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Preparation of enzyme calibration materials
Clinica Chimica Acta 278 (1998) 151–162
Preparation of enzyme calibration materials *, Jean-Marc Lessinger ´ Georges Ferard ´ , UFR des Sciences Pharmaceutiques, Universite´ Louis Pasteur de Laboratoire de Biochimie Appliquee ´ , France Strasbourg, 74 route du Rhin, B.P. 24, 67401 Illkirch Cedex
Abstract 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. 1998 Elsevier Science B.V. All rights reserved. Keywords: Purification; Homogeneity; Stability; Commutability; Catalytic properties; Certified reference material
Abbreviations: ACP, acid prostatic phosphatase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; ´ ´ AST, aspartate aminotransferase; BCR, Bureau Communautaire de Reference; CK, creatine kinase; CRM, certified reference material; EC, enzyme calibrator; GGT, gamma glutamyltransferase; IFCC, International Federation of Clinical Chemistry and Laboratory Medicine; IRMM, Institute for Reference Materials and Measurements; LDH, lactate dehydrogenase; MECRM, multienzyme certified reference material; RM, ´ ´ Franc¸aise de Biologie Clinique reference material; SFBC, Societe *Corresponding author. 0009-8981 / 98 / $ – see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00141-7
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´ , J. Lessinger / Clinica Chimica Acta 278 (1998) 151 – 162 G. Ferard
1. Introduction The continuous introduction of new procedures and analysers for the measurement of catalytic activity concentration of enzymes induces a wide dispersion of numerical values produced by the laboratories. Consequently, interpretation of results by the clinicians remains difficult. Recommended methods of measurement have been developed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to harmonize the results in clinical enzymology but they have not been implemented to any desirable extent. In addition, several enzyme reference materials (RM) have been produced, mostly by the Community Bureau of Reference (BCR, European Union, Brussels) and certified by the IFCC reference methods. These preparations are available as single-enzyme lyophilized materials. The combination of a unit of catalytic activity, an authoritative reference procedure of measurement and a reference material forms a reference system for a given quantity, here the catalytic activity concentration of a defined enzyme. It has already been demonstrated that at least four enzyme certified reference materials (CRM) could serve as enzyme calibrators (ECs) for several methods, including routine methods of measurement [1]. In this way, it was possible to assess and transfer trueness from the reference system to patients’ results and to establish traceability of results [2] (Fig. 1). This approach was named intermethod
Fig. 1. Model for the transfer of trueness from the reference system (see text) to patients’ results. CRM and validated products calibrators can be used for this purpose.
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calibration [3] and conditions for the validation of an EC were recently described by the IFCC working group ‘Calibrators in Clinical Enzymology’ [4]. However, enzyme CRMs are only available in limited amounts. Furthermore, the dependence of some new analytical systems on calibrators for enzyme activity measurements is more and more frequent. Thus, the demand for validated ECs is expected to increase with an improved standardisation in clinical enzymology. The objective of this paper is to review the possibilities and requirements for the preparation of materials intended to be used as EC.
2. Terminology
2.1. Catalytic activity of an enzyme Property of a biological component measured by the catalyzed substance rate of conversion of a given reaction under specified conditions. The coherent SI unit is mole per second (mol ? s 21 ), also called katal (kat). Another noncoherent unit (‘international unit’) is mmol per min and is symbolized by U.
2.2. Reference method Method which after exhaustive investigation has been shown to have negligible inaccuracy in comparison with its imprecision [5].
2.3. Reference material ( RM) Material or substance one or more of whose property values are sufficiently homogeneous and well established to be used for the calibration of an apparatus, the assessment of a measurement method, or for assigning values to materials [5,6].
2.4. Certified reference material ( CRM) Reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure which establishes its traceability to an accurate realization of the unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence [5]. All CRMs lie within the definition of ‘measurement standards’ or ‘etalons’ given in the ‘International Vocabulary of basic and general terms in Metrology’ [5,6].
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2.5. Traceability Property of the result of a measurement or the value of a standard whereby it can be related to stated references, usually national or international standards, through an unbroken chain of comparisons all having stated uncertainties [5].
2.6. Uncertainty of measurement Parameter associated with the result of a measurement, that characterizes the dispersion of values that could reasonably be attributed to the measurand [5,7].
