[Phalloidin syndrome: role of Elisa-based assay for the detection of alpha- and gamma-amanitins in urine. Preliminary results]. / Syndrome phalloïdien: quelle est la place du dosage des alpha- et gamma-amanitines par ELISA (Bühlmann)? Résultats préliminaires.

UNLABELLED: After consumption of mushrooms containing amatoxins (Amanita, Lepiota, and Galerina species), symptoms usually develop after a long delay (>6 h). Initial symptoms start as severe gastroenteritis, progressing to liver failure and possibly death as a result of hepatic coma. Since the survival rate of poisoned patients is claimed to depend on the time of beginning of efficient treatment, fast and reliable assays for amatoxins in biological fluids are essential. Described analytical methods for amatoxins include high performance liquid chromatography and radioimmunoassay (RIA). Recently, a new enzyme-linked immunosorbent assay (Bühlmann Amanitin ELISA kit) has been introduced as an alternative method to RIA. This ELISA-based assay offers several advantages: no complex extraction procedure is required (vs. HPLC) and no safety precautions concerning radioactivity have to be taken (vs. RIA). From August 2004 to October 2005, a pilot study was performed to test the practicability and the clinical utility of this method in emergency situations. RESULTS: ten urines, 9 serums and 1 faeces from 10 patients suffering from acute gastroenteritis after mushroom ingestions (7 contaminated meals) were analyzed. Definitive diagnosis of amatoxin poisoning was made in 4 cases (3 contaminated meals) on the basis of the anamnesis, laboratory results, and clinical course. A patient developed a severe amatoxin poisoning with urinary amanitins level < 1.5 microg/L (urines were collected more than 72 h after mushroom ingestion). Two patients were paucisymptomatic with urinary amanitins levels >10 microg/L (urines were collected before the 36th hour). CONCLUSION: Urine is the sample of choice for the determination of amatoxins. The most critical factor to invalidate the usefulness of this analysis is time. After 36 h, the sensitivity is unreliable.

[Antidigoxin Fab fragments and digoxin monitoring: a challenge for the biologist]. / Suivi des digoxinémies chez les patients traités par fragments Fab antidigitaliques: un défi pour le biologiste.

Following administration of anti-digoxin Fab fragments, monitoring unbound digoxin concentrations may help ensure appropriate dosing, and prevent recrudescent toxicity. Ultrafiltration by using Centrifree system and measurement of digoxin in the ultrafiltrate is considered as reference technique. However, ultrafiltration method is cumbersome, costly, and some immunoassays are affected by matrix differences. Another approach is to analyse the serum directly by digoxin immunoassays without ultrafiltering it. The validity of results obtained depends on the architecture of the immunoassay and the amount of Fab in the sample. The old radioimmunoassays and usually the other competitive immunoassays give inaccurate results. The fluorescence polarization immunoassay (FPIA) slightly underestimates the total digoxin concentrations. Total digoxin levels obtained at 24 hours and 48 hours after treatment permit measurement of the half-life of digoxin Fab complexes and can be used to estimate when the patient can be redigitalized, if necessary. The sequential immunoassays usually overestimate the free digoxin concentrations. The differences observed are >25% and cannot be explained solely by albumin binding (normal range, 20% +/- 5%). To date, ultrafiltration remains the best strategy for accurate determination of digoxin concentrations in the presence of antidigoxin Fab fragments.

European multicentre evaluation of the analytical performance of the Abbott AxSYM Abused Drugs Assays.

In accordance with the guidelines of the European Committee for Clinical Laboratory Standards (ECCLS), the performance of the Abbott AxSYM Abused Drugs assays were evaluated and compared with the results provided by the following systems: Syva Emit d.a.u./Roche Cobas Mira S Plus, Abbott TDx and ADx, Syva Emit d.a.u./Syva ETS Plus, Syva Emit II/Hitachi 717 and Roche Abuscreen OnLine/Roche Cobas Mira S Plus. The test analytes, cannabinoids, cocaine metabolites, opiates, benzodiazepines and barbiturates, were each investigated in three laboratories on different systems. The imprecision of all systems in the series and from day to day was good, with CVs less than 5% or 10%, respectively. The AxSYM calibration curves were stable for 3-4 months and none of the systems displayed any shift in the results of the analyses within one day or any faults caused by sample contamination. Within the framework of this study, a total of 1860 urine samples were investigated; 741 results were positive. All results which remained discrepant between AxSYM and the comparison systems after repeated analysis (n = 17) were subjected to further investigation using a reference method, with the exception of one barbiturate and two benzodiazepine samples. An additional test criterion was the practicability of the systems investigated and the versatility of the software. During this evaluation, the results provided by the Abbott AxSYM were excellent and were fully in line with the manufacturer's claims. The reliability of the FPIA technology that has been the subject of frequent investigation was also convincing during this evaluation. The possibility of semi-quantitative determination, the stability of the calibration curves, the ability to process an emergency sample without delay and its high suitability to routine operations are the convincing benefits offered by this system.

