[Historic Development of Clinical Biology Laboratories in Luxembourg]. / Evolution historique des laboratoires de biologie médicale au Luxembourg.

After a short overview on the development of diagnostic tools in clinical biology at an international level from Antiquity towards today, a history of the clinical biology including public and private institutions in Luxembourg will be outlined.

Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations in hair of alcoholics, social drinkers and teetotallers.

In previous investigations hair analysis for ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE) proved to be suitable for the detection of excessive alcohol consumption. The aim of this study was to compare EtG and FAEE concentrations in hair of alcoholics, social drinkers and teetotallers. Hair samples from 10 alcoholics in withdrawal treatment, 11 fatalities with documented excessive alcohol consumption, four moderate social drinkers who consumed up to 20 g ethanol per day, and three strict teetotallers were analysed. After external degreasing with n-heptane, extraction with a dimethyl sulfoxide/n-heptane mixture and headspace solid-phase microextraction of the extracts, four fatty acid ethyl esters (FAEEs) (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) were analysed by gas chromatography-mass spectrometry (GC-MS) with deuterated internal standards. EtG was determined by GC-MS/NCI after ultrasonication of the samples with H2O, cleanup by SPE with aminopropyl columns and PFP derivatisation. The following concentrations were measured for the four groups: teetotallers EtG < 0.002 ng/mg, FAEE 0.05-0.37 ng/mg, moderate social drinkers EtG < 0.002 ng/mg, FAEE 0.26-0.50 ng/mg, alcoholic patients EtG 0.030-0.415 ng/mg, FAEE 0.65-20.50 ng/mg and the fatalities with alcohol history EtG 0.072-3.380 ng/mg, FAEE 1.30-30.60 ng/mg. The results confirm that by using a cut-off value of the sum of FAEE > 1 ng/mg and/or a positive EtG result in hair, excessive alcohol consumption can be identified using hair analysis. However, no significant correlation between the EtG and FAEE concentrations in the positive cases could be shown. Segmental analysis of some of the specimens did not reveal the same distribution for EtG compared to FAEE in hair, and no chronological accordance compared to the self-reported alcohol consumption could be observed for both parameters. These different results of both methods are discussed in terms of differences between EtG and FAEE in mechanism of formation and incorporation into hair and elimination from hair.

Suicide with remifentanil and midazolam: a case report.

BACKGROUND: Remifentanil (Ultiva) is a recently introduced esterase-metabolized potent opioid (EMO) mu-receptor agonist with a rapid onset and offset of action for use as part of general anaesthesia in association with isoflurane or propofol or with midazolam (Dormicum) and in combination with mivacurium. CASE REPORT: A nurse from the anaesthesiology department of a hospital was found dead at home with a syringe and empty vials of Ultiva (2 mg) and of Dormicum (1 mg/mL). At autopsy several injection sites were found on her body. Usual toxicology screening operated in our laboratory revealed only the presence of benzodiazepines in blood. Regarding the case history, we decided to adapt a method specially designed for remifentanil and its inactive metabolite testing. The analytes were isolated by SPE on a Clean-Screen column. After silylation with BSTFA with 1% TMCS, the analytes were detected by GC/MS-EI operating in the SIM mode. No Remifentanil could be detected, whereas the metabolite of remifentanil GR90291 was determined in femoral blood, liver, kidney and lung. Furthermore, midazolam could be detected in femoral and cardiac blood. Hair analysis of a segment of 5 cm revealed the presence of diazepam and midazolam, whereas neither remifentanil nor its metabolite could be detected.

Suicide with remifentanil and midazolam : a case report.

Background : Remifentanil (Ultiva®) is a recently introduced esterase-metabolized potent opioid (EMO) µ-receptor agonist with a rapid onset and offset of action for use as part of general anaesthesia in association with isoflurane or propofol or with midazolam (Dormicum®) and in combination with mivacurium. Case Report : A nurse from the anaesthesiology department of a hospital was found dead at home with a syringe and empty vials of Ultiva® (2 mg) and of Dormicum ® (1 mg/mL). At autopsy several injection sites were found on her body. Usual toxicology screening operated in our laboratory revealed only the presence of benzodiazepines in blood. Regarding the case history, we decided to adapt a method specially designed for remifentanil and its inactive metabolite testing. The analytes were isolated by SPE on a Clean-Screen column. After silylation with BSTFA with 1% TMCS, the analytes were detected by GC/MS-EI operating in the SIM mode. No Remifentanil could be detected, whereas the metabolite of remifentanil GR90291 was determined in femoral blood, liver, kidney and lung. Furthermore, midazolam could be detected in femoral and cardiac blood. Hair analysis of a segment of 5 cm revealed the presence of diazepam and midazolam, whereas neither remifentanil nor its metabolite could be detected.

