[Recommendations for metrological control of measuring equipments in the clinical laboratories]. / Recommandations pour la maîtrise métrologique des équipements de mesure au laboratoire d'analyses de biologie médicale (Document D).

The control of the measuring equipment (balances, mass standards, micropipettes, dilutors, volumetric glassware, thermometers...) is essential to obtain reliable analytical results. This control requires knowledge and use of the main standards, instructions for use, metrological verifications and confirmations as well as creation of checking and standardizing certificates ensuring metrological traceability. These items should help to control the quality of the analytical performances (fidelity and trueness in particular).

[Sweat testing: review of technical requirements]. / Recommandations pour l'exécution et l'interprétation du test de la sueur.

The sweat test, a quantitative measurement of chloride in sweat, remains a key laboratory test to support the diagnosis of cystic fibrosis. However, because of its delicate execution, sweat test result should be interpreted with biological, clinical and genetic arguments. The following guidelines which we propose, were established in order to harmonize the practices of the sweat test. They are elaborated in a consensual way by biologists from cystic fibrosis reference centers and/or from the working group "Sweat Testing" of the National College of Biochemistry Hospital praticiens, according to the current state of knowledge on the subject, the experiment of the biologists and the recommendations established in the United States and in the United Kingdom.

[Recommendations for expressing uncertainty of measurement of quantitative results in laboratory medicine]. / Recommandations relatives à l'expression de l'incertitude de mesure des résultats quantitatifs en biologie médicale (Document F).

In laboratory medicine, the quantitative results of examinations are interpreted with regard to reference intervals, clinical decision limits or previous results of a patient, from which it is necessary to inform the clinician about the uncertainty of measurement linked to the value of the result. This document explains the problematic of the expression of the uncertainty of measurement. It proposes recommendations concerning a simple way to evaluate uncertainty of measurement using long term internal quality control data and the value of the uncertainty linked to the method calibration. It approaches the determination of analytical goals and the choice of methods and also the comments accompanying the record of results and a help to their interpretation.

Evaluation of GHb assays in France by national quality control surveys.

OBJECTIVE: To evaluate the state of the art concerning GHb assays through analysis of a large-scale quality control survey and to compare the results with those of previous surveys. RESEARCH DESIGN AND METHODS: A lyophilized hemolyzate prepared from human erythrocytes containing a physiological HbA1c level (5.5%) was sent to 3,500 French laboratories in February of 1995 and assayed as a patient's sample under routine conditions. Distribution of values was analyzed from the reported results for each method. The results were compared with the assigned value (acceptable range: +/- 20%) and with the upper value of the reference range currently used. RESULTS: Results were obtained from 2,674 laboratories, among which 39% used cation-exchange chromatography methods, 37.5% affinity chromatography, 16% immunological methods, and 7.5% electrophoresis. The number of laboratories using immunological methods increased from 100 to 400 between 1993 and 1995. The overall interlaboratory coefficient of variation (CV) was 20.2%, with within-method CVs ranging between 3.2 and 29.5%. Method-to-method accuracy varied dramatically, with mean HbA1c values ranging from 4.4 to 8.2%. Results from 75% of the laboratories were comprised in the acceptable range; 88% of them reported a value within the normal range of the method used. CONCLUSIONS: The interlaboratory variability of results illustrates the difficulties encountered by diabetologists in the follow-up of diabetic patients using results obtained from different laboratories. It demonstrates the usefulness of the internationally developed standardization process of GHb measurements and points out the need for laboratories to fulfill good practices.

Uremia-induced disturbances in hepatic carbohydrate metabolism: enhancement by sucrose feeding.