2.7. Commutability Ability of a material to show interassay properties comparable to those of human sera [8]. Another, somewhat wider definition has been proposed: ability of a material to yield the same numerical relationships between results of measurements by a given set of measurement procedures, purporting to measure the same quantity as those between the expectations of the relationships obtained when the same procedures are applied to other relevant types of material [9].
2.8. Calibrator Any substance, material intended to be used to establish the value(s) of one or more quantities.
2.9. Product calibrator This material is a single or multicalibrator in an appropriate matrix, certified by a reference method against the CRM. The commutability of the product calibrator for one or a set of procedures of measurement must be experimentally verified. The certification of the product calibrator is the responsibility of the manufacturer.
2.10. Set of measurement procedures All measurement procedures exhibiting the same analytical specificity for a given quantity.
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3. General remarks Some general remarks may be formulated to prepare enzyme materials intended to be used as calibrators. They concern the sources of the enzymes, the procedures of purification, the characterization of the materials, the evaluation of their stability, homogeneity and commutability, and their certification to assign a value by using a reference method of measurement. An EC, like a CRM, should be accompanied by a certificate indicating its main characteristics. A user can also ask the producer for more details. The content of the certificate is important to consider because it allows a proper use of ECs [10,11].
4. Enzyme sources Enzyme sources are until now either animal or human (Table 1). Animal tissues were the first sources used to prepare enzyme RM. The approach was convenient for the purification of GGT, ALP and ALT reference materials from pig tissues since it was demonstrated that their catalytic properties were similar to those of the corresponding enzyme in human samples [12–14]. Then, human tissues, cells, or secretions were most often used. However, due to ethical or legal problems, human tissues have a limited availability. They must also be handled with caution because of potential viral contaminations. Human gene transfer technologies were also successfully developed to prepare most of the enzymes relevant in clinical enzymology (GGT, lipase, AST, ALT, CK-MM, Table 1 Some BCR-RMs certified for one enzyme catalytic activity Enzyme
Code (CRM)
Organ
Form
Animal origin g-Glutamyltransferase Alkaline phosphatase Alanine aminotransferase
319 371 426
Pig kidney Pig kidney Pig heart
Light subunit Liver-bone-kidney Cytoplasmic isoenzyme
Human origin Creatine kinase Lactate dehydrogenase Acid phosphatase a-Amylase Creatine kinase Lipase a
299 404 410 476 608 675
Placenta Erythrocytes Prostate Pancreas Heart Pancreatic juice and human recombinant
Isoenzyme 1 Isoenzyme 1 Prostatic isoenzyme Pancreatic isoenzyme Isoenzyme 2 ‘Pancreatitis’ isoform
a
Only feasibility study, work in progress. ASAT was prepared from human erythrocytes by NIST; no more available.
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LDH-1 . . . ) by different groups [15–20]. This approach has the great advantage of ensuring batch-to-batch reproducibility [20]. As a conclusion, the source of enzyme is fundamental to obtain commutable materials. More and more often, enzyme CRMs and ECs will be of human source, and human recombinant technology is a promising approach for the production of ECs. Note that the source of enzyme(s) must be indicated in the certificate [10].
5. Purification procedures Purity of a material should be such that no substances are present which will interfere with the later intended measurements [11]. Usually, a high degree of purification is required for a CRM to obtain a well defined composition of the material. Difficulties of purifying enzymes have been long recognized. However, it was possible to purify several enzymes without significant alteration of their catalytic properties. Various amongst classical purification procedures were employed, most often in combination: precipitation by different reagents, mainly ammonium sulphate; chromatography on ion exchanger, gel filtration, preparative isoelectric focusing (Table 2). More recently, affinity chromatography with immobilized anti-enzyme monoclonal antibodies or competitive inhibitors was used to purify almost in one step some enzymes such as ACP [21] and lipase [20]. Affinity chromatography was also efficient for the removal of residual contaminating substances, including other enzyme catalytic activities [12,14]. Various compounds such as thioprotective agents [22,23], protease inhibitors, coenzyme such as pyridoxal phosphate [14], and albumin (bovine or human) have been added to improve the stability of enzymes during and after the purification process. The purity of a preparation may be checked by measuring the activities of potential enzyme contaminants, by polyacrylamide gel electrophoresis and by blotting transfer. When considering the purification procedures described in the BCR certification reports, their diversity is remarkable, demonstrating that several enzymes can be today partially or extensively purified as well as other proteins, provided that adapted procedures are carefully chosen. Table 2 Purification procedures for enzymes Selective precipitation Gel filtration Ion exchange chromatography Preparative isoelectric focusing Affinity chromatography
ALT, GGT, lipase . . . LD-1, amylase . . . LD-1, ALP, CK-2 . . . Lipase ACP, ALP, ALT, adenosine deaminase . . .