Compliance with cotrimoxazole prophylaxis for the prevention of opportunistic infections in HIV-positive tuberculosis patients in Thyolo district, Malawi.

OBJECTIVE: To verify compliance with cotrimoxazole prophylaxis in human immunodeficiency virus (HIV) infected tuberculosis (TB) patients during the continuation phase of anti-tuberculosis treatment, and to assess the sensitivity, specificity and positive predictive values of verbal verification and pill counts as methods of checking compliance. DESIGN: Cross-sectional study. METHODS: Cotrimoxazole compliance was assessed in a cohort of TB patients who were attending four TB follow-up centres during the continuation phase of anti-TB treatment between months 4 and 6. Verbal verification of drug intake, physical verification of pill count balance, and urine trimethoprim detection by gas chromatography and mass spectrometry were used for assessing compliance. RESULTS: Using urine trimethoprim detection as the gold standard for compliance, trimethoprim was detected in 82 (94%) of 87 patients in the cohort. Verbal verification of cotrimoxazole intake and objective pill count balances showed high sensitivity and positive predictive values compared with the gold standard of urine trimethoprim detection. CONCLUSIONS: In a rural district in Malawi, compliance with cotrimoxazole as an adjunct to anti-tuberculosis treatment in HIV-infected TB patients was good, and can be assessed simply and practically by verbal verification and pill counts.

The antispasmodic drug mebeverine leads to positive amphetamine results by fluorescence polarization immunoassay (FPIA)--studies on the toxicological analysis of urine by FPIA and GC-MS.

Mebeverine (Duspatal, MB), an antispasmodic drug, is the veratric acid ester of 4-[ethyl-[2-(4-methoxyphenyl)-1-methylethyl]amino]butan-1-ol (MB-OH), which is a N-substituted ethylamphetamine derivative. MB is metabolized via ester hydrolysis to MB alcohol (MB-OH) and veratric acid. N-Dehydroxybutylation leads to methoxyethylamphetamine (MO-EA) and, after O-demethylation, to hydroxy EA (HO-EA). N-Bisdealkylation leads to p-methoxyamphetamine (PMA). MO-EA and PMA are also known as designer drugs. Fluorescence polarization immunoassay (FPIA) and gas chromatographic-mass spectrometric studies on the toxicological analysis of MB after ingestion of a single 405-mg oral dose of MB were performed. We could show that intake of MB leads to positive FPIA results for amphetamine. The N-dehydroxybutyl metabolites of MB, MO-EA, HO-EA, and the bis-dealkyl metabolite PMA should be responsible for the positive immunoassay results. Using our systematic toxicological analysis procedure, every positive amphetamine immunoassay could be explained by detection of MO-EA, HO-EA, and/or PMA. Misinterpretation of the origin of MO-EA, HO-EA, or PMA can be avoided by detecting the specific (non-dehydroxybutylated) metabolites of MB, which are excreted for a much longer time after ingestion.

Threshold values in toxicology - useful or not?

In many fields of toxicology, numbers are used as threshold values, e.g. as "acceptable daily intake values" resulting in maximum permissible concentrations in food or in animal feed by using "safety factors"; maximal admissible concentrations of toxic substances in the air at the workplace; cut-off values in analytical toxicology; limit values for biological specimens in the case of driving under the influence of drugs, guidance values for environmental specimens, etc. The philosophy behind these values must be well understood and they should only be applied to real cases by persons with enough toxicological background. The bad use of these numbers in toxicology can have dramatic consequences. Especially in regulatory toxicology their use should be made with great care. Moreover, tremendous improvements in analytical methodology, e.g. the decreasing of the limits of detection for many potentially toxic substances in recent years, should not end up in an overestimation of risks to humans. To avoid these abuses careful interpretations of analytical findings by qualified toxicologists are of paramount importance. The use and abuse of some of these threshold values will be outlined in several applications from analytical toxicology, risk assessment issues, forensic toxicology in post-mortem cases, as well as from the drugs and driving cases. Generally, if threshold values are considered as guidance values and not as the "absolute truth" in toxicology, they may be very useful in the interpretation of toxicology data.

