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Exploring coagulation in newborns and children is a challenge due to the low levels of both procoagulant factors and inhibitors. Conventional coagulation tests might be inadequate to explore all of these changes. The aim of the study is to evaluate the thrombin generation assay, a global test to explore coagulation, in a pediatric population (n=586) compared to an adult population (n=166). The thrombin generation assays were performed using Calibrated Automated Thrombography with two different tissue factor concentrations (1 and 5 pM), with and without thrombomodulin (TM). In the absence of TM, the endogenous thrombin potential (ETP) is significantly lower in the pediatric population, reflecting the decrease in procoagulant factors. In the presence of TM, ETP values in pediatric subjects are within the reference range of adult values. The thrombin generation assay demonstrates that coagulation balance is maintained in the pediatric population.

In order to perform biological analysis, clinical laboratories apply the instructions of reagent suppliers. For culture media these instructions are often incomplete and poorly adapted to the variety of clinical samples and micro-organisms. The REMIC can help to overcome these shortcomings. Required time of incubation for culture media are proposed based on the nature of the sample and the type of micro-organism suspected. Nevertheless, they are most often expressed in multiple of 24 hours and they are often considered as minimal by the laboratories. As the samples are inoculated "continuously", while the readings are most often done at a single definite time of the day, we propose a strategy to optimize incubation duration of cultures medium. A time of incubation in the day so-called "limit" is defined. From this, the incubations are stopped or prolonged according to the results of the culture and the direct examination. As the instructions of suppliers of culture media are not adapted, it appears necessary that these suppliers relies on the repositories of professional societies as this is the case for agars medias used for antibiotic susceptibility testing.

Preliminary evaluation of qualitative and semi-quantitative clinical laboratory measurements is a prerequisite for the accreditation of clinical laboratories, according to the French committee of accreditation guidelines following the European reference standard EN ISO 15189. Numerous papers have been published regarding biochemistry, hematology or immunology. However, data are still lacking for molecular biology. We report our experience and present the performance characteristics of Allplex™ STI Essential real time PCR assay for the detection of seven pathogens, among which some are systematically responsible of sexually-transmitted infections. Moreover, a semi-quantitative determination of Ureaplasma urealyticum, Ureaplasma parvum and Mycoplasma hominis is proposed thanks to a calibration.

Manual blood smear review is required when an anomaly detected by the automated hematologic analyzer triggers a flag. Our will through this study is to optimize these flagging thresholds for manual slide review in order to limit workload, while insuring clinical care through no extra false-negative. Flagging causes of 4,373 samples were investigated by manual slide review, after having been run on ADVIA 2120i. A set of 6 user-adjustments is proposed. By implementing all recommendations that we made, false-positive rate falls from 81.8% to 58.6%, while PPV increases from 18.2% to 23.7%. Hence, use of such optimized thresholds enables us to maximize efficiency without altering clinical care, but each laboratory should establish its own criteria to take into consideration local distinctive features.

The purpose of this article is to describe the setting up of 15 blood gas analyzers GEM(®) Premier™ 4000 (IL) at Montpellier hospital. This experience includes analytical characterization (within and between run coefficient of variation) using GSE and GHE IL controls, correlation of 35 samples with a routinely used laboratory blood gas analyzer (Cobas b221, Roche(®)). We shall also develop the training, the habilitation and its follow-up for the user staff (450 people) of the different hospital's units in the aim of the accreditation.

Metrology resumes activities which allow the management of results measurement quality. In this paper, we present basic principles of metrology applied to the activities of laboratory medicine. We successively treat general concepts useful for laboratory staff, the interest in the definition of a metrological function and its activities field, the applications in measuring instruments necessary for the exams of medical biology, and the specific documentation control needed.