A high-sucrose (S) diet accentuates anorexia and stunts growth in uremic (U) rats, and an oral S load induces a greater hyperfructosemia in U rats than in control (C) rats. Four studies were performed to determine the roles of S feeding and an acute S load on liver carbohydrate (CHO) metabolism in U and C rats (eight to 10 rats per group). We also examined the plasma responses to either water or a S load. Levels of the main metabolites of glycolysis, gluconeogenesis, and glycogenesis were measured under basal conditions (7 hours' postmeal) in U and C rats fed either a cornstarch diet (study I) or S diet (study II) and at 30 and 60 minutes after an intragastric S load (studies III and IV) in s-fed U and C rats. The weight gain, food intake, and plasma creatinine and urea levels of the rats in the four studies were comparable. Weight gain and liver weight (g/100 g body weight) were lower in U than in C rats. In the plasma, baseline levels of lactate were decreased by uremia and S feeding and those of glucose (G) were increased by S feeding. The increases in plasma G and fructose (F) levels after a S load were greater in U rats than in C rats, whereas those of plasma lactate were comparable. In the liver under basal conditions, uremia markedly decreased levels of glycogen, F-1,6-diphosphate (F-1,6-diP), F-2,6-diP, 3-glycero-phosphate (3-glycero-P), dihydroxyacetone phosphate (DHAP), pyruvate, lactate, and adenosine triphosphate (ATP), and the phosphorylation state (ATP/adenosine diphosphate [ADP] x inorganic phosphorus [PI]), increased phosphoenolpyruvate (PEP), ADP, and Pi levels, but did not affect the cytosolic redox state (pyruvate/lactate). In addition to uremia, S feeding further decreased levels of glycogen, F-2,6-diP, 3-glycero-P, and ATP. After S loading, liver F levels increased more in U than in C rats, but glycogen and 3-glycero-P levels increased less in U than in C rats. Liver lactate and pyruvate levels increased more in U than in C rats, and the pyruvate/lactate and DHAP/3-glycero-P ratios were higher in U than in C rats after a S load. The ATP level and the phosphorylation state in U rats increased 30 minutes later in U than in C rats. Our findings indicate that uremia causes a depletion in liver glycogen, which is enhanced by S feeding and could be partially attributed to decreased glycogen synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Interassay calibration as a major contribution to the comparability of results in clinical enzymology.

OBJECTIVE: Factors contributing to the applicability of interassay calibration of methods measuring enzyme catalytic activities are described. Also discussed are the properties essential for such a material. Similarity of specificity for the methods to be calibrated as well as commutability between the material(s) intended to be used as calibrator are the main criteria to be satisfied. RESULT: Several examples demonstrated that interassay calibration is feasible but a multi-enzyme calibrator with a wide commutability for the most popular methods remains to be developed. This is the project of the IFCC Working Group on Calibrators in Clinical Enzymology (WG-CCE). Several experimental data are also presented that indicate that the temperature at which the reaction is carried out is not a limiting factor in the implementation of interassay calibration in clinical enzymology.

Validation of an enzyme calibrator--an IFCC guideline. International Federation of Clinical Chemistry.

OBJECTIVES: The objective of this guideline is to improve standardization in clinical enzymology in order to improve intermethod comparability of patients' results. DESIGN AND METHODS: The reference system, combination of the reference method and the reference material, is used to produce a reference value for a given catalytic activity. Sets of methods are formed of methods exhibiting the same analytical specificity. Materials intended to be used as enzyme calibrators are experimentally checked for their commutability. RESULTS: The transfer of accuracy from the reference value to patients' results is dependent on methods (analytical specificity) and on materials (experimentally assessed commutability). The feasibility of this approach was demonstrated with materials of high level for several enzymes and for each of them for several routine methods. CONCLUSION: Expected advantages of this approach in clinical enzymology are presented.

[In vitro supplementation of pyridoxal phosphate for the optimisation of the determination of the catalytic activity of alanine aminotransferase and aspartate aminotransferase in kidney transplant patients (author's transl)]. / Intérêt de l'addition in vitro de phosphate de pyridoxal dans la détermination de l'activité catalytique des amino-transférases dans une population de transplantés rénaux.

Pyridoxal phosphate (PLP) is the coenzyme of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Thus, in vitro supplementation with PLP is important for the optimisation of the determination of catalytic activity of both enzymes. It patients with kidney transplants, stimulation by PLP is very important for ALT activity, which could be affected by plasma PLP deficiency. Furthermore, in this population catalytic activities are more frequently found increased using methods with PLP supplementation than without PLP supplementation (56% and 71% of patients for AST and ALT, respectively). These differences are not related to HBs antigen.

Immunochemical assays of serum proteins: a European external quality assessment survey and the effects of calibration procedures on interlaboratory agreement.

An external quality assessment survey of immunochemical assays of 9 proteins (immunoglobulins G, A and M, complement components C3 and C4, alpha1-antitrypsin, orosomucoid, haptoglobin and transferrin) in 5 European countries (Austria, France, Hungary, Italy and UK) showed inter-country differences in the mean values obtained. Reprocessing of the results using one of the two specimens distributed as a 'calibrant' effectively eliminated or reduced substantially these differences. Consideration of the methods used by participants confirmed previous indications from national surveys that the differences were due to lack of agreement among commercial calibrants. Such interlaboratory variations were also minimised by the 'calibration' in this survey. The role of European working calibration materials in ensuring interlaboratory agreement on an international basis is discussed.

