Abstract

The effect of long-term room temperature storage on the stability of ethanol in whole blood specimens was investigated. One hundred and seventeen preserved whole blood case samples (110 of 117 with two tubes of blood in each case) were used for this study. One tube from each case was initially tested for blood alcohol concentration (BAC) for criminal driving under the influence proceedings. Cases positive for ethanol ranged in BAC from 0.023 to 0.281 g/dL. The second tube, if present, remained sealed. All blood samples were then stored at room temperature. After 5.4–10.3 years, the opened tubes were reanalyzed for BAC by the same laboratory that performed the initial testing using the same method and same instrumentation. After the same storage period, the unopened tubes were sent to a different laboratory, using a different method and different instrumentation, and reanalyzed for BAC after a total of 5.6–10.5 years of room temperature storage. Seven samples initially negative for alcohol remained negative. All samples initially positive for ethanol demonstrated a decrease in BAC over time with a statistically significant difference in loss observed based on blood sample volume and whether or not the tube had been previously opened. The decrease in BAC ranged from 0.005 to 0.234 g/dL. Tubes that were not previously opened and were more than half full demonstrated better BAC stability with 89% of these tubes demonstrating a loss of BAC between 0.01 and 0.05 g/dL.

Introduction

In criminal driving under the influence (DUI) proceedings, especially those involving fatalities, reanalysis of the blood specimen for alcohol concentration may be requested many years after the specimen was collected and initially tested. The reanalysis is usually performed by a different laboratory using different instrumentation. Oftentimes, the police agency that stores the blood specimen does not have the capacity to refrigerate blood specimens for long-term storage and, therefore, it is stored at room temperature.

The stability of ethanol in blood specimens has been studied fairly extensively over the years (1–14), but relatively few studies use authentic antemortem case samples and modern headspace (HS) gas chromatography (GC) techniques. Additionally, none have included an investigation of the use of a different laboratory to conduct the reanalysis after storage. In one study, an interlaboratory evaluation of the stability of ethanol in control materials prepared in sterile, treated whole blood was conducted (2). However, no study using a second laboratory to reanalyze authentic case samples has yet been reported. Since reanalysis of blood samples for ethanol in criminal cases is regularly performed by a different laboratory using different instrumentation than the initial analysis, it would be useful to evaluate the complication of these additional variables.

Ethanol concentration in stored antemortem blood samples will decrease over time (1–14). The mechanism of this loss has been attributed to microorganisms (4), evaporation (4) and oxyhemoglobin-mediated oxidation (3, 5, 13, 15). In sealed, preserved tubes, oxidation has been identified as the primary process (9, 12).

Time, temperature, preservative, airspace in the sample container and whether the tube has been previously opened have been identified as significant factors affecting the magnitude of ethanol loss in antemortem samples (1–14). At room temperature or colder storage conditions, ethanol is stable for at least 2 weeks regardless of whether or not a preservative is used (7). At elevated temperatures and/or extended periods of time, BAC will decrease in stored antemortem samples (1, 3, 6). The magnitude of the decrease will be greater in those tubes that have been previously opened as there is a greater supply of free oxygen for the oxyhemoglobin-mediated oxidation of ethanol (3, 5, 6, 12–14). Tubes with greater airspace would likewise be expected to display greater decreases in ethanol over time.

Experimental

One hundred and seventeen blood specimens (110 with two tubes of blood designated as Tubes A and B) in 10 mL gray-stoppered glass vacutainer style vials containing 20 mg potassium oxalate as an anticoagulant and 100 mg sodium fluoride as a preservative collected for the purpose of DUI investigations were used in this study. Initial analysis for blood alcohol concentration (BAC) was performed by the Florida Department of Law Enforcement Orlando Regional Operations Center (FDLE OROC) Toxicology Section on Tubes A using a method similar to one previously published (1) with slight modifications that are described below. The blood samples were submitted in the normal course of business for DUI investigations. After the cases were adjudicated by the court and the police agency was going to proceed with evidence destruction, FDLE OROC obtained the samples and used the original method for the reanalysis of BAC on all Tubes A after 5.4–10.3 years of storage at room temperature. Tubes B, available for 110 case samples, were previously unopened and transported to the Palm Beach County Sheriff's Office (PBSO) Toxicology Unit for BAC analysis after 5.6–10.5 years of storage at room temperature using a method previously published (16, 17) and described below. Upon receipt of Tubes B from FDLE OROC and throughout the experimental period, the blood was stored at room temperature.

Materials and methods used at FDLE OROC

Standards

For the initial analysis, commercially prepared aqueous ethanol standards at concentrations of 0.050, 0.100, 0.200 and 0.400 g/dL used as calibrators were obtained from EMD Chemicals, Inc. (Philadelphia, PA, USA) and a 0.020-g/dL calibrator was prepared from the 0.200-g/dL solution. For the reanalysis, equivalent aqueous ethanol standards at 0.020, 0.050, 0.100, 0.200 and 0.400 g/dL obtained from Cerilliant, Inc. (Round Rock, TX) were used as calibrators. Additional levels of 0.025, 0.080 and 0.300 g/dL used as controls for both initial testing and reanalysis were obtained from Cerilliant, Inc.

A whole blood volatiles control at an ethanol concentration of 0.12 g/dL used for both initial and reanalysis containing methanol, ethanol, isopropanol and acetone was prepared using laboratory verified blank blood from a blood bank, absolute ethanol and at least ACS grade isopropanol, methanol and acetone (Fisher Scientific, Pittsburgh, PA, USA). Normal propanol internal standard used for both initial and reanalysis was prepared at a concentration of 0.01% by volume (%v/v) with at least ACS grade n-propanol (Fisher Scientific) in deionized water.

