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.

Preparation of enzyme calibration materials.

Standardisation in clinical enzymology needs not only reference methods but also reference materials. While single-enzyme reference enzymes have been developed, a multienzyme certified reference material (MECRM) available in high amount remains to be produced. To transfer trueness from the value of the reference system to patients' results, validated enzyme calibrators (EC) are also needed. Both the MECRM and the ECs must exhibit the same catalytic properties as the corresponding enzymes in human plasma. Moreover, commutability of these materials with patients' samples must be experimentally tested for one or a set of methods defined by an analytical specificity equal to that of the reference method. Various experimental studies have shown that the commutability of an enzyme material depends on the source of enzyme and its purification process, the matrix (including cofactors, effectors, additives, stabilisers... ) and the mode of processing of the final material. To promote intermethod calibration in clinical enzymology, a collaborative programme between the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) and IFCC corporate members is in progress for the development of a MECRM containing amylase, ALT, AST, ALP, CK, GGT, LDH, and lipase and exhibiting a wide and defined commutability.

Catalytic properties and stability of lipase purified from human pancreatic juice.

Catalytic properties of a preparation of human pancreatic lipase purified from pancreatic juice have been compared to those of the enzyme present in pooled plasma from patients suffering from acute pancreatitis. They were very similar as regards influence of effectors (sodium deoxycholate, colipase and Ca2+), optimal pH and apparent KM in optimized conditions. The stability of the preparation appeared to be satisfactory. It was found to be stable for at least 200 days in a liquid form at +4 degrees C and predictive degradation rates per year of the lyophilized form at +4 degrees C and -20 degrees C were 0.06% and 0.00%, respectively. The close similarity of properties of this preparation with those of a recombinant human pancreatic lipase produced in V79 Chinese hamster lung cells suggests that both approaches (purification from human pancreatic juice and gene transfer technology) could be used to produce a suitable reference material for this enzyme.

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.

In vivo evaluation of a reverse water-in-fluorocarbon emulsion stabilized with a semifluorinated amphiphile as a drug delivery system through the pulmonary route.

The potential of a reverse water-in-fluorocarbon (w-in-FC) emulsion stabilized with a semifluorinated amphiphile, namely C8F17(CH2)11OP(O)[N(CH2CH2)2O]2 (F8H11DMP) for drug delivery through intrapulmonary administration was investigated in the mouse. This study involved assessment of the effect of single or repeated intranasal instillations of a plain emulsion on lung tissue integrity, and evaluation of blood glucose levels in mice treated with an insulin-loaded emulsion. When instilled intranasally to mice, the plain emulsion did not alter lung tissue integrity, as demonstrated by histological staining, and did not induce any airway inflammatory reaction. Treated mice exhibited decreased body weight within the 3-4 days that followed the first emulsion administration, but this decrease was reversible within few days. Mice instilled intranasally with the insulin-loaded emulsion displayed decreased blood glucose levels within the 20 min that followed the administration, thus demonstrating the potential of the reverse w-in-FC emulsion stabilized with F8H11DMP to systemically deliver drugs, including peptides, upon lung administration.

[Human pancreatic lipase activity: review of methods and general recommendations]. / L'activité de la lipase pancréatique humaine: revue des méthodes et recommandations générales.

The properties of human pancreatic lipase are described, especially as regards the influence of the kind and the presentation of the substrate, as well as the effects of bile pigments and colipase. The authors have made a classification of the described methods for the determination of lipase activity in serum or plasma and have proposed recommendations for this assay.

[Microanalysis on dry reactive supports]. / Microanalyse sur supports de réactifs secs.

Among actual technological developments in clinical chemistry, the use of dry reagents fixed on a solid support is particularly advantageous for assays in emergency. Reflectometry is used for the measurement and was analysed in terms of principles and measurement devices. To day, only one apparatus Seralyser is furnished by Ames and we have evaluated its performances. However, other apparatus are in evaluating. Thus, Ektachem Kodak 400, 700 or DT 60 employs multilayer slides. Reflotron from Boehringer allows the direct use of blood samples. Stratus with a fluorimetrie measurement is adapted to drug assays. This new technology some permits interesting applications derived from basic research but arises some problems for the realisation of the as well as the role of the clinical chemist.