Chemiluminescent measurement of oxalate in serum by detection of hydrogen peroxide generated through oxalate oxidase.

Today, chemiluminescence detection reactions have become popular in analytical biochemistry essentially due to their high sensitivity. A chemiluminescent synthetic system (luminol/porphyrin) was successfully used to measure serum oxalate by determination of hydrogen peroxide generated through oxalate oxidase (EC 1.2.3.4.). This new method is efficient and simple, highly sensitive and the results obtained in normal adult subjects are in good agreement with those of approved methods. This original application of such a chemiluminescent system allowed us to achieve a sensitive serum oxalate assay (detection limit of 0.2 mumol/L) characterized by a low serum volume (200 microL) required for analysis.

Serum oxalate microassay using chemiluminescence detection.

Determination of serum oxalate concentration is important for the diagnosis and monitoring of hyperoxalurias, and extends to patients with all types of renal disease. Approximately 5 to 10 ml of blood is required for each test by conventional methods, and the test is not adapted for use in children. We developed a highly sensitive method that limits the volume of blood required for the test. This new and sensitive tool to detect H2O2 can be successfully substituted for the conventional, and expensive, colorimetric reaction to accurately analyze oxalate concentration.

Automated Cobas Mira kinetic enzymatic assay for ethylene glycol applied to emergency situations.

An automated kinetic method for assaying ethylene glycol in serum using glycerol dehydrogenase with the multiparametric analyzer Cobas Mira is described. Initially, 5 microL of sample is mixed with tris-NAD buffer; after enzyme addition, the variation of the absorbance at 340 nm is automatically measured, and the instrument calculates the ethylene glycol concentration of the specimen. The method has good precision and specificity and is suitable for emergency screening. Some applications developed in our laboratory are also described.

Assessment of recently developed blood gas analysers: a multicentre evaluation.

Providing guidelines for testing expected inaccuracy and imprecision is still a matter under debate. The Expert Panel of the French Society of Clinical Chemistry has developed a protocol, which was based on a comparative multi-centre evaluation of four instruments: the Ciba-Corning 278, the Instrumentation Laboratory 1306, the Nova SP 5 and the ABL 330. The purpose was to evaluate the analytical performance and efficiency of the analysers. Another aim was to design a valid approach for evaluating any new system. As buffered aqueous solutions and fluorocarbon emulsions give only partial information, tonometered blood was used at different levels of gas mixture, even though it is both difficult and time-consuming. Comparisons have been established on patients' blood samples with the analysers currently used in the evaluation sites. The tests showed that the four analysers have the same degree of precision, and interinstrument comparisons demonstrated a very high degree of reliability.This analysis emphasizes that the evaluation of instruments for pH and blood gas analysis is neither easy nor is it often done, mainly due to the choice of a quality-control material and the lability of the measured parameters.

Ca2+ measurement with ion selective electrodes. The French coordinated evaluation of seven analyzers, for a better clinical relevance and acceptance.

The measurement of ionized calcium has evolved in the last decade, and can now be easily performed by clinical laboratories using direct potentiometric analyzers available from a number of manufacturers. An original protocol for a comparison of the analyzers was used through a parallel multicenter evaluation in France. Using newly developed aqueous buffered solutions, this study focused not only on the analytical performance and operational handling of analyzers, but also on possible interferences in biological samples and the clinical relevance of the measurement with respect to the techniques of sample collection. All the instruments exhibited good precision and linearity, and were easy to handle and robust for daily use. However, not all the models gave identical results on the same patient's specimen. The utility of some Ca2+ analyzers has been further enhanced by the ability to "correct" the results to pH 7.40, although care must be taken in the interpretation of these results. While there are a number of clear-cut situations in which Ca2+ measurement is more relevant than total calcium, it seems that chemical activity of Ca2+ in blood may sometimes be considered with great caution under pathological conditions. The role of Ca2+ measurement in routine will be discussed in relation to the potential benefits of the instruments in laboratories.