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6. Characterization of the purified product Molecular properties of the purified enzymes such as relative molecular mass, average isoelectric point should be compared to those of the corresponding enzyme in patients’ samples. Furthermore, catalytic properties such as the value of the optimal pH and apparent affinity constants of ECs, have also to be determined. Similarity in the catalytic properties between an EC and the corresponding enzyme in patients’ samples is a prerequisite for commutability of a material. This was well illustrated for a candidate RM of lipase and for some ECs [24,25]. As measurements may be carried out at different temperatures (30 or 37 8C), the effect of temperature on RMs and ECs must be compared to that of the corresponding enzyme catalytic activity in patients’ samples. The temperature effect is not only dependent on the enzyme, but also on the method of measurement [26]. It was demonstrated that amylase CRM (CRM476, BCR) from human pancreas has the same thermodependency in the presence of different substrates as plasma samples from patients suffering from acute pancreatitis [27]. Lessinger et al. have observed the same fact for GGT and ALP CRMs from BCR [28]. They also reported that many enzyme commercial preparations (calibrants and control materials) differed in their thermodependency in comparison with patients’ samples for three enzyme catalytic activities (amylase, ALP, GGT). Thus, these materials cannot be used as calibrators for methods carried out at 30 and at 37 8C. Also, such materials have limited value to evaluate interlaboratory coherency of results in clinical enzymology.
7. Choice of the matrix The choice of an adequate matrix is essential for stability and commutability of the materials. Because the purified enzyme is present at low mass concentration, albumin, bovine or human, is often chosen and constitutes the basis of the matrix. A buffer is also added to improve stability. Lipase is a good example that illustrates the importance of albumin (Fig. 2a and b). For this enzyme, low concentrations of bovine or human albumin are necessary for stability, but high concentrations (about 40 g / l) are generally chosen to mimic human serum, and thus to have commutable CRMs. The addition of albumin in sufficient amount is also required to give between-vial homogeneity. The nature of the buffer is also important to preserve catalytic properties and it is often a major point for ensuring commutability. Amylase CRM is a good example to illustrate this [27,29]. It has also been proposed to retain a natural matrix to prepare enzyme CRMs, for example a bovine or human serum-based matrix. A complete description of the used matrix, including buffer, effectors, stabilizers should be given in the certificate [10].
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Fig. 2. (a) Effect of temperature and albumin on the stability of human pancreatic lipase. (b) Effect of albumin concentration on the stability of human pancreatic lipase.
8. Viscosity Viscosity should be within the usual range for human sera at 37 8C i.e. 1.25 to 1.35 centipoise. This is particularly relevant for RMs and ECs used in automated procedures. Consequently, this information should be furnished to the users in the certificate because it can avoid some misuse of CRMs and ECs. However, we must recognize that information on viscosity is often lacking.
9. Stability Stability of CRMs and ECs must be clearly defined in terms of time delay and storage conditions. This information must be included in the certificate. Stability should be evaluated in liquid and solid forms (if relevant). As the temperature is
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Table 3 Estimated stability of some CRMs (stored in a solid form at 2 20 8C) GGT ALP ALT CK-2 LD-1 ACP Amylase
25 years 33 years . 50 years 10 years . 50 years . 50 years . 50 years
Estimated by accelerated degradation tests (ref. [30]).
expected to be the main factor influencing the stability of an enzyme CRM, the degradation rate is usually predicted using the Arrhenius model. For that purpose, the material is stored at different temperatures, for example at 2 20, 1 4, 1 20, 1 37 and 1 56 8C. A predicted degradation rate is derived from the Arrhenius equation [27,30]. Using this approach, the stability of enzyme CRMs is most often longer than 10 years when stored in a solid form at 2 20 8C (Table 3). Stability after reconstitution of material must also be documented. A multienzyme preparation in a frozen liquid form has been proposed as EC. Activities of this preparation were stable for at least 4 years at 2 20 8C and for 7 days at 5 8C. Apparently, it demonstrates that the preparation of a MECRM or a multienzyme calibrator is possible. However, the commutability of such a material remains to be evaluated extensively to define more precisely the interest of using this kind of material to calibrate a set of methods.
