The alcohol biomarker phosphatidylethanol (PEth) - test performance and experiences from routine analysis and external quality assessment.

Phosphatidylethanol (PEth) are membrane molecules formed from phosphatidylcholine and ethanol through transphosphatidylation catalyzed by phospholipase D. Measurement of the main PEth form 16:0/18:1 is used as a specific and sensitive alcohol biomarker, since its formation requires ethanol, it accumulates in the blood upon repeated ethanol exposure, and it is only slowly eliminated during abstinence. PEth formation correlates with alcohol intake at the population level, albeit with considerable inter-individual variation as for the half-life during withdrawal. Over the past decade, the use of PEth has increased significantly and the applications have broadened. In Sweden, routine decision limits and the interpretation of test results for PEth were harmonized in 2013, using < 0.05 µmol/L (∼35 µg/L) as the recommended lower reporting limit and values > 0.30 µmol/L (∼210 µg/L) to indicate regular high alcohol intake. Routine test results show a large variation with about half being < 0.05 µmol/L and some even exceeding 10 µmol/L. In 2013, an external quality assessment (EQA) scheme for PEth 16:0/18:1 measurement in whole blood was also started (Equalis, Uppsala, Sweden), presently involving 56 laboratories from 13 countries. The agreement of PEth results between the laboratories has gradually improved to a CV < 15%. The current clinical and scientific information suggests that PEth values below the lower reporting limit (typically ∼0.03-0.05 µmol/L, or ∼20-35 µg/L) indicates sobriety or only low or occasional alcohol consumption, while regular high alcohol intake at levels corresponding to harmful drinking is required in most cases to reach PEth values > 0.30 µmol/L.

[The alcohol biomarker phosphatidylethanol (PEth) - recommendations for use and interpretation of test results]. / Alkoholmarkören fosfatidyletanol (PEth) ­ så bedöms testresultatet.

Phosphatidylethanol (PEth) is a group of phospholipids that are formed in blood from the corresponding phosphatidylcholines in the presence of ethanol by action of phospholipase D. Since PEth formation requires ethanol, it is used as a specific alcohol biomarker. Use of PEth measurement in whole blood as an alcohol biomarker has risen sharply in recent years, increasing the demand for knowledge about how it should be utilized and test results evaluated. In Sweden, the use since 2013 of harmonized LC-MS analytical methods targeting the main form PEth 16:0/18:1, and confirmation of comparable test results between laboratories in the Equalis (Uppsala, Sweden) external quality control program (CV <15%), has enabled use of common decision limits. A measurable PEth result confirms ethanol exposure, but due to interindividual variations in test response to a given dose and elimination half-life during abstinence, it is not possible to indicate the exact amount or time of alcohol intake. However, a PEth level above 0.30 µmol/L (~210 µg/L) is a strong indicator of harmful drinking, while a test result below 0.05 µmol/L (~35 µg/L) excludes harmful drinking but does not confirm complete abstinence. According to current test statistics from two Swedish hospital laboratories, each performing > 60 000 routine PEth measurements annually, ~45-50% of the values were < 0.05 µmol/L, ~23-24% between 0.05-0.30 µmol/L, ~16-19% between 0.30-1.0 µmol/L, and ~10-12% > 1.0 µmol/L. Some PEth results even exceeded 10 µmol/L.

Women´s experiences of preeclampsia as a condition of uncertainty: a qualitative study.

BACKGROUND: Preeclampsia is a severe condition that annually affects about 3-8% of pregnancies worldwide. Preeclampsia is thereby one of the most common pregnancy complications for both mother and child. Despite that, there is limited research exploring the women´s perspective of experiencing preeclampsia. AIM: The aim of this study was to describe women´s experiences of preeclampsia to improve the support and care given during and after pregnancy. METHODS: A qualitative descriptive interview study was undertaken. Nine women, diagnosed with preeclampsia, were recruited from a maternity unit in southern Sweden. The descriptive phenomenological method according to Amadeo Giorgi was used to analyse the data. RESULTS: The women´s experiences of PE were expressed as A condition of uncertainty, meaning that it was an unexpected and unknown situation. This main result consisted of 1) incomprehensible diagnosis message, 2) ambivalent feeling when the unexpected happens, 3) confusing contradictory messages, 4) appreciated support from the midwife, 5) need for continuous information. The nature of preeclampsia can sometimes deteriorate rapidly both for the mother and/or the child, often resulting in conversion from a planned vaginal spontaneous delivery to an emergency Caesarean section. The women narrated diffuse symptoms, and they experienced that they got contradictory information from different health care professionals regarding the severity of their disease. Detailed and continuous information is requested throughout the course of the disease, and the postpartum period. CONCLUSION: This qualitative study reveal a need for improved clinical management. Health care professionals must be aware that women and their partners need detailed, consistent and repeated information about severity and prognosis to diminish the condition of uncertainty, confusion and fearful experience. The clinical implication would be a standardized preeclampsia education for pregnant women early on in the pregnancy, to raise awareness of preeclamptic symptoms. Furthermore, there is a need for harmonized guidelines and individualized support to the woman and her partner both at the antenatal care and the maternity ward and inpatient care at the hospital.