Analysis

The quantitative analyses of blood alcohol were performed utilizing a HS-GC method with dual column flame ionization detection similar to one described previously (1). All standards were allowed to come to room temperature and samples were mixed on a tube rotator prior to sampling. One hundred microliters of alcohol calibrators, controls and blood samples were transferred with 1 mL of internal standard into 20 mL HS vials utilizing a Cavro or Hamilton® Model 503A Diluter-Dispenser equipped with 1 and 0.1 mL syringes. Vials were sealed, crimped and then placed onto an autosampler for analysis. One aqueous standard at 0.020, 0.050, 0.100, 0.200 and 0.400 g/dL was used to generate the linear (origin not included) calibration curve. To verify the calibration, the whole blood volatiles control with a target ethanol concentration of 0.12 g/dL and aqueous standards at 0.025, 0.080 and 0.300 g/dL were prepared in duplicate. At least one internal standard blank-negative control was also prepared with deionized water. All case samples were prepared in duplicate and the results presented in this study were the average of duplicate analyses.

A Perkin-Elmer Autosystem or Autosystem XL GC equipped with dual flame ionization detectors and a Perkin-Elmer HS40 or HS40XL HS autosampler were used at FDLE OROC for the initial and reanalysis of Tubes A. The GCs were equipped with dual columns connected by a glass y-splitter: Column A was a DB-ALC1 (Agilent, Palo Alto, CA) fused-silica capillary column with dimensions of 30 m × 0.53 mm i.d. and a 3-µm film thickness, and Column B was a DB-ALC2 (Agilent) fused-silica capillary column with dimensions of 30 m × 0.53 mm i.d. and a 2-µm film thickness. Helium was used as the carrier gas. All gases were Ultra High Purity. The HS oven temperature was set at 35°C. The needle and transfer line temperatures were set at 90°C. Vial equilibration was set at 22 min. For the GC, a constant helium pressure of ∼9.5 psi was used. The isothermal GC run at 40°C was 6.5 min per sample. The flame ionization detector (FID) temperatures were maintained at 150°C with hydrogen and air pressures of 40 and 450 psi, respectively. Quantitation was performed using the response ratio of the FID response on Column A (DB-ALC1) of ethanol to n-propanol.

Materials and methods used at PBSO

Standards

Commercially prepared aqueous ethanol standards at concentrations of 0.020, 0.025, 0.100, 0.200, 0.300 and 0.500 g/dL were obtained from Cerilliant, Inc. A whole blood volatiles standard containing methanol, ethanol, acetone and isopropanol with an ethanol target concentration of 0.08 g/dL was purchased from Cliniqa Corporation (San Marcos, CA, USA). Normal propanol internal standard was prepared at a concentration of 0.01% v/v with n-propanol from Alfa Aesar (Ward Hill, MA, USA) in deionized water.

Analysis

The quantitative analyses of blood alcohol were performed utilizing a HS-GC method with simultaneous flame ionization and mass spectrometric detection described previously (16, 17). All standards were allowed to come to room temperature and samples were mixed on a tube rotator prior to sampling. One hundred microliters of alcohol calibrators, controls and blood samples were transferred with 1 mL of internal standard into 20 mL HS vials utilizing a Hamilton® Model 503A Diluter-Dispenser equipped with 1 and 0.1 mL syringes. Vials were sealed, crimped and then placed onto an autosampler for analysis. One aqueous standard from Cerilliant at 0.020, 0.100, 0.200 and 0.500 g/dL was used to generate the linear (origin not included) calibration curve. To verify the calibration, the whole blood volatiles control from Cliniqa with a target concentration of 0.08 g/dL ethanol and aqueous standards from Cerilliant at 0.025 and 0.300 g/dL were prepared in duplicate. One set of controls was analyzed prior to case samples and one set immediately after. An internal standard blank-negative control was also prepared with deionized water and analyzed after the 0.500-g/dL calibrator. All case samples were prepared in duplicate and the results presented in this study were the average of duplicate analyses.

The instrumentation used for analysis at PBSO of Tube B samples was an Agilent G1888 HS sampler with a 7890A series GC equipped with a Capillary Flow Technology (CFT) two-way splitter with makeup gas, FID and 5975C series mass spectrometer (MS). The analytical column used was a DB-ALC1 (Agilent) fused-silica capillary column with dimensions of 30 m × 0.32 mm i.d. and a 1.8-µm film thickness. The terminal end of the analytical column was connected to the CFT two-way splitter with makeup gas. From the CFT two-way splitter, deactivated fused-silica columns (restrictors) were connected to each detector. The CFT two-way splitter with makeup gas was configured using a 1 : 1 split ratio to the FID and MS according to the manufacturer's instructions, with the exception of the flow rate, using fused-silica capillary restrictors with dimensions of 1.06 m × 0.18 mm to the FID and 2.89 m × 0.18 mm to the MS. The makeup gas of the restrictors was set at a constant flow rate of 2 mL/min, not a constant pressure of 3.8 psi as recommended by the manufacturer. Helium was used as the carrier gas and makeup gas. All gases were Ultra High Purity.