Paraphenylenediamine is an aromatic amine used as a hair dye; it is responsible for poisoning characterized by respiratory distress involving life-threatening. The objective of this work is the development and validation of an assay of para-phenylenediamine in the whole blood. The method is based on the determination of paraphenylene diamine in whole blood by gas chromatography-mass spectrometry after liquid-liquid extraction and derivatization. The validation protocol has included the study of the recovery factor of extraction, the measurement range, accurency, repetability and intermediate precision. The calibration curve was linear between 98 and 1350 μg/L (r = 0.999), the limit of detection and quantification were 37 μg/L and 63 μg/L respectively. The accuracy were 94.7%. Coefficients of variation were (2.3/6.8/9.7%) for repeatability and (4.4/8.7/9.8%) for intermediate precision. The method is suitable for quantification of PPD in acute poisoning situations. A method for the determination of the paraphenylene diamine in the whole blood by gas chromatography coupled to mass spectrometry was developed. The validation of the method showed good linearity, good accuracy and low limit of quantification.

High-dose methotrexate treatment requires pharmacological monitoring in order to tailor administration of folinic acid to reduce side effects. The aim of the study was to validate the adaptation of the EMIT reagent on the l'Unicel DxC 600® Beckman Coulter. The establishment of two assays was necessary to obtain a quantification limit as low as possible (0.05 μmol/L). The linearity of the adapted methods extends from 0.05 to 0.25 μmol/L on the one hand, and from 0.25 to 1 μmol/L on the other hand. For each method, fidelity and accuracy were studied and the limits of detection and quantification were quantified. The correlation with the FPIA method was performed on the Abbott TDX(®). The results of all tests are satisfactory with coefficients of variation (CV) of repeatability and reproducibility of less than 6%. However the daily assays are heavy as 66% of blood samples require at least two dosages and 30% a manual dilution.

Accuracy of results depends on the traceability of the standards values to a reference system. According to the ISO 15189 requirements, the laboratory has to document the traceability of the value of the standards used.

The newly developed, high throughput, Vitros 3600 immunoassay system associates different technologies on board: MicroWell (analytes immunocapture), MicroSensor (visible interference analysis), and Intellicheck (real time process control for analytical error detection). During a three months period, Vitros 3600 system, ease of use, practicability and MicroSensor technology efficiency have been tested using the following assays: betaHCG, troponin, ferritin, aHBcT, HBsAg, aHCV, AFP and CEA. Within-day and within-batch, between-calibration precisions are acceptable. Overall assay correlations versus the methods in use in our laboratory are good, despite small numerical differences that never affect clinical interpretation of the results. We have appreciated the system reliability, reinforced by the ease of use (simple software, on-board VDocs, continuous access to reagent and sample loading, reagent stability, very low noise (60 dB)), the quality of the results, MicroSensor and Intellicheck technologies efficiency, the short maintenance time and its traceability, and the very low liquid waste volume (5 L of liquid waste for 1600 tests). In conclusion and considering its practicability, the Vitros 3600 system is fully validated and well suited for our laboratory.

L-carnitine is a quaternary ammonium compound that facilitates long-chain fatty acids crossing through the mitochondrial membrane allowing their beta-oxidation. Human pathologies relatives to carnitine are mainly deficiencies, with myopathy and lipids metabolism disorders. The aim of this study was to validate the free and total carnitine plasmatic level measurement using the Dimension HM, X-Pand model analyzer (Dade Behring). Free carnitine was directly determined on plasma samples, deproteinized by using ultrafiltration, whereas total carnitine was measured after alkaline hydrolysis. Within-day and between-day imprecision were < 4,00 and 9,00 % respectively. The method is linear through 250,00 micromol/L and the limit of quantification is 3,00 micromol/L. Our method was correlated to tandem mass spectrometry (r = 0,956 for free and total carnitine). Recovery of free and total carnitine from spiked plasmas was > 99 %. On board stability of cartridge (5 days) and of calibration (at least 30 days) are in agreement with a method easily adaptable in routine laboratories.

The treatment with methotrexate at high doses is responsible of many side effects. It's necessary to evaluate serum methotrexate rate to monitor the administration of the methotrexate antagonist, the folinic acid. The aim of this study is to validate the determination of methotrexate in plasma using the automate Xpand Dimension (Dade Behring). Assay results were linearly related to the concentration for the wide range which was examinated (0.15 - 1.4 micromol/L). We report the precision, accuracy, linearity, sensitivity of this assay. The CV was less than 10%. We present the results of correlations with Aca (Dade Behring) and Cobas Mira (Roche). The reagent cartridge in the instrument and the calibration are stable during 28 days.