[Increased availability needed for analysis of anabolic androgenic steroids outside of sports - proposal of a uniform clinical analysis method in Sweden]. / Ökad analys av anabola androgena steroider utanför idrotten behövs.

The abuse of anabolic androgenic steroids (AAS) outside of sports is a far greater societal problem than the abuse in sports. Therefore, there is an increasing need for a suitable clinical method for analysis of AAS in urine samples, but only three clinical laboratories in Sweden currently perform analyses of AAS outside of sports. There is a need for harmonization regarding which substances to be analyzed,  which analytes to measure and which concentration thresholds (¼cut-offs«) to us. Based on data from the three analyzing clinical laboratories, and data from the Swedish Customs Service and National Forensic Centre, a list of suggested substances, analytes and thresholds is presented. The proposed list allows detection of at least 95% of the positive samples outside of sports.

[Simplified laboratory analysis of ethylene glycol allows improved management of poisoning cases]. / Förenklad analys av etylenglykol kan ge snabbare diagnos vid förgiftningsfall.

Toxicological analysis constitutes an important part of the acute treatment of poisonings. Timely laboratory results are essential for the patient to be diagnosed and treated appropriately, but also to exclude poisoning and avoid unnecessary overtreatment. Ingestion of ethylene glycol may cause acute kidney injury and, in severe cases, death, unless treated early with an antidote (ethanol infusion or fomepizole) to inhibit the formation of toxic metabolites. Diagnosis of poisoning is based on detection of ethylene glycol in plasma or serum, but a challenge remains that acute toxicology service is only available at major hospital laboratories using gas chromatography. A simple enzymatic method for the quantification of ethylene glycol (Catachem) was evaluated as a complement to currently used methods and demonstrated to provide fast and accurate measurement in a clinically relevant concentration range (1-80 mmol/l) with a minimal risk of analytical interference. The method is suitable for use on several automated clinical chemistry analyzers. Use of the enzymatic method can improve availability of acute toxicology service for ethylene glycol and contribute to better healthcare from both a patient and health resource perspective.

Inter Individual Variation and Factors Regulating the Formation of Phosphatidylethanol.

BACKGROUND: Alcohol use disorders are a major but often unrecognized health problem. Alcohol markers can therefore be of great value for diagnosis, follow-up, and treatment evaluation. Phosphatidylethanol in blood (B-PEth) is an alcohol biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers but has shown a considerable interindividual variation in relation to reported consumption. METHODS: An in vitro system was used to investigate factors, which may affect the formation rate of PEth or which may give rise to interindividual variation in the rate of formation. In this system, isolated erythrocytes from 31 individuals were incubated in the presence of various concentrations of ethanol (EtOH). The concentration of PEth and phosphatidylcholine (PC), the parent molecule of PEth, was determined by chromatographic methods. RESULTS: Time, EtOH, and PC concentration were major factors determining the amount of PEth formed. The interindividual variation in PEth formation rate, calculated at an EtOH concentration of 50 mmol/l, showed a coefficient of variation (CV) from 23 to 31% for the different PEth forms studied (PEth 16:0/18:2, total PEth and PEth 16:0/18:1). The concentration of PC was found to be an important determinant of this variation. The formation rate for PEth 16:0/18:2 was somewhat higher than for PEth 16:0/18:1. The formation of PEth 16:0/18:1 but not PEth 16:0/18:2 showed a positive correlation to the concentration of PEth at baseline (endogenous PEth). Calculation of enzyme kinetics for the reaction resulting in the formation of PEth 16:0/18:1 or PEth 16:0/18:2 showed an apparent Km (Michaelis constant) of approximately 160 to 170 mmol/l. CONCLUSIONS: Interindividual variation in the formation rate of PEth appears to be a significant but relatively modest source of variation in the relation between B-PEth and reported consumption. Correction for interindividual variation in PC concentrations might substantially reduce the interindividual variability in PEth formation and consequently in B-PEth.