[A progress in the standardization in clinical enzymology using calibrators adapted to several techniques]. / Un progrès dans la standardisation en enzymologie clinique par l'utilisation de calibrateurs adaptés a plusieurs techniques.

Results in enzymology obtained in routine conditions, differ considerably according to the measurement procedures, and the use of conversion factors is not an advisable solution. Some studies show that between-laboratory agreement of results can be improved by using validated enzyme calibrators. The conditions, which are required to define a strategy for the development of such calibrators, are described in a first part. The example of lipase activity, which is measured in routine conditions with important between-method discrepancy, is studied in a second part. This example emphasised the need of an a priori control of the validity of the calibrators. Under these conditions, between-method agreement is in fact considerably improved. With the collaboration of manufacturers for the development of validated enzyme multicalibrators, it will be possible to improve the efficiency of the information transmitted by clinical chemists to clinicians. Thus, enzyme activities measurements could benefit from the same improvement as immunoassay of proteins with the use of CRM 470 by manufacturers to calibrate their standards.

[Interference of hemolysis on haptoglobin determined by kinetic immunonephelometry and comparison between phenotypes]. / Interférence de l'hémolyse sur la détermination de l'haptoglobine en immunonéphélémétrie cinétique et comparaison selon les phénotypes.

The aim of the study was to check if a slight and non visible hemolysis to naked eye such as that induced by blood coagulation could interfere in the immunonephelometric measurement of haptoglobin, and if this interference was dependent on the protein phenotype. Results confirmed that blood coagulation induced a non visible hemolysis whose intensity markedly varied from one specimen to another. Under our conditions (kinetic measurement with a Beckman Coulter immunonephelometer), we observed with the sera a negative interference linked to the hemolysis induced by blood coagulation when compared to corresponding plasmas obtained with lithium heparinate (p < 0,005). It was checked also that this anticoagulant did not induce a positive interference. Hemolysis interference was not found phenotype dependent. These results lead us to recommend to perform haptoglobin measurements on heparinised plasmas.

[Harmonization of practices: application to the measurement of enzymatic activities used in prevention, screening, diagnosis and therapeutic monitoring]. / Harmonisation des pratiques: application à la mesure d'activités enzymatiques utilisées en prévention, dépistage, diagnostic et surveillance thérapeutique.

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.

[1995: a decisive progress for the interlaboratory transferability of results by using enzyme and protein reference materials]. / 1995: un progrès décisif dans la transférabilité interlaboratoire des résultats grâce aux matériaux de référence d'enzymes et de protéines.

Transferability of results in clinical enzymology and immunochemistry may be improved by using validated calibrators. For that purpose, certified reference materials (CRM) have been developed. Authors emphasize the need to make known and to use these tools properly, and on the potential efficiency of this approach in immunochemistry with CRM 470, certified for 14 plasma proteins and with enzyme CRMs produced at the international level. They recall the necessity to validate calibrators for specified techniques of measurement used in routine. They also indicate that this approach, if properly conducted, should allow to ensure the transferability of results in enzymology and immunochemistry and thus the clinical efficiency of laboratory results.

Spectrophotometric determination of lipase activity in the presence of increased triolein concentration.

A novel approach for the determination of pancreatic lipase (EC 3.1.1.3) activity by a spectrophotometric method is described. Enzyme activity is measured in the presence of 1 mmol/l triolein, a concentration much higher than usually employed in turbidimetry. The primary reaction medium is optimized as regards bile salt and colipase concentrations. The released fatty acids are enzymatically determined via a secondary reaction scheme using the constituents of a commercially available kit. The proposed method is easy to perform and may prove useful in determining lipase activity of standard lipase preparations, which is required for indirect assays. In addition to satisfactory precision and linearity as well as close correlation with other lipase assays, the procedure described in the present paper by-passes a number of short-comings (eg uncontrolled increase in absorbance) inherent to the turbidimetric methods.