10. Homogeneity Every material is to some extent inhomogeneous. Thus, it is necessary to evaluate inhomogeneity between units of a batch for the certified quantity. For this purpose, a method of measurement highly repeatable rather than accurate must be chosen [10]. At first glance, homogeneity seems not to be a crucial point because the filling process and lyophilisation are today well controlled. However, between-vials homogeneity must be carefully evaluated and must be taken into account to evaluate uncertainty.
11. Additional remarks The certificate which accompanies an enzyme CRM should contain all the information which is essential for correct use of the CRM i.e. the certified value with the corresponding uncertainty, and the method(s) used to determine the
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certified value(s), also details concerning the way in which the material should be stored (temperature, exposure to light), transported, the mode of reconstitution (if any) and delay before use (if relevant) must be extensively described, as well as other data concerning the CRM stability [10]. The certificate must also indicate the intended use of the material, for example to evaluate the comparability of results from laboratories using the measurement procedure retained for the certification of the material. Information on the safety of a CRM should also be provided to the user, also potential biological dangers. When commutability studies have been performed, information concerning the methods of measurement for which the CRM is commutable should also be provided. This is essential not only to promote intermethod calibration, but also to allow a correct use of this approach. The same applies to control materials when they are used for the control of trueness.
12. Description of the IFCC MECRM project The IFCC working group ‘Calibrators in Clinical Enzymology’ has a fourstep project. The first is to revise IFCC recommended methods to 37 8C; secondly, monoenzyme CRMs will be recertified at 37 8C with these methods by several laboratories. Thirdly, two single materials (ALP and AST) will be prepared and certified at 37 8C. Finally, taking into account the available information presented in the BCR certification reports, the members of IFCC working group ‘Calibrators in Clinical Enzymology’ have the project to prepare in collaboration with IRMM an MECRM containing eight purified enzymes: amylase, ALT, AST, ALP, CK, GGT, LDH, and lipase. Activity concentrations will be near to those of the single-material CRM from BCR. Also the choice concerning the sources of enzymes will be directed according to the same principles, except when significantly improved sources have been recently identified. Matrix will be constituted by buffered bovine albumin, added with some effectors. Beside purity and stability, commutability will be extensively evaluated in order to promote an intercalibration approach. Implementation of IFCC recommended methods in the laboratories participating in the certification campaign will be assessed by using single-enzyme CRMs when available.
13. Conclusions It is now possible to purify enzymes without significant alteration of their catalytic properties. Stability was shown to be satisfactory since it was evaluated to at least 10 years when stored at 2 20 8C in a solid form. However, these single enzyme CRMs are expensive and available in limited amounts. It is
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expected that in the future, a MECRM exhibiting a wide commutability will be prepared and that recommendation concerning the proper use of this material will be provided to the users to improve standardisation in clinical enzymology.