A high-performance liquid chromatography method was developed for the rapid determination of ciprofloxacin in human serum. After protein precipitation, chromatographic separation of ciprofloxacin in plasma was achieved with an octyl column and 20 mmol/L phosphate buffer, methanol and acetonitrile 76/12/12 (v/v/v), pH 4, as mobile phase. Quantitative determination was monitored on a fluorescence detector using an excitation and emission wavelength of 278 and 450 nm, respectively. Enrofloxacin was used as internal standard. Calibration curve is linear over the range 0,12 to 20 microg/mL (r=0,9992). Limit of detection and limit of quantification are 0,04 and 0,12 microg/mL, respectively. Within-day coefficient of variation (2,0 to 3,1 %) and day-to-day coefficient of variation (4,3 to 5,5 %) at three different concentrations are sufficient. Relative recovery ranged from 91,1 to 94,0 % at three different concentrations. Each analysis requires no longer than 7 min.

The Advia 70 hematology system is a new automated analyser manufactured in Texas (USA) by MWI-DANAM and marketed by Bayer- Diagnostics in France. The throughput is 70 complete blood counts (CBC) with leukocyte differential counts (diff) per hour. Three sample modes are proposed: normal mode (180 muL of whole blood), sample saver mode (90 muL) or automatic sampler (180 muL). The principles used on Advia 70 are: 1) electrical impedance for cell counting (WBC, RBC, platelets, MCV) and RBC/plt sizing; 2) multi-dimensional optical system and absorbance for white cell differential. The Advia 70 has been evaluated in our laboratory over a three-week period. Within-run variations of blood counting (CBC & diff) were determined on normal and abnormal patient specimens whereas between run-variations were determined on normal controls. Linearity and detection limits were also tested. The Advia 70 system results were reproducible and reliable. The correlations between the various sample modes were very good. Contamination and store assays were performed. Thirty normal and abnormal samples were successively analysed on the Advia 70 system and on the Advia 120 system which methodologies are different (myeloperoxidase staining coupled with flow cytometry-nuclear lobularity analysis for WBC/Diff, flow cytometry and laser diffraction for RBC/platelets), and the results were compared: despite the different technologies used on both analysers, the correlations were very good (CBC and diff parameters). Finally, we evaluated the leukocyte differential flags on 72 patients samples with abnormal cells, using the blood smear examination as the reference method. In conclusion, the Advia 70 can be used in private and hospital laboratories.

Having had the opportunity of testing two of the three commercially available single use unit systems (i-STAT and OPTI-1), we have listed a series of performance limiting factors which they share, even though, their detectors being based on different methodologies, they have specific causes of dysfunction. PO2 detectors face the worse problems for different reasons. Temperature control is critical: the cassette or at last the detectors, the internal solution and the specimen have to be heated to 37 C, thermostabilisation to one tenth of degree centigrade is a must. Gas equilibration between liquid phases, the outside ambiance and the plastic material can raise problems: a decrease of the external barometric pressure alters the result, an absence of oxygen buffer capacity can induce modifications of the PO2 in the solutions. The transition of the detectors from a dry to a wet stage can provoke changes of the final result in case of accidental pre-humidication of the cassette. The presence of a single internal solution generates problems linked to a single point calibrage procedure which is a source of variability. The general quality of the cassette can only be evaluated by numerous internal electronic checks: increasing the severity of the controls improves quality but increases the number of rejected cassettes. External quality control through classical procedures are not applicable and finally the quality of the cassettes depends primarily on rigorous manufacturing conditions.