Production and certification of an enzyme reference material for pancreatic alpha-amylase (CRM 476).

We describe the preparation of a lyophilized material containing purified human pancreatic alpha-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was > 99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic alpha-amylase had a molar mass of 57,500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified alpha-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/l and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20 degrees C. The certified value for alpha-amylase catalytic concentration in the reconstituted reference material is 555 U/l +/- 11 U/l when measured by the specified method at 37 degrees C. The material can be used to verify the comparability of results from laboratories, for intra-laboratory quality control or for calibration of alpha-amylase catalytic concentration measurements.

Glycolytic profile in normal brain tissue and gliomas determined by a micro-method analysis.

High aerobic glycolysis is frequent in cancers. Glucose phosphorylation is under control of hexokinase which is found in the cytosol or bound to mitochondria. Glycolysis parameters (glucose, pyruvate and lactate) and hexokinase were evaluated in extracts of 15 human gliomas and of normal brain tissue. Extracts were run and analysed for glucose lactate and pyruvate content using a centrifugal automatic analyzer, Mitochondria fractions were separated from total extracts and hexokinase enzymatic activities were measured in both, using an original micro-method. Conditions of hexokinase assay were standardized in terms of substrate concentration and linearity. Mean hexokinase activity in gliomas was variable, 3 times lower than in normal tissues and mainly bound to mitochondria, although lactate/pyruvate ratios were found to be 3.5 to 5.4 times higher. Glycolytic profile of tumor tissues can be rapidly assayed and evalated glycolysis in tumors could constitute a basis for therapy using antiglycolytic strategies.

Tolerance to starvation in children on long-term total parenteral nutrition.

To evaluate the consequences of long-term cyclic total parenteral nutrition (TPN) on metabolic pathways which contribute to energy metabolism, adaptation to starvation was studied in a group of seven children 2-7 years old, on long-term cyclic TPN. In addition to clinical monitoring, the following biological parameters were measured: blood levels of glucose, free fatty acids, ketone bodies and carnitine, and urinary excretion of dicarboxylic acids. Five of the seven children had good clinical tolerance up to 30 h of fasting. This indicated that metabolic changes arising from prolonged cyclic TPN are easily reversed when such children are fasting. The other two children had to be refed after 22 and 24 h, respectively. Both had metabolic signs of impaired hepatic fatty acid oxidation or ketogenesis. These metabolic changes might reflect the liver failure caused by TPN in these children, and stresses the fact that prolonged starvation has to be carefully monitored in patients with liver dysfunction secondary to TPN.

Between-country comparability of clinical chemistry results: an international quality assessment survey of 17 analytes in six European countries through existing national schemes.

Two lyophilized control sera were distributed through seven national external quality assessment schemes in six European countries--Belgium, Switzerland, France, The Netherlands, Sweden and the United Kingdom--participated in the study. The results for 17 routine analytes were obtained from almost 5000 laboratories for the two sera. The organizers of the schemes were asked to process the results according to a common outlier removal procedure, and submit method-related data if available. The two sera were also distributed through the external/internal scheme of The Netherlands, and the within-laboratory standard deviations calculated in this scheme have been used in a scaling procedure for the external mean values and between-laboratory standard deviations of the participating countries. The results show remarkable agreement in the national mean values for practically all analytes, but considerable differences in the between-laboratory variation. Data from comparable method groups was obtained for 12 analytes from Belgium, France, The Netherlands and the UK. Though revealing some specific differences between methods and countries, the method-related data are generally in agreement with the all-method data. In this study reference method values were only available for cholesterol. The high degree of agreement found suggests, however, that mutual recognition of all-method mean values in national schemes could be acceptable, especially for analytes for which reliable reference methods are not available. The major element of variation is between-laboratory rather than between-country.

[Evaluation according to the NCCLS protocol of the Kodak-Ektachem procedure applied to the estimation of glucose and urea in plasma (author's transl)]. / Evaluation d'après le protocole NCCLS du procédé Kodak-Ektachem appliqué au dosage du glucose et de l'urée dans le plasma.