All B tube samples were analyzed on the HS-GC-FID-MS instrumentation described above with a HS oven temperature of 50°C. The HS loop and transfer line temperatures were set at 70 and 90°C, respectively. Vial equilibration was set at 20 min. The vial pressurization was set at 15 psi for 0.15 min. Injection, loop fill and loop equilibration times were set at 0.50, 0.15 and 0.05 min, respectively. Multi HS extraction and vial shaking were set to off. The GC cycle time was set at 13.5 min. For the GC, a constant helium flow rate of 3 mL/min was used. The injection port temperature was maintained at 90°C with a 5 : 1 split injection of the HS and a septum purge flow of 3 mL/min. The initial GC oven temperature of 35°C was held for 2 min and then ramped at 25°C per minute to a final temperature of 90°C, which was held for 4.3 min. The total GC run time was 8.5 min per sample. The FID temperature was maintained at 300°C with hydrogen, air and constant column plus helium makeup pressures of 40, 450 and 50 psi, respectively. The FID signal was zeroed at 0 min with a data collection rate of 10 Hz. The MS transfer line was maintained at 280°C. The MS source and quadrupole were maintained at 230 and 150°C, respectively. The MS electron multiplier voltage was set to a gain factor of 1 (tuned using Agilent Chemstation Gain Tune followed by Low Mass Auto Tune). The scan range was set at 20–200 m/z with a threshold of 150 and a sample number of 4 which resulted in a scan rate of 2.02 scans/s. The trace ion detection feature was turned on. Quantitation was performed using the response ratio of the FID response of ethanol to n-propanol.

Validation of the method included evaluation of HS oven thermostat time, thermostat stability, sensitivity, linearity, matrix effects, carryover, repeatability, drift/bias, specificity and a crossover case comparison study. The LOD and LOQ were determined to be 0.005 and 0.010 g/dL, respectively. The CV of replicate analyses was <3.1%. Quantitative accuracy was within ±8, ±6, ±3 and ±1.5% at concentrations of 0.010, 0.025, 0.080 and 0.300 g/dL, respectively. Further details on the method validation have already been discussed elsewhere (16, 17).

Results and discussion

Table I lists the original and reanalysis results for both Tubes A and B, and Table II summarizes the change in BAC for cases originally positive for ethanol. Seven of the cases were initially negative; six had both Tubes A and B. All tubes which were originally negative remained negative when retested after storage. Of the 110 previously opened tubes (Tubes A) initially positive for ethanol, all showed a decrease in concentration with a mean decrease of 0.051 g/dL. There were 104 previously unopened tubes (Tubes B) initially positive for ethanol that also showed a decrease in concentration that was lower than the previously opened tubes with a mean decrease of 0.035 g/dL. A paired Student's t-test demonstrated that the reanalysis results for both Tubes A and B were significantly different from the original results at a confidence level (CL) of 99.9% (P < 0.001). Identical lots of 0.025 and 0.300 g/dL standards from Cerilliant were analyzed along with the experimental samples for a total of 16 replicates by each method. There was no significant difference in the results of the standards at either level (CL 99.9%, P > 0.001). The stated accuracy of both methods of BAC analysis was 0.005 g/dL or 5%, whichever is larger (1). The changes in BAC for 9 of 104 of the B tubes were within the stated accuracy of the method. However, for all the A tubes and 95 of the B tubes, the deviations were greater than the accuracy of the method, indicating that the differences in BAC results were true losses in BAC rather than due to normal variation in the measurement. None of the blood samples, either for Tubes A or B, showed any increase in BAC. These findings are consistent with previously published studies on ethanol stability in antemortem blood (1, 3, 6, 7, 10, 12).

Table I

Change in BAC After Long-Term Storage at Room Temperature

Case Approximate tube volume (mL, if ≤6 mL)
 
Storage time (years)
 
Averaged results (g/dL)
 
Change in BAC (g/dL)
 