References ´ [1] Ferard G, Edwards J, Kanno T, Lessinger JM, Moss DW, Schiele F, Tietz NW, Vassault A. Interassay calibration as a major contribution to the comparability of results in clinical enzymology. Clin Biochem, 1998;31:489–94. ´ [2] Lessinger JM, Ferard G, Grafmeyer D, et al. Usefulness of reference materials in calibration of enzyme activities. Eur J Clin Chem Clin Biochem 1995;33:858–64. [3] Moss DW. Enzyme reference materials. Their place in diagnostic enzymology. JIFCC 1994;6:6–9. ´ [4] Ferard G, Edwards J, Kanno T, Lessinger JM, Moss DW, Schiele F, Tietz NW, Vassault A. Validation of an enzyme calibrator — an IFCC guideline. Clin Biochem, 1998;31:495–500. [5] International Vocabulary of Basic and General Terms in Metrology, 2nd ed. ISO, 1993. [6] ISO guide 32. Calibration in analytical chemistry using certified reference materials (in press). [7] Guide for the expression of uncertainty in measurement (GUM). BIPM, 1993. [8] Fasce CF, Rej R, Copeland WH, Vanderlinde RE. A discussion of enzyme reference materials: application and specifications. Clin Chem 1973;19:5–9. [9] Dybkaer R. Vocabulary for use in measurement procedures and description of reference materials in laboratory medicine. Eur J Clin Chem Clin Biochem 1997;35:141–73. [10] Contents of certificates of reference materials, ISO Guide 31, 1st ed. ISO, 1981. [11] Guidelines for the production and certification of BCR reference materials. Part A. Recommendations to proposers of reference material projects. Doc BCR / 01 / 97, Standards, Measurements and Testing Programme. [12] Schiele F, Muller J, Colinet E, Siest G. Production and certification of an enzyme reference material for g-glutamyltransferase (CRM 319). Part 1: Preparation and characterization. Clin Chem 1987;33:1971–7. [13] Schiele F, Muller J, Colinet E, Siest G. Certification of an enzyme reference material for alkaline phosphatase (CRM371). Clin Biochem 1991;24:159–68. [14] Schiele F, Muller J, Colinet E, et al. Interlaboratory study of the IFCC method for alanine aminotransferase performed with use of a partly purified reference material. Clin Chem 1992;38:2365–71. [15] Eto A, Shiki A, Chikaura Y, Oka T, Nakano NI. Multienzyme control serum (Seraclear-HE) containing human enzymes from established cell lines and other sources. 1: Preparation and properties. Clin Chem 1995;41:872–80. [16] Nakano NI, Eto A, Chikaura Y, Shiki A. Multienzyme control serum (Seraclear-HE) containing human enzymes from established cell lines and other sources. 2: Evaluation as candidate working enzyme reference material for alanine and aspartate aminotransferase. Clin Chem 1995;41:881–91. [17] Marui Y, Yayashi C, Matsuda Y, et al. Multi-enzyme reference material from established human cell lines and human sources. Clin Chim Acta 1995;233:19–38. [18] Siest G, Oster T, Visvikis A, et al. Application of gene transfer technologies to the production of enzyme reference materials: example of g-glutamyltransferase. Clin Chem 1993;39:1573– 89.
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[19] Canalias F, Visvikis A, Thioudellet C, Siest G. Stable expression of enzymatically active human pancreatic lipase in V79 cells: purification and characterization of the recombinant enzyme. Clin Chem 1994;40:1251–7. [20] Oster T, Visvikis A, Schiele F, Wellman-Rousseau M, Siest G. Gene transfer technologies for the production of enzyme and protein reference materials. Clin Chim Acta 1997;257:3–23. [21] Francis JM, Moss DW, Colinet E, Calam DH, Bullock DG. A reference preparation of human prostatic acid phosphatase: purification, characterisation and field trials. Ann Clin Biochem 1992;29:176–83. ´ [22] Lessinger JM, Tavridou A, Arzoglou P, Ferard G. Interest of using a purified, stable and commutable preparation of human pancreatic lipase in indirect assays. Anal Lett 1992;25:1453–68. ¨ [23] Mathieu M, Steghens JP, Horder M, et al. A reference preparation for creatine kinase BB. Clin Chem 1993;39:1894–8. ´ [24] Lessinger JM, Ferard G, Mignot V, Calam DH, Gaines Das R, Dourson JL. Catalytic properties and stability of lipase purified from human pancreatic juice. Clin Chim Acta 1996;251:119–29. ´ [25] Lessinger JM, Dourson JL, Ferard G. Importance of standardization of lipase assays by using appropriate calibrators. Clin Chem 1996;42:1979–83. ´ [26] Lessinger JM, Dourson JL, Ferard G. Importance of the definition of catalytic properties for the commutability of an enzyme reference material: example of lipase. Fres J Anal Chem 1998;360:494–7. [27] Moss DW, Maire I, Calam DH, et al. Reference materials in clinical enzymology: preparation, requirements and practical interests. Ann Biol Clin 1994;54:189–98. ´ [28] Lessinger JM, Ferard G, Frei J, Grafmeyer D, Labbe´ D, Maire I, Mathieu M, Schiele F, Vassault A, Vialle A. Pratique quotidienne en enzymologie clinique: du danger de l’utilisation ´ a` la temperature ´ des facteurs de conversion lies de mesure. Ann Biol Clin 1998;56:359–61. [29] Gubern G, Canalias F, Gella FJ. Determination of a-amylase activity: methods comparison and commutability study of several control materials. Clin Chem 1995;41:435–8. [30] Tydeman MS, Kirkwood TBL. Design and analysis of accelerated degradation tests for the stability of biological standards. J Biol Stand 1984;12:195–224.