Frontline immunochromatographic device for on-site urine testing of amphetamines: laboratory validation using authentic specimens.

We evaluated a new test device for amphetamines and methamphetamines (Frontline, cut-off limit 300 ng/mL) using authentic clinical and forensic specimens. The device is based on immunochromatography and is dipped into urine and read visually by comparison with a colour scale after a few minutes. A total of 658 specimens were tested by comparing results of the screening procedure with established immunoassays. Discordant results were further investigated by gas chromatography mass spectrometry or gas chromatography (with flame ionization detector). The Frontline device had a sensitivity of 93% and a specificity of 98%. When specimens were classified by urine amphetamine concentration, close agreement was obtained at concentrations below 150 ng/mL and above 1000 ng/mL. A small number of specimens with amphetamine concentrations between 300 and 1000 ng/mL tested negative in the Frontline test. This finding could to some extent be explained by the enantioselectivity of the antibodies in the Frontline test to d-amphetamine. We conclude that the performance of the Frontline test device for amphetamines is adequate for presumptive clinical and forensic screening.

Structural elucidation of an uncommon phenylethylamine analogue in urine responsible for discordant amphetamine immunoassay results.

The present paper describes investigations following the analysis of a urine specimen containing important amounts of an unknown substance detected by gas chromatography-mass spectrometry (GC-MS) analysis. FPIA analysis was positive (cutoff 0.3 mg/L) and Triage 8 rapid test was negative (cutoff 1 mg/L) for amphetamines. Considering the GC-MS spectrum, two different molecules, for example, N-ethyl-1-(3,4-methylenedioxyphenyl)ethylamine (1) or N-ethyl-4-methoxyamphetamine (2), have been suspected. Synthesis of these two compounds was carried out together with spectral (MS, 1H and 13C NMR, IR, UV) and chromatographic (GC) characterization as well as determination of immunological cross reactivities (FPIA and Triage 8). The unknown compound present in the urine specimen has been finally identified as N-ethyl-4-methoxyamphetamine (2), an uncommon amphetamine analogue.

Potential problems with the interpretation of hair analysis results.

Due to differences in hair growth rate depending on anatomical region, age, gender, ethnicity and interindividual variability, interpretation of parent drug or/and metabolite concentrations in hair is not easy. Furthermore, as drug incorporation mechanisms into hair matrix is not yet fully understood, it is rather difficult to extrapolate details on time and dose from hair segment analysis. If incorporation sources other than from bloodstream (skin secretions and/or external/environmental contamination) are considered, interpretation becomes even more complicated. For evaluating possible passive contamination, it is essential to consider specific identification of metabolites, use of metabolite-to-parent drug ratios, assays of decontamination washes and analysis of specimens collected from other body parts. Cosmetic hair treatment, natural and artificial hair colour, differences in hair structure and specificity of analytical methodology may represent other bias sources affecting concentrations of drugs in hair. A suitable cut-off level related to the LOD will allow correct identification of drugs or metabolites in hair. Regarding the performance of different hair testing laboratories, little information is available at this time to what extent test results are comparable and their interpretation is consistent. Frequency of drug consumption and time intervals between multiple consumption or lag time between consumption and appearance in the hair has not been fully investigated and needs further research.

Influence of bleaching on stability of benzodiazepines in hair.

In order to study the influence of hair bleaching on benzodiazepines concentrations, hair was treated with a bleaching product (Poly Blonde, Schwarzkopf & Henkel) for 20 min. The treated hair specimen was obtained from a person who died after an overdose of several illicit drugs associated with benzodiazepines. Bleached and non bleached hair were washed (acetone and water), pulverised and then incubated for 2 h in a thioglycolic solution. In the extracts obtained by solid-phase extraction on C18 columns, the different drugs with the corresponding deuterated standards were derivatized and determined by GC-MS in a SIM mode. These results show that the concentrations of all the drug detected decreased in bleached hair in comparison with non treated hair. Whereas the diminution was less important for cocaine and benzoylecgonine (decrease of 24.6 and 36.4%, respectively), concentrations for codeine, 6-monoacetylmorphine and morphine decreased more significantly (decrease of 57.5, 88.6 and 67.4%, respectively) as well as those of diazepam, nordazepam and 7-aminoflunitrazepam (decrease of 39.7, 67.7 and 61.8%, respectively). The results in this study agree with those of other authors that bleaching affects the stability of cocaine and opiates incorporated in hair. These findings also point out that bleaching influences the stability of entrapped benzodiazepines in hair. Finally, these results reconfirm that it is very important to consider the cosmetic history of a hair sample in the interpretation of hair analysis results.