% Change in BAC
 
Tube A Tube B Tube A Tube B Initial (Tube A) Tube A reanalysis Tube B reanalysis Tube A reanalysis Tube B reanalysis Tube A reanalysis Tube B reanalysis 
0.5 N/A 10.3 N/A 0.023 Negative N/A −0.023 N/A −100 N/A 
  10.3 10.5 Negative Negative Negative N/A N/A N/A N/A 
  10.3 10.4 0.199 0.159 0.189 −0.040 −0.011 −20 −5 
  10.3 10.4 0.093 0.060 0.077 −0.033 −0.016 −35 −17 
  10.2 10.3 0.179 0.141 0.166 −0.038 −0.014 −21 −8 
  10.1 10.3 0.274 0.214 0.268 −0.060 −0.006 −22 −2 
 10.1 10.3 0.046 Negative 0.011a −0.046 −0.035 −100 −76 
10.1 10.3 0.276 0.205 0.130 −0.071 −0.146 −26 −53 
 10.2 10.3 0.203 0.157 0.155 −0.046 −0.048 −22 −23 
10   10.0 10.2 0.154 0.109 0.122 −0.045 −0.032 −29 −21 
11   10.0 10.2 Negative Negative Negative N/A N/A N/A N/A 
12   10.0 10.2 0.107 0.069 0.090 −0.038 −0.017 −35 −16 
13  10.0 10.2 Negative Negative Negative N/A N/A N/A N/A 
14   9.9 10.1 0.232 0.186 0.209 −0.046 −0.023 −20 −10 
15 9.9 10.1 0.177 0.081 0.103 −0.096 −0.074 −54 −42 
16   9.9 10.1 0.066 0.022 0.036 −0.044 −0.030 −67 −45 
17   9.8 10.0 0.323 0.273 0.291 −0.050 −0.032 −15 −10 
18 2.5 N/A 9.8 N/A 0.141 0.112 N/A −0.029 N/A −21 N/A 
19 9.8 9.9 Negative Negative Negative N/A N/A N/A N/A 
20   9.8 9.9 0.118 0.070 0.080 −0.048 −0.038 −41 −32 
21   9.8 9.9 0.125 0.076 0.092 −0.049 −0.034 −39 −27 
22   9.8 9.9 0.233 0.197 0.226 −0.036 −0.008 −15 −3 
23  9.8 9.9 0.162 0.106 0.128 −0.056 −0.034 −34 −21 
24   9.7 9.8 0.170 0.118 0.123 −0.052 −0.047 −30 −27 
25   9.7 9.8 0.278 0.231 0.253 −0.047 −0.025 −17 −9 
26   9.7 9.8 0.169 0.129 0.148 −0.040 −0.021 −24 −12 
27  9.7 9.8 0.121 0.076 0.073 −0.045 −0.048 −37 −40 
28   9.7 9.8 0.179 0.149 0.170 −0.030 −0.009 −17 −5 
29 1.5  9.6 9.8 0.067 Negative 0.028 −0.067 −0.039 −100 −58 
30  9.6 9.8 0.229 0.187 0.093 −0.042 −0.136 −18 −59 
31   9.6 9.8 0.174 0.126 0.141 −0.048 −0.034 −28 −19 
32  9.6 9.8 0.151 0.099 0.076 −0.052 −0.075 −34 −50 
33   9.6 9.8 0.082 0.041 0.053 −0.041 −0.029 −50 −35 
34   9.6 9.8 0.180 0.130 0.145 −0.050 −0.035 −28 −19 
35 N/A 9.6 N/A Negative Negative N/A N/A N/A N/A N/A 
36   9.5 9.7 0.214 0.162 0.175 −0.052 −0.039 −24 −18 
37   9.5 9.7 0.183 0.135 0.153 −0.048 −0.030 −26 −16 
38   9.5 9.7 0.260 0.207 0.245 −0.053 −0.015 −20 −6 
39  9.4 9.6 0.213 0.150 0.172 −0.063 −0.041 −29 −19 
40   9.4 9.6 0.187 0.140 0.157 −0.047 −0.031 −25 −16 
41   9.4 9.6 0.174 0.134 0.153 −0.040 −0.021 −23 −12 
42 9.4 9.6 0.142 0.064 0.082 −0.078 −0.060 −55 −42 
43  9.4 9.6 0.111 0.074 0.098 −0.037 −0.013 −33 −12 
44   9.3 9.5 0.184 0.132 0.150 −0.052 −0.034 −28 −18 
45   9.3 9.5 0.258 0.205 0.215 −0.053 −0.043 −21 −17 
46  9.3 9.5 0.090 0.054 Negative −0.036 −0.090 −40 −100 
47   9.3 9.4 0.178 0.136 0.134 −0.042 −0.045 −24 −25 
48   9.3 9.4 0.055 0.013a 0.020 −0.042 −0.035 −76 −64 
49  9.3 9.4 0.226 0.166 0.189 −0.060 −0.037 −27 −16 
50   9.3 9.4 0.064 0.021 0.031 −0.043 −0.033 −67 −51 
51   9.2 9.3 0.211 0.160 0.177 −0.051 −0.034 −24 −16 
52   9.2 9.3 0.199 0.147 0.152 −0.052 −0.047 −26 −24 
53   9.2 9.3 0.222 0.192 0.212 −0.030 −0.010 −13 −5 
54   9.2 9.3 0.147 0.106 0.128 −0.041 −0.020 −28 −13 
55   9.2 9.3 0.170 0.123 0.142 −0.047 −0.028 −27 −17 
56   9.2 9.3 0.166 0.117 0.139 −0.049 −0.027 −30 −16 
57   9.2 9.3 0.135 0.090 0.106 −0.045 −0.029 −33 −22 