High-Throughput LC-MS/MS Method for Determination of the Alcohol Use Biomarker Phosphatidylethanol in Clinical Samples by Use of a Simple Automated Extraction Procedure-Preanalytical and Analytical Conditions.

BACKGROUND: Phosphatidylethanol (PEth) is an alcohol use biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers. Since its introduction as a clinical alcohol-marker in 2006, the number of samples sent to our laboratory for the determination of PEth has shown a strong annual increase. This has prompted the need to develop a cost-effective and reliable analytical procedure with high capacity. METHODS: An LC-MS/MS method for the determination of PEth 16:0/18:1 with a short turnaround time (3 min) has been evaluated with respect to accuracy, sensitivity, and precision. We compared this method with a previously used HPLC method, as well as a manual and a simplified automated method for sample workup, and investigated potential causes of analytic and preanalytic errors. RESULTS: The method shows limits of detection and quantification of 0.0075 µmol/L (5.2 ng/mL) and <0.05 µmol/L (<35 ng/mL), respectively. During a 2.1-year period, the method has shown a total CV < 8% for control samples (n = 2808) in the range of 0.10 (70) to 3.5 µmol/L (2461 ng/mL). The simplified automated method for sample preparation works equally well as the manual one. No specific and clinically significant causes of preanalytic errors were found. CONCLUSIONS: This LC-MS/MS method with automated sample workup is well suited for a clinical laboratory with LC-MS/MS experience and has the capability, proven from several years of use, to produce reliable PEth results in a high-volume laboratory (>50000 clinical samples/year).

[Access to rapid laboratory analytical services in cases of acute poisoning provides better and safer patient care]. / Tillgång till snabb laboratorieanalys vid akut förgiftning ger bättre och säkrare vård - Men möjligheten till akuta prov­­svar varierar över landet.

Access to rapid laboratory analytical services in cases of acute poisoning provides better and safer patient care The Swedish Poisons Information Centre, a nationwide 24/7 service to healthcare providers and the public, answers many questions about serious cases of acute poisoning. In some of these, prompt and proper treatment recommendations can be crucial for the clinical outcome. In cases where self-reported information is missing or considered unreliable, more emphasis is placed on the clinical symptoms and results of toxicological analyses. However, rapid access to toxicological analysis for the most common set of poisoning agents varies between hospitals and laboratories. A priority list of toxic agents for which improved analytical techniques could offer a more widespread availability and rapid access to clinically important test results is presented.

Detectability of designer benzodiazepines in CEDIA, EMIT II Plus, HEIA, and KIMS II immunochemical screening assays.

The emerging new psychoactive substances made available for recreational drug use have recently started to include designer benzodiazepines. As a consequence, the routine immunoassay drug testing for benzodiazepines may become less effective, due to an increased occurrence of 'false negative' and 'false positive' results. This work aimed to extend the knowledge of analytical cross-reactivity of 13 designer benzodiazepines in the CEDIA, EMIT II Plus, HEIA, and KIMS II immunoassays. Urine standards were prepared by spiking blank urine with clonazolam, deschloroetizolam, diclazepam, estazolam, etizolam, flubromazepam, flubromazolam, flutazolam, 3-hydroxyphenazepam, meclonazepam, nifoxipam, phenazepam, and pyrazolam. Authentic urine samples from intoxication cases identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were also investigated. For the spiked standard samples, the 13 designer benzodiazepines generally showed a high cross-reactivity in all assays. This was further confirmed when investigating their detectability in authentic urine samples from cases of drug intake. The test responses also indicated additional reactivity from metabolites. The lowest detectability in spiked samples was observed for flutazolam, which shows the most divergent chemical structure compared with the other benzodiazepines. Overall, the KIMS II and CEDIA immunoassays, which both include enzymatic hydrolysis of conjugated forms, showed the highest, and EMIT II Plus the lowest degree of reactivity, for spiked parent substances and authentic urine specimens. The results of this study demonstrated that designer benzodiazepines can be detected in standard urine immunoassay drug screening and this should be taken into consideration when performing confirmation analysis. Copyright © 2016 John Wiley & Sons, Ltd.

