ice chest were found. Conversion of the serum alcohol concentration using a 1:1.16 ratio and truncation gives a whole blood equivalent value of 0.19 g/dL. The two values, 0.19 and 0.16 g/dL, are for samples taken approximately 1.5 hours apart. This is consistent with a subject in a post-absorptive state who is eliminating alcohol at a reasonable rate of 0.020 g/dL/hour.

Case 2. A blood sample was drawn from a suspect at 0600 hours, following an accident that occurred at 0425 hours; the phlebotomist used isopropyl alcohol disinfectant. The reported result of an enzymatic serum analysis on an Axsym autoanalyzer was 116 mg/dL. A second specimen was drawn at 0803 hours, again with isopropyl alcohol disinfectant. The result of the second Axsym analysis was 57 mg/dL. A third specimen was drawn at 0805 hours, and that time a povidine swab was used. The results by gas chromatographic analysis of whole blood were 0.0379 and 0.0368 g/dL, which were reported as 0.03 g/dL. According to additional data provided, the suspect was infused with most of a 1L bag of Lactated Ringer's solution; all blood sample draws and the intravenous infusion were believed to be in the left arm (the right arm was broken). A calculated hematocrit at 0555 hours was 43.1%. Some empty beer cans were found strewn about the scene, but conclusive evidence of an open container was absent. The fact that alcohol swabs were used for the first two specimens certainly clouds the issue of alcohol concentration. Conversion to whole blood equivalents using a ratio of 1:1.16 gives 0.100 and 0.049 g/dL for samples taken two hours apart. This would predict an elimination rate of 0.025  g/dL/hour. However, comparison of the second and third specimens, taken almost simultaneously, shows a difference of 0.012 g/dL. If the first and third results are compared (0.100 and  0.037  g/dL), the predicted elimination rate becomes 0.031 g/dL/hour. The hematocrit value, 43.1% at 0555 hours, is in the normal range; thus blood dilution at that time is not suggested. As a further trial complication, a defense expert testified that infusion of the patient with Ringer's Lactate solution would elevate the lactate concentration to such a high level that the enzymatic alcohol analysis would be meaningless.

Case 3. After an accident at 0234 hours, a blood specimen was drawn at 0411 (or 0400) hours for medical purposes. Enzymatic analysis (autoanalyzer unspecified) revealed an alcohol concentration of 51.1 mg/dL. A mandatory blood specimen was drawn for law enforcement purposes at 0431 hours. Subsequent analysis by gas chromatographic analysis produced a result of 0.08 g/dL. No records were provided as to intravenous solutions prior to arrival. The medical records indicate that the suspect received a Lactated Ringer's infusion in the left arm (wide open) at 0338 hours, a normal saline in the right arm at 0342 hours (wide open), an additional Lactated Ringer's at 0405 hours (wide open, infusion site not recorded), and an additional normal saline at 0412 (wide open, site not recorded). No hematocrit result was available. No open container was found in the suspect's vehicle.