[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.

[Influence of pyridoxal phosphate in measuring aminotransferases activities in patients with viral hepatitis]. / Effet du phosphate de pyridoxal dans la mesure des activités aminotransférases chez les patients avec hépatite virale.

Effect of a pyridoxal phosphate (PP) supplementation of reagents used for ALT and AST measurement was studied in serum of 20 patients suffering from viral hepatitis. Measurements of enzyme activities were carried out at 37 degrees C, using an automate (AU 600, Olympus). Significant differences (p < 0.0001) were observed both for ALT and AST, meanwhile they were more marked for ALT than for AST. This difference was associated with a strong interindividual variability regarding PP activation effect on ALT. In conclusion, aminotransferase measurements should be carried out with a reagent supplemented with PP, when the enzyme activity is used to evaluate a cytolysis. The same recommendation applies when ALT results are integrated into various combinations developed for the evaluation of liver status.

Reference materials in clinical enzymology: preparation, requirements and practical interests.

Five enzyme materials (gamma-glutamyltransferase, alkaline phosphatase, creatine kinase, alanine aminotransferase and prostatic acid phosphatase) are currently certified using a reference method. Furthermore, feasibility studies have been performed for four other enzymes (aspartate aminotransferase, lactate dehydrogenase, amylase and lipase). They indicated that these enzymes can be purified and stabilized, but the materials have not yet been certified. This shows that the most important enzymes in clinical laboratories can be purified, and stabilized, without significant alteration of their catalytic properties. By carefully choosing a matrix, the commutability of these enzyme preparations and patients' samples between some methods, including routine methods, may be preserved. Thus, these materials can be used to calibrate the routine methods in terms of the corresponding reference methods after commutability has been verified. Current studies suggest that this objective can be reached, provided three criteria are satisfied: i) the calibrated and reference methods must be of equal specificity; ii) the enzyme calibrator should be, as closely as possible, identical to the human analyte enzyme in its native matrix (eg serum); iii) and the inter-method ratio should be constant (within the limits of experimental error) for the enzyme calibrator and for all patients' samples.

[Enzyme calibrators: principle and practical use]. / Le calibrage en enzymologie clinique: principe, conditions d'application et résultats.

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.

Diagnostic accuracy of intra-articular C-reactive protein assay in periprosthetic knee joint infection--a preliminary study.

BACKGROUND: Periprosthetic joint infection often raises diagnostic challenges, as the published criteria are heterogeneous. New markers for predicting periprosthetic infection have been evaluated. Here, we assessed one of these markers, C-reactive protein (CRP), in joint fluid. HYPOTHESIS: We hypothesised that intra-articular CRP levels would perform better than serum CRP concentrations in diagnosing knee prosthesis infection. PATIENTS AND METHODS: We prospectively included 30 patients including 10 with native-knee effusions, 11 with prosthetic-knee aseptic effusions, and 11 with prosthetic-knee infection defined using 2011 Musculoskeletal Society criteria. Serum CRP was assayed using turbidimetry or nephelometry and intra-articular CRP using nephelometry. Appropriate statistical tests were performed to compare the three groups; P values < 0.05 were considered significant. RESULTS: Serum and intra-articular CRP levels were 5- to 16-fold higher in the group with periprosthetic infection than in the other two groups. Although the areas under the ROC curves were not significantly different, the likelihood ratios associated with the selected cut-offs suggested superiority of intra-articular CRP: a value > 2.78 mg/L suggested possible infection (100% sensitivity and 82% specificity) and a value > 5.37 mg/L probable infection (90% sensitivity and 91% specificity). DISCUSSION: Our findings suggest a possible role for intra-articular CRP assay in diagnosing knee prosthesis infection and perhaps periprosthetic infection at any site. LEVEL OF EVIDENCE: Level III, diagnostic study, development of a diagnostic criterion in consecutive patients comparatively to a reference standard.