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��@@@�@@@,,,,,,����Impact of blood collection tubes on erroneous 1-propanol detection and on forensic ethanol analysis




Vassiliki A. Boumba*, Theodore Vougiouklakis







*Corresponding author: 
Laboratory of Forensic Medicine & Toxicology,
 Faculty of Medicine, 
School of Health Sciences, 
University of Ioannina, 
45110 Ioannina, 
Greece    
vboumba@cc.uoi.gr



Abstract
Introduction: Forensic alcohol analysis in blood from living subjects is performed in various situations, such as driving under the influence, is usually related to consumption of alcoholic beverages and often results to the detection of ethanol and/or congener alcohols. Alternatively, ethanol and congeners, such as 1-propanol, can be microbially produced especially in autopsy samples. 
Aim: The present study aims to emphasize the importance of selecting the proper blood collection tubes for the right intended use in order to avoid erroneous results, especially in reference to forensic alcohol analysis.
Methods:  Sixty eight blood samples from living subjects, all drivers involved in traffic accidents, contained in different collection tubes, were included in the study. Volatiles were detected with a validated HS-GC-FID method. Samples of normal human blood, spiked with ethanol and/or 1-propanol, dispersed in different tubes were treated and analyzed as the authentic blood samples in order to test the suitability of the collection tubes.
Results: Authentic blood samples, contained in certain tubes with separator gel, had high concentrations of �1-propanol�. It was shown that the separator gel released a substance that was recognized as �1-propanol� during analysis by HS-GC-FID at 600C (routine conditions). The erroneous �1-propanol� was separated from authentic 1-propanol during analysis at 580C.
Discussion: The separator gel of certain blood collection tubes compromises a source of pre-analytical error that results to erroneous concentrations of 1-propanol. This is unfavorable in forensic cases and can complicate interpretation of the origin (exogenous or microbial) of the detected ethanol. 


Keywords: forensic; ethanol; 1-propanol; blood; interpretation; pre-analytical error.
Introduction
Ethanol analysis is the most frequent test performed in forensic toxicology laboratories. Ethanol testing is performed in a variety of situations, including postmortem cases, driving under the influence (DUI), drug facilitated sexual assault (DFSA) cases, workplace testing and probation monitoring and in various specimens, both biological fluids (e.g. blood) and tissues [1, 2]. Typically, ethanol analysis is performed in whole blood. However, in forensic cases, it is common to determine ethanol concentration in matrices other than blood and, then, to compare the determined value to the blood alcohol concentration (BAC) [3]. Currently, the most applied analytical technique for measuring ethanol with high analytical standards, in terms of accuracy, specificity, precision and selectivity, is head-space gas chromatography with flame ionization detection (HS-GC-FID) which allows the simultaneous determination of many other volatile compounds [2, 4].
 	Detection of ethanol in blood from living subjects is usually related with consumption of alcoholic beverages.  Amounts of 1-propanol are often determined among the volatiles during congener analysis, since it can originate from sources such as food, beverages, disinfectants or solvents [5-9].  On the other hand, 1-propanol is the most common other volatile - beyond ethanol - that can be detected, in considerable concentrations, during ethanol analysis in blood samples where microbial ethanol production has occurred [4, 10-13]. In the latter cases, its presence can complicate seriously the interpretation of ethanol analysis results [10, 11]. Needless to say that detection of high amounts of 1-propanol during forensic ethanol analysis of living subjects is a challenging issue that can question the integrity of the sample and debate the results. 
Finally, the possible interference of the blood collection tubes on clinical chemistry assays, and its impact on the pre-analytical phase it is recorded in many relevant reports [14-25]. In the forensic field, it is well recognized the importance of adding anticoagulant and preservative, in blood samples submitted for ethanol analysis in order to avoid clotting and microbial activity, respectively [3]. However, to the best of our knowledge, there is no evidence, so far, for the impact of separator gel of certain blood tubes on the blood ethanol, or other alcohol, analysis. 
The present study aims to indicate the separator gel in blood collection tubes as a possible source of pre-analytical error during the forensic alcohol analysis, which can lead to erroneous 1-propanol detection complicating this way the interpretation of results.