[Diagnosis of acute parathion intoxication and medical-legal consequences]. / Diagnostic d'une intoxication aiguë au parathion et conséquences médico-légales.

During a toxicological screening of a 16-year-old girl, in a clinical toxicological case, a parathion-intoxication has been detected. GC/MS revealed the presence of ethyl parathion in the gastric content and p-nitrophenol in urine. p-Nitrophenol is a major metabolite of ethyl-parathion. A serum concentration of 0.07 mg/L of ethyl parathion was measured by GC/MS-SIM. During hospitalisation the severity of parathion intoxication was monitored by measurement of pseudo-cholines-terase activities in serum. The young girl recovered well of her intoxication after a few days, without any apparent sequellae. A preliminary inquiry revealed that the adolescent had consumed a bowl of soup at the home of a 67-year-old female neighbour, who died the same day. Her corpse was found at her home. The certificate of death, written by the doctor in charge, attested a heart failure. Due to the results of the toxicological analyses from the kid, the juridical authorities ordered an autopsy of the corpse of the lady. Ethyl parathion was found in the gastric content and p-nitrophenol in urine. Even after several temptations it has not been possible to detect ethyl parathion neither in blood nor in some organs. But it was possible to detect traces of p-nitrophenol her blood.

Classification of medicines according to their influence on driving ability.

Within the scope of an information campaign of the Belgian Institute for Road Safety an attempt was made to classify 179 medicinal drugs from 9 therapeutic groups, listed in the Belgian "Commented Repertory of Drugs--1997", according to their effect on driving performance. The categorisation was based on literature data from about 500 references and used the system proposed by Wolschrijn et al [1]: 7 classes ranging from no effect (I) over minor and moderate (II.1,II.2) to severe effects (III), completed with the respective* categories (I*,II*,III*) for presumed classes with insufficient scientific data. Forty-two drugs (24%) were considered having severe effects (III/III*). Only 28/179 molecules (16%) were classed in I/I*: no hypnotics-sedatives (33), anticonvulsants (10), antidepressants (25), neuroleptics (29), nor narcotic analgesics and antitussives (18) were listed in this no-effect category, while for 7/24 antihistamines (5/20 H1 and 2/4 H2), 12/20 beta blockers and 9/10 central stimulants the effect was considered negligible. Antidiabetics were not classified, as the danger lies in the risk of hypoglycemia due to inadequate use. The classification of the molecules proved to be problematic due to the lack of study data (42% of molecules in presumed categories) and the diversity in the study protocols. The effect on driving ability is dose-dependent and time-related, which makes the use of a single category inadequate; the effect further depends on co-ingestion of other medicines or alcohol, the development of tolerance and the condition of the subject. Physicians and pharmacists can use the proposed categorisation as a scientific base for guiding their patients, but should take into account the factors involved for each patient when estimating the driving ability.

Determination of lysergide (LSD) and phencyclidine in biosamples.

Lysergic acid diethylamide (LSD) is difficult to detect and to quantify in biosamples because of its very low active dose. Although there are a number of tests available, routine analysis of LSD is rarely performed. Immunoassays largely vary in their specificity and cross-reactivities with other molecules often make these tests unreliable. Because of its low concentration and the instability of the derivatives (e.g. trimethylsilyl-LSD), routine gas chromatography-mass spectrometry (GC-MS) detection and quantitation of LSD remains a difficult task. The most promising procedures for LSD determination seems to be liquid chromatography-MS analysis using electrospray ionisation and selected ion monitoring (SIM). Extraction, derivatization, GC or high-performance liquid chromatography conditions and the different detection modes will be summarised. Phencyclidine (PCP) is an abused drug seldom found outside the United States. Well established detection and quantitation procedures include radioisotopic and nonradioisotopic immunoassays and GC-MS analysis using SIM mode with deuterated PCP as internal standard. Alternatively, GC with nitrogen-phosphorus detection or capillary electrophoresis has been used. Recent progress in PCP analysis will be summarised.