Preparation of enzyme calibration materials *, Jean-Marc Lessinger ´ Georges Ferard ´ , UFR des Sciences Pharmaceutiques, Universite´ Louis Pasteur de Laboratoire de Biochimie Appliquee ´ , France Strasbourg, 74 route du Rhin, B.P. 24, 67401 Illkirch Cedex
Abstract 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. 1998 Elsevier Science B.V. All rights reserved. Keywords: Purification; Homogeneity; Stability; Commutability; Catalytic properties; Certified reference material
Abbreviations: ACP, acid prostatic phosphatase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; ´ ´ AST, aspartate aminotransferase; BCR, Bureau Communautaire de Reference; CK, creatine kinase; CRM, certified reference material; EC, enzyme calibrator; GGT, gamma glutamyltransferase; IFCC, International Federation of Clinical Chemistry and Laboratory Medicine; IRMM, Institute for Reference Materials and Measurements; LDH, lactate dehydrogenase; MECRM, multienzyme certified reference material; RM, ´ ´ Franc¸aise de Biologie Clinique reference material; SFBC, Societe *Corresponding author. 0009-8981 / 98 / $ – see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00141-7
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´ , J. Lessinger / Clinica Chimica Acta 278 (1998) 151 – 162 G. Ferard
1. Introduction The continuous introduction of new procedures and analysers for the measurement of catalytic activity concentration of enzymes induces a wide dispersion of numerical values produced by the laboratories. Consequently, interpretation of results by the clinicians remains difficult. Recommended methods of measurement have been developed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to harmonize the results in clinical enzymology but they have not been implemented to any desirable extent. In addition, several enzyme reference materials (RM) have been produced, mostly by the Community Bureau of Reference (BCR, European Union, Brussels) and certified by the IFCC reference methods. These preparations are available as single-enzyme lyophilized materials. The combination of a unit of catalytic activity, an authoritative reference procedure of measurement and a reference material forms a reference system for a given quantity, here the catalytic activity concentration of a defined enzyme. It has already been demonstrated that at least four enzyme certified reference materials (CRM) could serve as enzyme calibrators (ECs) for several methods, including routine methods of measurement [1]. In this way, it was possible to assess and transfer trueness from the reference system to patients’ results and to establish traceability of results [2] (Fig. 1). This approach was named intermethod
Fig. 1. Model for the transfer of trueness from the reference system (see text) to patients’ results. CRM and validated products calibrators can be used for this purpose.
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calibration [3] and conditions for the validation of an EC were recently described by the IFCC working group ‘Calibrators in Clinical Enzymology’ [4]. However, enzyme CRMs are only available in limited amounts. Furthermore, the dependence of some new analytical systems on calibrators for enzyme activity measurements is more and more frequent. Thus, the demand for validated ECs is expected to increase with an improved standardisation in clinical enzymology. The objective of this paper is to review the possibilities and requirements for the preparation of materials intended to be used as EC.
2. Terminology
2.1. Catalytic activity of an enzyme Property of a biological component measured by the catalyzed substance rate of conversion of a given reaction under specified conditions. The coherent SI unit is mole per second (mol ? s 21 ), also called katal (kat). Another noncoherent unit (‘international unit’) is mmol per min and is symbolized by U.
2.2. Reference method Method which after exhaustive investigation has been shown to have negligible inaccuracy in comparison with its imprecision [5].
2.3. Reference material ( RM) Material or substance one or more of whose property values are sufficiently homogeneous and well established to be used for the calibration of an apparatus, the assessment of a measurement method, or for assigning values to materials [5,6].
2.4. Certified reference material ( CRM) Reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure which establishes its traceability to an accurate realization of the unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence [5]. All CRMs lie within the definition of ‘measurement standards’ or ‘etalons’ given in the ‘International Vocabulary of basic and general terms in Metrology’ [5,6].