58  9.1 9.3 0.204 0.140 0.176 −0.064 −0.029 −31 −14 
59   9.1 9.3 0.198 0.135 0.172 −0.063 −0.027 −32 −13 
60 9.1 9.3 0.081 Negative Negative −0.081 −0.081 −100 −100 
61   9.1 9.3 0.112 0.064 0.069 −0.048 −0.043 −43 −38 
62  9.1 9.3 0.204 0.151 0.153 −0.053 −0.051 −26 −25 
63   9.0 9.2 0.167 0.123 0.148 −0.044 −0.020 −26 −12 
64   9.0 9.2 0.211 0.177 0.201 −0.034 −0.009 −16 −5 
65   9.0 9.2 0.206 0.159 0.174 −0.047 −0.032 −23 −16 
66   9.0 9.2 0.150 0.092 0.112 −0.058 −0.038 −38 −25 
67  9.0 9.2 0.100 0.025 0.069 −0.075 −0.031 −75 −31 
68 4.5  9.0 9.2 Negative Negative Negative N/A N/A N/A N/A 
69   9.0 9.2 0.104 0.050 0.067 −0.054 −0.038 −52 −36 
70   9.0 9.2 0.218 0.170 0.183 −0.048 −0.035 −22 −16 
71  N/A 8.9 N/A 0.136 0.090 N/A −0.046 N/A −34 N/A 
72 8.9 9.1 0.086 0.015a 0.039 −0.071 −0.047 −83 −55 
73  8.9 9.1 0.082 0.038 0.042 −0.044 −0.040 −53 −48 
74 N/A 8.8 N/A 0.276 0.080 N/A −0.196 N/A −71 N/A 
75   8.8 0.176 0.131 0.137 −0.045 −0.039 −26 −22 
76   8.8 8.9 0.148 0.102 0.102 −0.046 −0.046 −31 −31 
77   8.8 8.9 0.176 0.133 0.141 −0.043 −0.036 −24 −20 
78   8.7 8.8 0.155 0.117 0.128 −0.038 −0.027 −24 −17 
79   8.6 8.8 0.221 0.175 0.192 −0.046 −0.029 −21 −13 
80   8.6 8.8 0.218 0.173 0.189 −0.045 −0.029 −20 −13 
81   8.6 8.8 0.211 0.166 0.170 −0.045 −0.042 −21 −20 
82   8.6 8.8 0.150 0.113 0.128 −0.037 −0.022 −24 −15 
83 8.5 8.7 0.172 0.092 0.111 −0.080 −0.061 −46 −35 
84 N/A 8.5 N/A 0.093 Negative N/A −0.093 N/A −100 N/A 
85   8.5 8.6 0.246 0.199 0.228 −0.047 −0.018 −19 −7 
86   8.5 8.6 0.189 0.146 0.161 −0.043 −0.028 −23 −15 
87   8.4 8.5 0.130 0.087 0.100 −0.043 −0.030 −33 −23 
88   8.4 8.5 0.278 0.231 0.268 −0.047 −0.010 −17 −3 
89   8.4 8.5 0.180 0.132 0.145 −0.048 −0.035 −26 −19 
90   8.4 8.5 0.215 0.168 0.179 −0.047 −0.037 −22 −17 
91   8.4 8.5 0.187 0.159 0.175 −0.028 −0.012 −15 −6 
92   8.4 8.5 0.196 0.161 0.170 −0.035 −0.027 −18 −14 
93  8.3 8.4 0.242 0.212 0.199 −0.030 −0.043 −12 −18 
94 1.5 N/A 8.3 N/A 0.234 Negative N/A −0.234 N/A −100 N/A 
95 8.3 8.3 0.186 0.122 0.121 −0.064 −0.065 −34 −35 
96   8.3 8.3 0.119 0.076 0.092 −0.043 −0.027 −36 −23 
97   8.3 8.3 0.094 0.053 0.064 −0.041 −0.031 −44 −32 
98   8.2 8.3 0.443 0.386 0.405 −0.057 −0.038 −13 −9 
99   8.2 8.3 0.264 0.215 0.229 −0.049 −0.035 −19 −13 
100   8.0 8.1 0.198 0.153 0.163 −0.045 −0.035 −23 −17 
101   8.0 8.2 0.222 0.174 0.198 −0.048 −0.024 −21 −11 
102   7.9 8.1 0.124 0.081 0.099 −0.043 −0.026 −35 −21 
103   7.9 8.1 Negative Negative Negative N/A N/A N/A N/A 
104   7.7 7.8 0.145 0.108 0.126 −0.037 −0.019 −26 −13 
105   7.6 7.8 0.235 0.182 0.221 −0.053 −0.014 −23 −6 
106 5.5 7.6 7.8 0.120 0.072 0.061 −0.048 −0.059 −40 −49 
107   7.6 7.8 0.221 0.185 0.203 −0.036 −0.018 −16 −8 
108   7.5 7.7 0.103 0.065 0.088 −0.038 −0.015 −37 −14 
109 7.5 7.7 0.149 0.082 Negative −0.067 −0.149 −45 −100 
110   7.5 7.7 0.150 0.111 0.120 −0.039 −0.030 −26 −20 
111   7.5 7.7 0.134 0.097 0.109 −0.037 −0.025 −27 −18 
112   6.3 6.4 0.158 0.131 0.152 −0.027 −0.006 −17 −4 
113 5.5 6.3 6.4 0.180 0.125 0.141 −0.055 −0.039 −30 −21 
114   5.8 6.0 0.306 0.281 0.301 −0.025 −0.005 −8 −1 
115 5.5  5.8 5.9 0.232 0.178 0.217 −0.054 −0.016 −23 −7 
116   5.7 5.8 0.102 0.065 0.095 −0.037 −0.007 −36 −7 
117   5.4 5.6 0.281 0.248 0.270 −0.033 −0.011 −12 −4 
Case Approximate tube volume (mL, if ≤6 mL)
 