Materials and Methods
Subjects / Samples - Blood tubes
Sixty eight blood samples received from living subjects, all being drivers involved in traffic accidents during the period 2008-2010, were included in this study. Blood samples were routinely submitted for ethanol analyses at the Laboratory of Forensic Medicine & Toxicology, University of Ioannina. Fifty blood samples were contained in 5-mL tubes with KF as preservative and Na2EDTA as anticoagulant, orange-caped (LP Italiana SPA, Milano, Italy); eleven blood samples were contained in 5-mL Vacutainer tubes with K2EDTA or K3EDTA as anticoagulant, lavender-caped, and; seven blood samples were contained in 5-mL BD Vacutainer SST II Advance tubes, with acrylic based separator gel, gold-caped (Becton Dickinson). After admission to the laboratory, samples from SST II tubes were centrifuged at 2500 rpm for 10 min; the supernatant serum was pipetted into polystyrene tubes, capped, and processed for alcohol analysis. Samples from other tubes were analyzed as whole blood (they were inverted gently 8�10 times before being processed). 

Alcohols determination
All chemicals were purchased in the highest possible purity and used without any further purification. Ammonium sulfate, ethanol (99.7%), 1-propanol, were purchased from Merck in the higher available purity (Darmstadt, Germany). All aqueous solutions were prepared using double distilled (DD) water, which was obtained using an Aquatron A400D (Bibby Sterilin, Staffordshine, UK). Stock aqueous standard solutions were prepared in concentration 4.00% (w/v) for ethanol and 0.40% (w/v) for 1-propanol. Acetonitrile was used as internal standard for the determination of volatiles in aqueous solution of 100 mg/dL. GC analyses were performed on a Shimadzu GC 17A gas chromatograph equipped with a SUPELCOWAX -10 fused silica capillary column (30 m x 0.25 mm, film thickness 0.25 mm) and with a FID. The GC was fitted with a Shimadzu AOC-5000 headspace-GC automated sample pretreatment and injection system. Ethanol and 1-propanol were detected with a HS-GC-FID method as previously described [4]. Limits of detection (LOD) and limits of quantification (LOQ) were 0.01 and 0.03 mg/dL for ethanol, and, 0.02 and 0.04 mg/dL for 1-propanol, respectively. 

Influence of blood tubes on �1-propanol� detection 
1st Experiment:  Aliquots of 3.0 mL of normal human blood from a pool blood preparation, free from ethanol or 1-propanol, were put in orange-, lavender- or SST II tubes (two tubes of each type), and they were spiked with ethanol and/ or 1-propanol at concentrations 0.5 g/L and 0.5 mg/dL, respectively. Samples after having remained for 30 min at room temperature were processed properly and analyzed by HS-GC-FID at 600C and at 580C, respectively.
2nd Experiment: Three series of 16 blood tubes each, orange-caped, lavender-caped - or SST II were prepared. An aliquot of 3.0 mL of normal human blood from a pool blood preparation, free from ethanol or 1-propanol, was dispensed into each tube. Blood samples were spiked with ethanol to a final concentration of 0.5 g/L. Then tubes were capped and stored at 40C for 4, 24, 48, 72, 96, 120 and 148 hours, respectively. Two blood tubes for each storing period and each type of tube were analyzed by HS-GC-FID. As previously described samples from orange and lavender-cap tubes were analyzed as whole blood, while samples from SST II tubes were analyzed as serum after centrifugation at 2500 rpm for 10 min. Results
Sixty eight blood samples from living subjects, routinely submitted for ethanol determination, were included in this study. They were contained either in blood tubes with anticoagulant and preservative (50 samples), or, in tubes with anticoagulant (11 samples), or, in tubes with separator gel (7 samples). Analyses were performed 4 h to 3 days after blood collection (due to differences in admission time by the authorities because of holidays, personnel availability etc) with a validated HS-GC-FID method [4].
The results for the measured concentrations of ethanol and 1-propanol are presented in Table 1. Ethanol was determined in all the samples, irrespectively of the tube type. Blood samples contained in tubes with preservative and anticoagulant had no detectable 1-propanol. Negligible amounts of 1-propanol were detected in blood samples contained in tubes with anticoagulant alone. Interestingly, �1-propanol� was detected in high concentrations exclusively in samples contained in SST II tubes and the differences in concentration were statistically significant (p<0.05). The high 1-propanol concentrations determined in samples from SST II tubes with separator gel looked suspicious. Therefore, our effort was focused to find out whether this result was �a false-positive� or a true-positive� one. 
In order to test whether the gel was the source of the �1-propanol� like compound the experimental design described as 1st experiment in the materials and methods section was performed. The blood samples from SST II tubes that were spiked only with ethanol when subjected to routine GC analysis at 600C (as serum), resulted to a chromatogram where a peak recognized as �1-propanol� was detected along with the peaks corresponding to ethanol and the internal standard (acetonitrile) (Figure 1A). The GC analysis at 600C of the blood samples from SST II tubes that were spiked with ethanol and 1-propanol resulted to a chromatogram where a second compound was co-eluted (as left shoulder) with the original 1-propanol peak at the 1-propanol�s retention time (Figure 1B). This �1-propanol�-like compound was well separated from the original 1-propanol when the GC analysis of the spiked samples (with ethanol and 1-propanol) from SST II tubes was performed at 580C (Figure 1C). This �1-propanol� peak was absent when the respective blood samples that were spiked only with ethanol from the tubes with orange- and lavender-cap were analyzed under the same conditions (not shown). Similarly, the blood samples, spiked with ethanol and 1-propanol, from the other tested types of tubes resulted in chromatograms where the �1-propanol� peak did not appear (as shoulder-peak). These results showed that the �1-propanol� like compound had eluted directly from tube gel-material during blood processing and, confirmed that gel was the source of this compound.
Furthermore, we tested whether the duration of storing blood samples contained in SST II tubes at 40C could affect the quantity of the eluted impurity. The gel-derived impurity was quantified as �1-propanol� equivalent concentration during HS-GC-FID analysis at routine conditions (600C) of the blood samples prepared as described in the materials and methods section (2nd experiment). It was found that the levels of the gel-derived impurity increased gradually for three days and then remained stable (Figure 2). In the two series of spiked (with ethanol and 1-propanol) blood samples from orange-cap or, lavender-cap tubes, only the original 1-propanol�s peak was detected (no shoulder-type peak appeared) without any significant changes in the concentrations of ethanol and 1-propanol over the storage period (not shown).