Development and evaluation of immunochromatographic rapid tests for screening of cannabinoids, cocaine, and opiates in urine.

The test principle and the optimization of the reactive ingredients are described for the one-step dip and-read immunochromatographic FRONTLINE rapid tests for drugs-of-abuse testing in urine samples. In a multicenter evaluation the rapid tests were compared with FPIA and EMIT immunoassays. Discrepant results were further analyzed by gas chromatography-mass spectrometry methods. In the comparison of the cannabinoids rapid tests versus both immunoassays using clinical and forensic urine samples (399 versus FPIA and 755 versus EMIT), sensitivities and specificities were 97% or better for both comparisons. For cocaine, a sensitivity of 100% versus both routine technologies was obtained, whereas the specificity was reduced somewhat to 91% because of some cross-reactivity with metabolites of methadone and of clozapine. Specificity was very high for the cocaine rapid tests (98-100%) when applied to urine samples of persons not in a methadone maintenance program. Sensitivities and specificities for the opiates rapid tests were 99% or better at all sites when compared with the routine methods. In the screening of about 1200 clinical urine samples for cannabinoids, cocaine or opiates misuse only six samples would have stayed undetected by rapid test analyzes. These results show the FRONTLINE assays allow a reliable and immediate screening for drugs of abuse.

Detection of benzodiazepines and other psychotropic drugs in human hair by GC/MS.

For the detection of psychotropic drugs in human hair we collected hair obtained from 21 corpses that died from an overdose of legal or illicit drugs. These persons were known to have taken psychotropic drugs prior to their death as determined by post-mortem toxicological analysis in blood. After washing, cutting hair into segments of 3 cm, pulverization, enzymatic hydrolysis with beta-glucuronidase/arylsulfatase and solid phase extraction, drugs were identified by GC/MS. For quantification of flunitrazepam we used its metabolite amino-flunitrazepam; for oxazepam and lorazepam we used the hydrolysed forms of the corresponding drugs. In the hair of 21 subjects tested we found in 20 cases nordazepam, in 15 cases diazepam, in 15 cases oxazepam and in eight cases flunitrazepam with maximal concentrations of 1.8, 2.2, 3.4 and 9.5 ng/mg hair respectively. In addition to these compounds, in subject 11 to 21 we also analyzed for and detected amitriptyline (seven positive), carbamazepine (eight positive), lormetazepam (three positive) and lorazepam (one positive) and found maximal concentrations of 106.0, 13.5, 29.0 and 4.9 ng/mg hair respectively. The comparison of hair analysis versus post-mortem blood and tissues analysis of all the drugs studied shows that in 40 cases, where a positive result was found in blood, the corresponding drug could also be detected in hair in 37 cases. Our results show that hair testing is complementary to classical post-mortem analysis in forensic toxicology.

Quantification of dextropropoxyphene and its metabolite by HPLC in hair of overdose cases.

Hair samples taken from 11 persons suspected to have died from an overdose of legal or illicit drugs were analysed. These hair samples were selected, because classical post-mortem toxicological investigations in blood revealed the presence of dextropropoxyphene (PPX) and its major metabolite norpropoxyphene (NPPX). For the hair analysis, hair strands were cut into segments of about 3 cm, washed with water and acetone, dried and pulverised. An aliquot of about 40 mg of those segments was incubated with acetate buffer pH 4.0 and beta-glucuronidase/arylsulfatase. After liquid-liquid extraction with hexane-3-methylbutanol (99:1), drugs were identified and measured by HPLC using piritramide as an internal standard. Preliminary assays showed that the limit of detection for PPX is below 1.0 ng/mg hair, the limit of detection for the metabolite NPPX being significantly higher (1.5 ng/mg). GC/MS, usually the method of choice for this kind of determination, was not chosen, because of the thermolability of PPX and its unspecific mass spectrum. From the hair of 11 persons, 24 segments were prepared and processed. Our results show that ten persons were found positive for PPX; moreover, when regarding the 24 segments, only three were found negative. PPX and NPPX concentrations were detected at maximal concentrations of 26.4 and 71.0 ng/mg hair respectively. Considering the ratio of PPX to NPPX for each person, we found more PPX than NPPX for three persons, whereas for seven persons we found more of the metabolite than its parent drug.