The study carried out consists of evaluating the results obtained for estimation of glucose and urea with a procedure the originality of which resides mainly in the use of preconditioned reagents, in the dry form, laid out on a support where the colour develops, proceeding to a reading by reflectometry and linearise the response by a procedure of calculation carried out by a microprocessor. The evaluation of the results obtained with the Kodak-Ektachem prototype for estimations of glucose and urea in the plasma was carried out according to the NCCLS protocol in the field of a coordinated European study. The imprecision was estimated at several levels of concentration after a study of intraserial and day to day reproducibility with and without contamination correction, the variation coefficients obtained from day to day are less than 3% when the concentrations of glucose and urea are higher than 5 mmol.l, the lower concentrations lead to less precision. No contamination was demonstrated. The inaccuracy is evaluated by comparison of the results supplied by the testing apparatus with those obtained by techniques chosen as reference. (Direct hexokinase with deduction of a blank sample and final reading at 340 nm for glucose, a colorimetric technique using monoxime diacetyl adapted to continuous flow for urea). The correlations between these methods are good : (mmol.l1) glucose : (EKTA) = 0.07 + 1,025 (HK); r = 0,993. Urea : (EKTA) = = 0,498 + 1,046 (DAM); r = 0,995. Further studies on the influence of proteins and a few physiological and drug substances liable to interfere have shown that a routine use in the laboratory may be considered favourably.

[Advantages and constraints of recent multichannel selective analyzers]. / Avantages et contraintes des analyseurs sélectifs multiparamétriques récents.

The analyzers have been defined as follows: the analyzers are multiparametric (at least 5 parameters available simultaneously), selective, operating patient by patient, list price on May 1, 1984 below 1,000,000 francs, and distributed in France after January 1, 1981. This definition applies in particular to systems already widely used in French Laboratories, such as the Technicon RA 1000, and the Hitachi Boehringer 705. It also applies to systems distributed more recently, such as the Kone "Progress" and the Coultronics "Dacos". Based on data provided by the manufacturers (technical instructions, operating instructions, evaluation reports, etc) and data provided by a certain number of users, we have tried to appraise the advantages and constraints of these analyzers with respect to the following points: required environment (installation, electrical current, temperature), training of personnel, ease of adaptation, work organization, rhythms, operating cost, ease of use, verification of correct function, format of results, maintenance, etc. Lastly, we discuss the problem of the reliability of the results (precision, accuracy), in particular the problems of referencing in substrate assays, and the problems of accuracy in the measurement of enzymatic activities, considering the current norms of standardization.

[Evaluation of the technique of CKMB assay using Ektachem (500 and 700)]. / Evaluation de la technique de mesure de la CKMB de l'Ektachem (500 et 700).

The aim of this study was to compare the dry-chemistry CKMB Ektachem method to a liquid immunoinhibition method (Merck/Cobas Bio) and to the electrophoretic method (Helena France), both on patients (n = 95) and control (4 specimens from different commercial origin) sera. The Ektachem method was found linear in the range tested (7 to 97 U/l). Within run imprecision tested with low, medium and high control sera were satisfying (CV 3-4%) as well as between run imprecision (CV < 10%), for CKMB activities of at least 30 U/l. As compared to the liquid immunoinhibition method (Merck), the results of patient sera were very close but slightly lower (y = 0.97x-1; r = 0.74; y = Ektachem, x = Merck). As compared to the electrophoretic method, the Kodak Ektachem method showed a specificity of 74% and a sensitivity of 85% (n = 95). A close agreement (r = 0.73) between these two methods was obtained for samples with a total CK activity at least 3 times over the upper limit of normal range. We therefore conclude that the Kodak Ektachem CKMB method allows a rapid and easy determination of CKMB activity for samples undergoing total CK activity 3 times over the upper limit of normal range. Like all liquid immunoinhibition methods, the Kodak Ektachem method shows some lack of specificity and does not show a better concordance with the electrophoretic method than does the Merck/Cobas Bio method.

[Improvement of result coherence in clinical enzymology: multicenter study of gamma-glutamyltransferase, alkaline phosphatase and amylase activities]. / Amélioration de la cohérence des résultats en enzymologie clinique: étude multicentrique concernant les activités gammaglutamyltransférase, phosphatase alcaline et amylase.