Phosphatidylethanol is superior to carbohydrate-deficient transferrin and γ-glutamyltransferase as an alcohol marker and is a reliable estimate of alcohol consumption level.

BACKGROUND: In clinical practice as well as research situations, it is of great importance to get reliable information about a patient's alcohol consumption. The aim of the study was to investigate the correlation of alcohol biomarkers (phosphatidylethanol [PEth], carbohydrate-deficient transferrin [CDT], γ-glutamyltransferase, aspartate aminotransferase, and alanine aminotransferase) to retrospective as well as diary-based alcohol self-reports and to examine whether it is possible to correlate a biomarker result to a more precise level of alcohol consumption. METHODS: One hundred and sixty alcohol-dependent patients were included in a randomized, placebo-controlled clinical trial of pharmacotherapy for alcohol dependence, of which 115 (76 men and 39 women) completed the study. Retrospective alcohol consumption data were collected at baseline, and alcohol diaries were used during the study. Blood samples for determination of alcohol biomarkers were collected on 5 occasions during the study. RESULTS: PEth and CDT showed a better correlation with alcohol consumption documented in the diary (PEth rs = 0.56 and CDT rs = 0.35) than with retrospective consumption data (PEth rs = 0.23 and CDT rs = 0.22). An even higher correlation (rs = 0.63) was seen between the 2 alcohol biomarkers PEth and CDT. At all consumption levels, PEth had the highest sensitivity of all biomarkers studied. CONCLUSIONS: PEth was the biomarker with the best correlation to self-reported alcohol consumption. PEth was superior to CDT owing to its substantially higher sensitivity but also due to its closer correlation to self-report. PEth values can be translated into an approximate level of alcohol consumption and PEth appears to be a more reliable measure of alcohol consumption than self-reports.

[Uniform analyzes of drugs in urine needed for rule of law]. / Enhetliga analyser av narkotika i urin krävs för rättssäkerheten.

Drugs of abuse testing is used in various areas of society for detection and follow-up of drug use. In routine laboratory drug testing, immunoassays are employed for initial screening of specimens to indicate the presence of drugs. To confirm a positive screening test, a secondary analysis by mass spectrometry is performed. The "cut-off" is the pre-defined concentration threshold of a drug or drug metabolite above which the sample is considered positive. A reading below this level implies a negative test result. Swedish drug testing laboratories currently employ varying cut-offs to distinguish between a positive and a negative test result. Because a positive drug test may have serious legal consequences to the individual, it is of importance that testing is performed and judged equally, regardless of where it is performed. A national harmonization of cut-offs is therefore warranted. Based on data from four major Swedish drug testing laboratories, and considering the recommendations in international guidelines, a proposal for national harmonization of urine cut-offs for the most common set of drugs of abuse is presented.

Phosphatidylethanol Compared with Other Blood Tests as a Biomarker of Moderate Alcohol Consumption in Healthy Volunteers: A Prospective Randomized Study.

AIM: It is generally agreed that traditional alcohol biomarkers lack in sensitivity to detect hazardous alcohol consumption. The present study was undertaken to evaluate the ability of phosphatidylethanol (PEth) and traditional alcohol markers to detect moderate alcohol consumption and to distinguish between moderate alcohol consumption and abstinence. METHODS: Forty-four subjects, 32 females and 12 males, were included in the study. They were randomized to alcohol abstention or to alcohol consumption. Female participants consumed 150 ml of red wine (equivalent to 16 g of alcohol) per 24 h and the male participants double the amount. The study lasted for 3 months. Blood samples were drawn at the start and at the end of the study period. Blood samples were analysed for PEth, carbohydrate-deficient transferrin (CDT), mean corpuscular volume (MCV), γ-glutamyltransferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). RESULTS: ROC curves for the various biochemical markers were plotted in order to assess their ability to discriminate between abstention and moderate daily consumption of alcohol. PEth and CDT were the only markers with AUROCs significantly higher than 0.5, and PEth was detected in all participants randomized to alcohol consumption. CONCLUSION: PEth was the only marker that could detect moderate intake and the present results also indicate that PEth probably can distinguish moderate alcohol consumption from abstinence.

