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

Abstract

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.

Introduction

In clinical chemistry, enzymes are mostly measured in terms of their catalytic activities for reason of low cost and high speed. Catalytic activities depend not only on the amount of active enzyme present, but also on a number of other factors, such as the nature and concentration of the substrate(s) used, cofactors, effectors, pH, temperature, ionic strength of the reaction medium, etc. Thus, the numerical results of catalytic activity assays are method-dependent, and the comparison of results continues to cause problems for their clinical interpretation, as well as for results obtained in external quality assessment schemes. Two approaches to standardization in clinical enzymology can be considered. The first one has been the development of reference methods by the Expert Panel on Enzymes of the International Federation of Clinical Chemistry (IFCC). At present, reference methods are available for GGT (1), ALP (2), ALT (3), AST (4), CK (5), LD (6), and soon for amylase. This effort of standardization has had considerable success in improving the quality of enzyme results as attested by numerous external quality control assessments. However, it must be recognized that the comparability of results is not achieved, as is shown by the variety of methods of measurement still in use in clinical laboratories. To illustrate this, at least 21 different substrates are currently distributed for the measurement of catalytic activity of alpha-amylase. Considering also that most of the measurement procedures can be performed at different temperatures, about 60 different procedures are used in the clinical laboratories for the assay of amylase catalytic activity.

A complementary approach can be proposed to achieve standardization and improve comparability of results in clinical enzymology, i.e., the use of an enzyme certified reference material with value assigned by a reference method. A such material can be used to calibrate one or more methods, including routine methods. The aim of this article is to present some data demonstrating the feasibility, the interest, and the limits of the intermethod calibration approach to standardize enzyme catalytic activity assays. Considerable progress have been made in enzyme technology during the past two decades 7, 8. Various possibilities have been explored to produce, purify, stabilize several enzymes. Gene transfer technology was successfully used to prepare human recombinant GGT 9, 10, pancreatic lipase 10, 11, ALT, ALP, and CK (10). Limited enzymatic proteolysis was retained to remove the hydrophobic domain of some membrane enzymes (12), saturation by pyridoxal phosphate was used to stabilize ALT (13), as was the addition of thiol agents to protect CK (14) and albumin for lipase (15). Currently, several enzymes have been purified and stabilized without significant alteration of their catalytic activities. Some of them have been certified at the international level (Table 1). For example, their stability is longer than 10 years when the lyophilisates are stored at −20 °C (7). Reference systems are now available for ALT, CK, GGT, ALP, LD and soon will be for amylase. In our meaning, a reference system consists in the combination of a certified reference material (CRM) with value assigned by a reference method, allowing assignment of a numerical value of a defined enzyme catalytic activity to a material. This system has to be recognized at the international level. However, these useful monospecific enzyme CRMs are expensive to produce, available in limited amounts, and cannot be used routinely to calibrate enzyme assays. Consequently, other materials are needed to transfer accuracy and insure comparability of results in clinical enzymology. The first goal of WG-CCE was to produce a guideline for the validation of calibrators in clinical enzymology (16).