Storage time (years)
 
Averaged results (g/dL)
 
Change in BAC (g/dL)
 
% Change in BAC
 
Tube A Tube B Tube A Tube B Initial (Tube A) Tube A reanalysis Tube B reanalysis Tube A reanalysis Tube B reanalysis Tube A reanalysis Tube B reanalysis 
0.5 N/A 10.3 N/A 0.023 Negative N/A −0.023 N/A −100 N/A 
  10.3 10.5 Negative Negative Negative N/A N/A N/A N/A 
  10.3 10.4 0.199 0.159 0.189 −0.040 −0.011 −20 −5 
  10.3 10.4 0.093 0.060 0.077 −0.033 −0.016 −35 −17 
  10.2 10.3 0.179 0.141 0.166 −0.038 −0.014 −21 −8 
  10.1 10.3 0.274 0.214 0.268 −0.060 −0.006 −22 −2 
 10.1 10.3 0.046 Negative 0.011a −0.046 −0.035 −100 −76 
10.1 10.3 0.276 0.205 0.130 −0.071 −0.146 −26 −53 
 10.2 10.3 0.203 0.157 0.155 −0.046 −0.048 −22 −23 
10   10.0 10.2 0.154 0.109 0.122 −0.045 −0.032 −29 −21 
11   10.0 10.2 Negative Negative Negative N/A N/A N/A N/A 
12   10.0 10.2 0.107 0.069 0.090 −0.038 −0.017 −35 −16 
13  10.0 10.2 Negative Negative Negative N/A N/A N/A N/A 
14   9.9 10.1 0.232 0.186 0.209 −0.046 −0.023 −20 −10 
15 9.9 10.1 0.177 0.081 0.103 −0.096 −0.074 −54 −42 
16   9.9 10.1 0.066 0.022 0.036 −0.044 −0.030 −67 −45 
17   9.8 10.0 0.323 0.273 0.291 −0.050 −0.032 −15 −10 
18 2.5 N/A 9.8 N/A 0.141 0.112 N/A −0.029 N/A −21 N/A 
19 9.8 9.9 Negative Negative Negative N/A N/A N/A N/A 
20   9.8 9.9 0.118 0.070 0.080 −0.048 −0.038 −41 −32 
21   9.8 9.9 0.125 0.076 0.092 −0.049 −0.034 −39 −27 
22   9.8 9.9 0.233 0.197 0.226 −0.036 −0.008 −15 −3 
23  9.8 9.9 0.162 0.106 0.128 −0.056 −0.034 −34 −21 
24   9.7 9.8 0.170 0.118 0.123 −0.052 −0.047 −30 −27 
25   9.7 9.8 0.278 0.231 0.253 −0.047 −0.025 −17 −9 
26   9.7 9.8 0.169 0.129 0.148 −0.040 −0.021 −24 −12 
27  9.7 9.8 0.121 0.076 0.073 −0.045 −0.048 −37 −40 
28   9.7 9.8 0.179 0.149 0.170 −0.030 −0.009 −17 −5 
29 1.5  9.6 9.8 0.067 Negative 0.028 −0.067 −0.039 −100 −58 
30  9.6 9.8 0.229 0.187 0.093 −0.042 −0.136 −18 −59 
31   9.6 9.8 0.174 0.126 0.141 −0.048 −0.034 −28 −19 
32  9.6 9.8 0.151 0.099 0.076 −0.052 −0.075 −34 −50 
33   9.6 9.8 0.082 0.041 0.053 −0.041 −0.029 −50 −35 
34   9.6 9.8 0.180 0.130 0.145 −0.050 −0.035 −28 −19 
35 N/A 9.6 N/A Negative Negative N/A N/A N/A N/A N/A 
36   9.5 9.7 0.214 0.162 0.175 −0.052 −0.039 −24 −18 
37   9.5 9.7 0.183 0.135 0.153 −0.048 −0.030 −26 −16 
38   9.5 9.7 0.260 0.207 0.245 −0.053 −0.015 −20 −6 
39  9.4 9.6 0.213 0.150 0.172 −0.063 −0.041 −29 −19 
40   9.4 9.6 0.187 0.140 0.157 −0.047 −0.031 −25 −16 
41   9.4 9.6 0.174 0.134 0.153 −0.040 −0.021 −23 −12 
42 9.4 9.6 0.142 0.064 0.082 −0.078 −0.060 −55 −42 
43  9.4 9.6 0.111 0.074 0.098 −0.037 −0.013 −33 −12 
44   9.3 9.5 0.184 0.132 0.150 −0.052 −0.034 −28 −18 
45   9.3 9.5 0.258 0.205 0.215 −0.053 −0.043 −21 −17 
46  9.3 9.5 0.090 0.054 Negative −0.036 −0.090 −40 −100 
47   9.3 9.4 0.178 0.136 0.134 −0.042 −0.045 −24 −25 
48   9.3 9.4 0.055 0.013a 0.020 −0.042 −0.035 −76 −64 
49  9.3 9.4 0.226 0.166 0.189 −0.060 −0.037 −27 −16 
50   9.3 9.4 0.064 0.021 0.031 −0.043 −0.033 −67 −51 
51   9.2 9.3 0.211 0.160 0.177 −0.051 −0.034 −24 −16 
52   9.2 9.3 0.199 0.147 0.152 −0.052 −0.047 −26 −24 
53   9.2 9.3 0.222 0.192 0.212 −0.030 −0.010 −13 −5 
54   9.2 9.3 0.147 0.106 0.128 −0.041 −0.020 −28 −13 
55   9.2 9.3 0.170 0.123 0.142 −0.047 −0.028 −27 −17 
56   9.2 9.3 0.166 0.117 0.139 −0.049 −0.027 −30 −16 