Detectability of new psychoactive substances, 'legal highs', in CEDIA, EMIT, and KIMS immunochemical screening assays for drugs of abuse.

The increasing number of new psychoactive substances made available for recreational drug use has created a challenge for clinical toxicology and drug testing laboratories. As a consequence, the routine immunoassay drug testing may become less effective due to an increased occurrence of false negative and false positive screening results. This work aimed to extend the knowledge about analytical cross-reactivity of new substances in selected CEDIA, EMIT, and KIMS immunoassays for drugs-of-abuse screening. Urine standards were prepared by spiking blank urine with 45 new substances. Authentic urine samples from intoxication cases identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were also studied. Several new psychoactive substances were demonstrated to display cross-reactivity in the immunoassays. CEDIA Amphetamine/Ecstasy and EMIT d.a.u. Amphetamine Class tests showed the highest reactivity towards the new drugs, which was expected since many have amphetamine-like structure and activity. In the samples from authentic cases, five new substances displayed 100% detection rate in the CEDIA Amphetamine/Ecstasy test. In conclusion, cross-reactivity data in routine urine drug screening immunoassays for a number of new psychoactive substances not studied before were reported. In both spiked and authentic urine samples, some new substances showed significant cross-reactivity and are thus detectable in the routine screening methods.

Detection of phosphatidylethanol (PEth) in the blood of drivers in an alcohol ignition interlock program.

OBJECTIVE: The rate of failed interlock blood alcohol content (BAC) tests is a strong predictor of recidivism post-interlock and a partial proxy for alcohol use. Alcohol biomarkers measured at the start of an interlock program are known to correlate well with rates of failed BAC tests over months of interlock use. This study evaluates 2 methods of measuring low blood levels of the biomarker phosphatidylethanol (PEth). PEth is a 100 percent alcohol-specific biomarker and strongly intercorrelated with several independent indicators of drinking driving risk, including 8 other biomarkers, 3 psychometric assessments, and the rate of failed interlock BAC tests during many months of interlock use. Does a more sensitive method of measuring PEth at program entry detect drinking even among those who subsequently log no failed interlock tests? METHODS: In a sample of 281 driver blood samples, PEth was measured by both high-performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LCMSMS) in order to compare sensitivity and accuracy. The average rate of failed interlock BAC tests was the criterion measure for marker sensitivity. LCMSMS, calibrated to detect low levels of drinking as a possible measure of abstinence violation, was judged relative to the standard HPLC assay for PEth measured up to 4 µmol/L. RESULTS: The 2 methods showed a good quantitative relationship (r(2)> .86). LCMSMS detected positive PEth levels in samples that were below the limit of detection of the HPLC method. PEth measured by LCMSMS was positive for a higher proportion of driving under the influence (DUI) offenders who logged zero failed interlock BAC tests than were detected by HPLC. CONCLUSION: Although HPLC is the widely used standard for measuring PEth in clinical alcoholism samples, the LCMSMS method, when calibrated to detect trace amounts of the major component of PEth, can detect abstinence levels of alcohol near zero intake and still correlate strongly with other indicators related to alcohol use and road safety.

Phosphatidylethanol in blood (B-PEth): a marker for alcohol use and abuse.

Phosphatidylethanol (PEth) represents a group of glycerophospholipid homologues where ethanol by phospholipase D has been bound at the position that normally contains an amino-alcohol. Since the formation of PEth is specifically dependent on ethanol, the diagnostic specificity of PEth as an alcohol biomarker is theoretically 100%. The half-life of PEth in blood is approximately 4 days. The amount of alcohol consumed correlates to blood concentration of PEth and PEth has been shown to be a more sensitive indicator of alcohol consumption than traditional alcohol markers, such as CDT (carbohydrate-deficient transferrin), GGT (γ-glutamyl transferase), and MCV (mean corpuscular volume) or a combination of these. Almost all clinical data so far available are based on a high performance liquid chromatography (HPLC) method with limited analytical sensitivity. With the advent of methods with considerably higher analytical sensitivity (e.g. mass spectrometric methods), clinical sensitivity will increase correspondingly. The possibility of determining very low concentrations of PEth by new sensitive analytical techniques may, however, have both ethical and legal consequences that have to be considered.