We report here on the results of a multicenter study of three enzyme activities (gamma-glutamyltransferase, alkaline phosphatase and amylase). For each activity, measurements were performed in two laboratories on different series of patients' specimens under routine conditions, at 30 and 37 degrees C, with techniques frequently used in France and with the IFCC reference method, when it exists. For each technique, precision was acceptable, but results differed considerably according to the technique used. The study also showed that for different techniques it is not possible to use a single transformation factor for activities between 30 and 37 degrees C. Patients' results determined by two techniques often showed a constant relationship. Groups of techniques that determined the same catalytic activity in patients' specimens were identified, whereas other techniques did not have this property. Several preparations, including reference materials produced by the Community Bureau of Reference (European Community, Brussels) and ten commercial secondary materials were tested for similar behaviour as compared to patients' samples. Results show the commutability of reference materials within a group of techniques indicating that they can be used as calibrators. This was seldom the case for the commercial secondary materials and we did not find any such material suitable for calibration of the three enzymatic activities. The present study demonstrates that with defined techniques and validated calibrators it is possible to reduce considerably differences between results obtained with different techniques at different temperatures and in different laboratories.

[Quality specifications and allowable standards for validation of methods used in clinical biochemistry]. / Analyses de biologie médicale: spécifications et normes d'acceptabilité à l'usage de la validation de techniques.

The purpose of this work is to provide, for a large number of analysis in the field of clinical biochemistry, appropriate criteria for the evaluation of the performance of in vitro diagnostic methods. Based on a first set of data established in 1986 and the experience cumulated by organisers in charge of internal and external quality assessment surveys, an expert group has proposed acceptable limits for a large list of analysis (n = 116). Data are reported and presented in tables divided into 7 chapters including: general biochemistry, enzymes, proteins, tumour markers, hormones, drugs (and toxic), and urinary analysis. For each analysis are given: analytical and reference ranges, three concentration levels for control specimens to be used during evaluations and the range of values within which they can be chosen, reproducibility and repeatability limits expressed as CV%. Maximal tolerable systematic error and inaccuracy are given for control and biological specimens and compared to those obtained using a reference or validated method. These data are essential for evaluations using the protocol designed by the SFBC and can serve as quality criteria for the choice and validation of in vitro diagnostic systems.

[Recent aspects of liver biochemistry: from physiopathology to functional liver exploration]. / Aspects récents de la biochimie hépatique: de la physiopathologie á l'exploration functionnelle du foie.

The advent of liver transplantation and the availability of effective medical therapeutics have recently made possible treatments of chronic liver diseases. These improvements have evidenced new needs for evaluation of the treated patients. In this review, authors present new biochemical liver tests proposed for a better monitoring in the course of the disease, to assess the therapeutic response in clinical trials and to reduce the number of liver biopsies. The different aspects of this paper concern the evaluation of hepatic uptake and biliary elimination, cholestasis, jaundice, cellular injury, fibrosis and liver tumor.

[Serum creatinine assay: results of a multicentric study with 16 analytical systems]. / Dosage de la créatinine sérique: résultats d'une étude multicentrique de 16 systèmes analytiques.

During a multicenter evaluation, 16 methods for creatinine measurement have been tested according to the guidelines of the Société française de biologie clinique (SFBC) protocol. Kinetic Jaffé methods, widely used in France, performed on different analytical systems (Astra Beckman, IL 508, RA 1000 Technicon, Hitachi 704, 705, 717 Boehringer, Fara Roche, Progress Kone, Kem-O-Mat Coulter, Perspective France Monitor) have been compared to a continuous flow method with aqueous standards, to enzymatic methods using creatinine amidohydrolase with a colorimetric measurement (Boehringer and Ektachem Kodak) and to an HPLC method. Reproducibility, estimated with four different control sera, proved to be unsatisfactory in some cases as compared to current criteria for imprecision (less than +/- 10 mumol/l for intralaboratory and less than +/- 20 mumol/l for interlaboratory imprecision). The same selected patients sera covering the whole range of physiopathological concentrations have been analyzed with each method, and compared with the continuous flow results. Differences are more dependent on the sample than on the calibrators. The influences of haemolysis, bilirubin, acetoacetate, albumin, lipids, glucose, and some cephalosporins have been evaluated with spiked human sera. Haemolysed, turbid and jaundiced patient samples have been analyzed as well. The results vary according to the analytical procedure. This study took place in the implementation of a selected method for routine purpose with special regards to interferences and an acceptable imprecision. The method must satisfy the physicians' demands in the renal function exploration, especially in kidney-transplant patients.