Discussion
In this report we show that the separator gel of certain blood tubes releases a substance that co-elutes with 1-propanol and it is recognized as �1-propanol� during the forensic ethanol analysis by HS-GC-FID at routine conditions. The use of this type of blood tubes for collecting and storing blood samples compromises a source of pre-analytical error that results to erroneous �1-propanol� detection. 1-Propanol can be detected in low quantities in blood samples from living subjects either after the consumption of alcoholic beverages or as of a metabolic product or following dermal or pulmonary absorption of solvents [6-9]. However, considerable blood concentrations of 1-propanol, along with ethanol, have been correlated with microbial ethanol production, in samples either from autopsy cases with putrefaction [10, 11, 13], or from experimental studies [4, 12]. Generally, 1-propanol is considered the volatile most correlated to microbial activity and if present in considerable concentrations in a blood sample may set suspicious and questionable the determined concentration of ethanol [10, 11].
The original blood samples included in this study were received from drivers involved and survived from traffic accidents by the personnel of local hospitals under the auspice of police officers. Early in 2008, our laboratory started to perform forensic ethanol analysis for those DUI in the Region of Epirus. By this time local hospitals were not aware about the correct sampling tubes for BAC, and therefore the personnel used tubes available for other laboratory analyses.  Later on, we conducted the hospital personnel and we informed them about the selection of the proper tubes for collecting blood samples for forensic purposes and in particular for ethanol analysis. Gradually from late 2008 and afterwards till present, blood samples submitted for forensic ethanol analysis are sampled exclusively in tubes containing anticoagulant and preservative, as it is suggested [3].  
Blood collection and processing are two major steps in pre-analytical laboratory testing while proper tube selection and handling are needed to ensure test reliability. Several reports from the clinical laboratory testing field showed that separator gels can affect analytic concentrations, mainly through absorption of hydrophobic drugs to the gel�s hydrophobic matrix [19-23]. Furthermore, separator gels may contain materials that have been shown to interfere with analytical assays [17, 18, 24, 25], in different ways, such as, by affecting the extraction of certain drugs [17], or, by increasing the concentrations of certain analytes [18]. The present experimental study contributes to the evidence stating that most errors in the total laboratory testing process fall outside the analytical phase, and the pre-analytical steps may have the most vulnerable impact on results [14].
In the forensic testing, there is currently a real paucity of data on the possible pre-analytical errors. In addition to the medical consequences, the legal ramification of these errors creates a sense of urgency for the medical /scientific community to highlight key opportunities for improvement. Selection of the right blood collection tubes for the right intended use is critical for valid results in combination with accurate measurement of alcohol concentration in human blood samples. In parallel, the unequivocal etiology of the ethanol's origin must be absolutely documented and stated whether it has resulted from alcohol consumption or possible microbial production, or both. Probably, an erroneous �1-propanol� concentration in a human sample is an unfavorable finding as it can complicate the interpretation of ethanol analysis results, making possible the attribution of ethanol�s origin to microbial activity.  In the forensic casework such finding can put the chain-of-custody of the analyzed specimens (their source and integrity) under question in possible later court proceedings, with possible serious adverse consequences, in administrating civil or legal responsibility. 
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