Results of catalytic activities of enzymes are highly dependent on the measurement procedures and on local conditions. Thus, only poorly marked improvement of interlaboratory comparability of results have been observed in clinical enzymology. To solve this problem, SFBC and IFCC have proposed to use "validated enzyme calibrators". Standardised operating procedures adapted to 37 C have been developed by IFCC for the most commonly used enzymes in clinical chemistry, and will be soon published. Reference materials which have been certified with these SOPs can be used as calibrators for a set of measurement methods which exhibit the same analytical specificity. Calibrators must be commutable, a property that must be checked experimentally. It is possible to produce stable and commutable materials for the calibration of a set of methods. Interest of this approach has been demonstrated for several enzymes. Results of two studies presented here show that the comparison of results to the upper limit of reference ranges does not improve the interlaboratory comparability of results in contrast to the calibration of different methods by a common calibrator which allowed to reach an interlaboratory CV close to 4% for ALT and gammaGT.

To validate the use of the i-STAT portable point-of-care instrument, performance checks have been conducted to verify its accuracy for PCO2/PO2 (tonometry) and precision for pH/PCO2/PO2 (control solutions). Results obtained with the i-STAT (pH/PCO2/PO2/Hct) were also compared to those provided by the Bayer Rapidlab 865 routinely used in our laboratory. All these measurements have been conducted under the same conditions using three types of i-STAT cartridges (G3, EG6, EG7) with two of different life duration to each cartridge with a view to observing their potential variability between types and any effect that ageing may have on analytical performances. Differences observed in this respect are not clear enough to draw firm conclusions. On the other hand, we consider the level of PCO2 and particularly PO2 performances not sufficient to warranty their use in the absence of a very strict quality control coupled with a highly developed clinical sense. No real use can be made of values noted for hematocrit (1998 survey).

The large metrological variation (CV, about 25%) observed between laboratories, at the national French level, for the measurement of enzymatic activities results in a loss of efficiency in using laboratory results. Current data show that the standardisation of methods is insufficient to solve this problem and needs to be completed by an harmonisation of the practices including the use of a common reference (calibrator). The present work, carried out by the joint working group between laboratories of the Centres for Periodic Health Examination and the French Society of Clinical Biology (SFBC), deals mainly with the feasibility and evaluation of the improvement of the consistency of the results. Twenty laboratories participated in this study. Five independent surveys were conducted during an height month period. Two enzymes were selected because of their clinical importance and their interest in prevention, screening, diagnosis or epidemiology: ALT (alanine aminotransferase) and GGT (gamma-glutamyltransferase). In each survey three kinds of samples i.e. control sera, candidate calibrators and human serum pools, each of them at two levels of activity (one physiological and the other pathological) were measured in duplicate. The low intra-laboratory imprecision and the high degree of the standardisation of used methods, due to an important effort previously done in this field, permitted to consider a common calibration. The stability and mainly the commutability, i.e. the ability for the candidate calibrator to show a behaviour similar to that of human samples towards the used methods, allowed to reduce the inter-laboratory variation by a half to two third-fold, reaching a coefficient of variation < 5% similar to those observed for cholesterolemia or glycemia. This level of consistency should permit to use common reference limits and common decision limits, after validation of this approach in real practice. The consequences of the harmonisation of practices, extended to the all laboratories, exceed largely the scope of this study. The reduction of the uncertainty and a better approach of the accuracy for the measurement of enzymatic activities should led to a real benefit for the patients in terms of prevention, screening, diagnosis or therapeutic monitoring and consequently for the public health.

Adherence receptors are essential for heterotypic (endothelial cell, platelet) polymorphonuclear neutrophil (PMN) interaction. Determination of their expression level give information about activation state and functionality of PMN. Use of flow cytometry associated with an immunolabeling standard, represented by beads coated by a determined amount of immunoglobulins (Qifikit, Dako), allows analysis of specific antibody binding capacity and gives information about antigen density. Using this methodology, the exploration of surface adherence receptors, L-selectin (CD62L) and b2-integrins (CD11a-c/CD18) from PMN unstimulated and incubated with pro-inflammatory stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) and tumor necrosis factor a (TNFa), allows, on the one hand, the establishment of basal expression values on resting PNN and on the other hand, the study of PNN reactivity. This method of quantification can be applied to clinical studies as adherence receptor deficiency syndromes or inflammatory, infectious and vascular diseases.