57   9.2 9.3 0.135 0.090 0.106 −0.045 −0.029 −33 −22 
58  9.1 9.3 0.204 0.140 0.176 −0.064 −0.029 −31 −14 
59   9.1 9.3 0.198 0.135 0.172 −0.063 −0.027 −32 −13 
60 9.1 9.3 0.081 Negative Negative −0.081 −0.081 −100 −100 
61   9.1 9.3 0.112 0.064 0.069 −0.048 −0.043 −43 −38 
62  9.1 9.3 0.204 0.151 0.153 −0.053 −0.051 −26 −25 
63   9.0 9.2 0.167 0.123 0.148 −0.044 −0.020 −26 −12 
64   9.0 9.2 0.211 0.177 0.201 −0.034 −0.009 −16 −5 
65   9.0 9.2 0.206 0.159 0.174 −0.047 −0.032 −23 −16 
66   9.0 9.2 0.150 0.092 0.112 −0.058 −0.038 −38 −25 
67  9.0 9.2 0.100 0.025 0.069 −0.075 −0.031 −75 −31 
68 4.5  9.0 9.2 Negative Negative Negative N/A N/A N/A N/A 
69   9.0 9.2 0.104 0.050 0.067 −0.054 −0.038 −52 −36 
70   9.0 9.2 0.218 0.170 0.183 −0.048 −0.035 −22 −16 
71  N/A 8.9 N/A 0.136 0.090 N/A −0.046 N/A −34 N/A 
72 8.9 9.1 0.086 0.015a 0.039 −0.071 −0.047 −83 −55 
73  8.9 9.1 0.082 0.038 0.042 −0.044 −0.040 −53 −48 
74 N/A 8.8 N/A 0.276 0.080 N/A −0.196 N/A −71 N/A 
75   8.8 0.176 0.131 0.137 −0.045 −0.039 −26 −22 
76   8.8 8.9 0.148 0.102 0.102 −0.046 −0.046 −31 −31 
77   8.8 8.9 0.176 0.133 0.141 −0.043 −0.036 −24 −20 
78   8.7 8.8 0.155 0.117 0.128 −0.038 −0.027 −24 −17 
79   8.6 8.8 0.221 0.175 0.192 −0.046 −0.029 −21 −13 
80   8.6 8.8 0.218 0.173 0.189 −0.045 −0.029 −20 −13 
81   8.6 8.8 0.211 0.166 0.170 −0.045 −0.042 −21 −20 
82   8.6 8.8 0.150 0.113 0.128 −0.037 −0.022 −24 −15 
83 8.5 8.7 0.172 0.092 0.111 −0.080 −0.061 −46 −35 
84 N/A 8.5 N/A 0.093 Negative N/A −0.093 N/A −100 N/A 
85   8.5 8.6 0.246 0.199 0.228 −0.047 −0.018 −19 −7 
86   8.5 8.6 0.189 0.146 0.161 −0.043 −0.028 −23 −15 
87   8.4 8.5 0.130 0.087 0.100 −0.043 −0.030 −33 −23 
88   8.4 8.5 0.278 0.231 0.268 −0.047 −0.010 −17 −3 
89   8.4 8.5 0.180 0.132 0.145 −0.048 −0.035 −26 −19 
90   8.4 8.5 0.215 0.168 0.179 −0.047 −0.037 −22 −17 
91   8.4 8.5 0.187 0.159 0.175 −0.028 −0.012 −15 −6 
92   8.4 8.5 0.196 0.161 0.170 −0.035 −0.027 −18 −14 
93  8.3 8.4 0.242 0.212 0.199 −0.030 −0.043 −12 −18 
94 1.5 N/A 8.3 N/A 0.234 Negative N/A −0.234 N/A −100 N/A 
95 8.3 8.3 0.186 0.122 0.121 −0.064 −0.065 −34 −35 
96   8.3 8.3 0.119 0.076 0.092 −0.043 −0.027 −36 −23 
97   8.3 8.3 0.094 0.053 0.064 −0.041 −0.031 −44 −32 
98   8.2 8.3 0.443 0.386 0.405 −0.057 −0.038 −13 −9 
99   8.2 8.3 0.264 0.215 0.229 −0.049 −0.035 −19 −13 
100   8.0 8.1 0.198 0.153 0.163 −0.045 −0.035 −23 −17 
101   8.0 8.2 0.222 0.174 0.198 −0.048 −0.024 −21 −11 
102   7.9 8.1 0.124 0.081 0.099 −0.043 −0.026 −35 −21 
103   7.9 8.1 Negative Negative Negative N/A N/A N/A N/A 
104   7.7 7.8 0.145 0.108 0.126 −0.037 −0.019 −26 −13 
105   7.6 7.8 0.235 0.182 0.221 −0.053 −0.014 −23 −6 
106 5.5 7.6 7.8 0.120 0.072 0.061 −0.048 −0.059 −40 −49 
107   7.6 7.8 0.221 0.185 0.203 −0.036 −0.018 −16 −8 
108   7.5 7.7 0.103 0.065 0.088 −0.038 −0.015 −37 −14 
109 7.5 7.7 0.149 0.082 Negative −0.067 −0.149 −45 −100 
110   7.5 7.7 0.150 0.111 0.120 −0.039 −0.030 −26 −20 
111   7.5 7.7 0.134 0.097 0.109 −0.037 −0.025 −27 −18 
112   6.3 6.4 0.158 0.131 0.152 −0.027 −0.006 −17 −4 
113 5.5 6.3 6.4 0.180 0.125 0.141 −0.055 −0.039 −30 −21 
114   5.8 6.0 0.306 0.281 0.301 −0.025 −0.005 −8 −1 
115 5.5  5.8 5.9 0.232 0.178 0.217 −0.054 −0.016 −23 −7 
116   5.7 5.8 0.102 0.065 0.095 −0.037 −0.007 −36 −7 
117   5.4 5.6 0.281 0.248 0.270 −0.033 −0.011 −12 −4 

N/A, not applicable, no B tube available.

aOutside calibration range. Approximate concentration.

Table II

Summary of Change in BAC for Cases Originally Positive for Ethanol

 Storage time (years)
 
Change in BAC
 
% Change in BAC
 
Tube A Tube B Tube A Tube B Tube A Tube B 
Mean 8.9 9.0 −0.051 −0.035 −34 −24 
Maximum 10.3 10.5 −0.234 −0.149 −100 −100 
Minimum 5.4 5.6 −0.023 −0.005 −8 −1 
n (Tube A) 110      
n (Tube B) 104      
 Storage time (years)
 
Change in BAC
 
% Change in BAC
 
Tube A Tube B Tube A Tube B Tube A Tube B 
Mean 8.9 9.0 −0.051 −0.035 −34 −24 
Maximum 10.3 10.5 −0.234 −0.149 −100 −100 
Minimum 5.4 5.6 −0.023 −0.005 −8 −1 
n (Tube A) 110      
n (Tube B) 104      

There was a significant difference in the loss of ethanol between 100 pairs of matched Tubes A (mean 0.047 g/dL loss) and Tubes B (mean 0.035 g/dL loss) by a paired Student's t-test (CL 99.9%, P < 0.001), demonstrating that tubes that have been previously opened had a greater loss of ethanol over long-term storage. This was consistent with observations in previous studies (3, 12) and may be due to introduction of more oxygen into the sample to be used in the oxyhemoglobin-mediated oxidation of ethanol in whole blood (5, 15). There was also a significant difference in ethanol loss in tubes that were less than half full (∼6 mL or less) compared with those that were more than half full (unpaired Student's t-test, CL 99.9%, P < 0.001). Eighteen percent (20/110) of Tubes A originally positive for ethanol had ≤6 mL of blood and had a mean ethanol loss of 0.080 g/dL, compared with a mean ethanol loss of 0.044 g/dL in Tubes A containing >6 mL of blood. The mean storage time for Tubes A with ≤6 mL of blood was 8.7 years, compared with 8.9 years for Tubes A with >6 mL of blood. Twenty-one percent (22/104) of Tubes B originally positive for ethanol had ≤6 mL of blood and had a mean ethanol loss of 0.065 g/dL, compared with a mean ethanol loss of 0.027 g/dL in Tubes B containing >6 mL of blood. The mean storage time for Tubes B with ≤6 mL of blood was 9.2 years, compared with 9.0 years for Tubes B with >6 mL of blood. Six of 110 (5%) of Tubes A and 3 of 104 (3%) of Tubes B that were originally BAC positive were negative after storage. Eight of these nine tubes were less than half full. This observation is consistent with previous research on ethanol stability on postmortem samples in which the percentage of air chamber in the storage container was found to be more significant than storage temperature on ethanol degradation (13) as well as other reports (5, 6, 14). Further study relating total sample volume (or the percentage of sample volume in the container) and ethanol loss would be beneficial as the data indicated that extremely low volumes led to extreme BAC loss as demonstrated in samples 1, 8, 15, 29, 30, 32, 46, 60, 74, 84, 94 and 109.

There was a lack of correlation between BAC loss and original BAC for all tubes as is demonstrated in Figure 1. This is consistent with previous studies by Chang et al. (3) and Shan et al. (1). There was also a lack of correlation between BAC loss and storage time as demonstrated in Figure 2. This is not consistent with a previous study by Jones (12), although storage times in the Jones study were much shorter in duration. After the extended periods of storage of the current study of 5–10 years, ethanol loss seems to diminish as the free oxygen available for the oxyhemoglobin-mediated loss of ethanol is exhausted.

Figure 1.

Lack of correlation between BAC loss and original BAC.

Figure 1.

Lack of correlation between BAC loss and original BAC.

Figure 2.

Lack of correlation between BAC loss and storage time.

Figure 2.

Lack of correlation between BAC loss and storage time.

Table III summarizes the data and Figures 3–6 are a helpful visual snapshot of the BAC loss observed under four different sample conditions, opened tubes with ≤6 mL of blood, opened tubes with >6 mL of blood, unopened tubes with ≤6 mL of blood and unopened tubes with >6 mL of blood. Comparing tubes that were over half full (Figures 3 and 5) with those that were less than half full (Figures 4 and 6) clearly identifies a more predictable, narrower range of ethanol loss for those tubes that were over half full (contained >6 mL). Figures 3–6 demonstrate that the ethanol loss was lowest in tubes that were previously unopened and were over half full (contained >6 mL). Similar to the total loss in unopened tubes reported by Chang et al. (3) of 0.02–0.04 g/dL for ∼65% of samples studied after long-term storage, the total loss observed in this study was between 0.02 and 0.04 g/dL for 62% and between 0.01 and 0.05 g/dL for 89% of the unopened tubes with >6 mL of blood that were originally positive for ethanol (n = 82).

Table III

Summary of Change in BAC (g/dL) for Cases Originally Positive for Ethanol Under Different Conditions

 n Mean Minimum Maximum 
Previously opened tubes (Tubes A) >6 mL 90 −0.044 −0.025 −0.063 
Previously opened tubes (Tubes A) ≤6 mL 20 −0.080 −0.023 −0.234 
Unopened tubes (Tubes B) >6 mL 82 −0.027 −0.005 −0.047 
Unopened tubes (Tubes B) ≤6 mL 22 −0.065 −0.013 −0.149 
 n Mean Minimum Maximum 
Previously opened tubes (Tubes A) >6 mL 90 −0.044 −0.025 −0.063 
Previously opened tubes (Tubes A) ≤6 mL 20 −0.080 −0.023 −0.234 
Unopened tubes (Tubes B) >6 mL 82 −0.027 −0.005 −0.047 
Unopened tubes (Tubes B) ≤6 mL 22 −0.065 −0.013 −0.149 
Figure 3.

BAC loss of previously opened tubes containing >6 mL (n = 90).

Figure 3.

BAC loss of previously opened tubes containing >6 mL (n = 90).

Figure 4.

BAC loss of previously opened tubes containing ≤6 mL (n = 20).

Figure 4.

BAC loss of previously opened tubes containing ≤6 mL (n = 20).

Figure 5.

BAC loss of unopened tubes containing >6 mL (n = 82).

Figure 5.

BAC loss of unopened tubes containing >6 mL (n = 82).

Figure 6.

BAC loss of unopened tubes containing ≤6 mL (n = 22).

Figure 6.

BAC loss of unopened tubes containing ≤6 mL (n = 22).

Conclusion

The study reported here on the stability of ethanol after long-term unrefrigerated storage of authentic DUI whole blood samples demonstrated that: When blood samples are reanalyzed after long-term room temperature storage, the results should be interpreted with these conclusions in mind. If the tube was unopened and more than half full, the sample should demonstrate a predictable loss in ethanol concentration. The expected ethanol loss would be in the range of 0.01 and 0.05 g/dL in most cases regardless of the original concentration.

  • alcohol negative preserved antemortem blood samples remained negative during long-term storage at room temperature regardless of blood volume or whether or not the sample tube was previously opened;

  • long-term storage decreased BAC and that decrease was greater if the tubes had been previously opened or were less than half full;

  • the loss of BAC was independent of the original concentration and

  • if the tube was unopened and more than half full, the loss of BAC was between 0.01 and 0.05 g/dL for 89% of case samples after long-term unrefrigerated storage.

Acknowledgments

The authors thank Russell Miller, Diana Lawrence, Dr Amber Kohl, Marcus Warner and Dr Cecelia Crouse for their helpful review of this manuscript, and the Florida Department of Law Enforcement Orlando Regional Operations Center and the Palm Beach County Sheriff's Office for supporting this work.

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