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2.5. Traceability Property of the result of a measurement or the value of a standard whereby it can be related to stated references, usually national or international standards, through an unbroken chain of comparisons all having stated uncertainties [5].
2.6. Uncertainty of measurement Parameter associated with the result of a measurement, that characterizes the dispersion of values that could reasonably be attributed to the measurand [5,7].
2.7. Commutability Ability of a material to show interassay properties comparable to those of human sera [8]. Another, somewhat wider definition has been proposed: ability of a material to yield the same numerical relationships between results of measurements by a given set of measurement procedures, purporting to measure the same quantity as those between the expectations of the relationships obtained when the same procedures are applied to other relevant types of material [9].
2.8. Calibrator Any substance, material intended to be used to establish the value(s) of one or more quantities.
2.9. Product calibrator This material is a single or multicalibrator in an appropriate matrix, certified by a reference method against the CRM. The commutability of the product calibrator for one or a set of procedures of measurement must be experimentally verified. The certification of the product calibrator is the responsibility of the manufacturer.
2.10. Set of measurement procedures All measurement procedures exhibiting the same analytical specificity for a given quantity.
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3. General remarks Some general remarks may be formulated to prepare enzyme materials intended to be used as calibrators. They concern the sources of the enzymes, the procedures of purification, the characterization of the materials, the evaluation of their stability, homogeneity and commutability, and their certification to assign a value by using a reference method of measurement. An EC, like a CRM, should be accompanied by a certificate indicating its main characteristics. A user can also ask the producer for more details. The content of the certificate is important to consider because it allows a proper use of ECs [10,11].
4. Enzyme sources Enzyme sources are until now either animal or human (Table 1). Animal tissues were the first sources used to prepare enzyme RM. The approach was convenient for the purification of GGT, ALP and ALT reference materials from pig tissues since it was demonstrated that their catalytic properties were similar to those of the corresponding enzyme in human samples [12–14]. Then, human tissues, cells, or secretions were most often used. However, due to ethical or legal problems, human tissues have a limited availability. They must also be handled with caution because of potential viral contaminations. Human gene transfer technologies were also successfully developed to prepare most of the enzymes relevant in clinical enzymology (GGT, lipase, AST, ALT, CK-MM, Table 1 Some BCR-RMs certified for one enzyme catalytic activity Enzyme
Code (CRM)
Organ
Form
Animal origin g-Glutamyltransferase Alkaline phosphatase Alanine aminotransferase
319 371 426
Pig kidney Pig kidney Pig heart
Light subunit Liver-bone-kidney Cytoplasmic isoenzyme
Human origin Creatine kinase Lactate dehydrogenase Acid phosphatase a-Amylase Creatine kinase Lipase a
299 404 410 476 608 675
Placenta Erythrocytes Prostate Pancreas Heart Pancreatic juice and human recombinant
Isoenzyme 1 Isoenzyme 1 Prostatic isoenzyme Pancreatic isoenzyme Isoenzyme 2 ‘Pancreatitis’ isoform
a
Only feasibility study, work in progress. ASAT was prepared from human erythrocytes by NIST; no more available.
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LDH-1 . . . ) by different groups [15–20]. This approach has the great advantage of ensuring batch-to-batch reproducibility [20]. As a conclusion, the source of enzyme is fundamental to obtain commutable materials. More and more often, enzyme CRMs and ECs will be of human source, and human recombinant technology is a promising approach for the production of ECs. Note that the source of enzyme(s) must be indicated in the certificate [10].
5. Purification procedures Purity of a material should be such that no substances are present which will interfere with the later intended measurements [11]. Usually, a high degree of purification is required for a CRM to obtain a well defined composition of the material. Difficulties of purifying enzymes have been long recognized. However, it was possible to purify several enzymes without significant alteration of their catalytic properties. Various amongst classical purification procedures were employed, most often in combination: precipitation by different reagents, mainly ammonium sulphate; chromatography on ion exchanger, gel filtration, preparative isoelectric focusing (Table 2). More recently, affinity chromatography with immobilized anti-enzyme monoclonal antibodies or competitive inhibitors was used to purify almost in one step some enzymes such as ACP [21] and lipase [20]. Affinity chromatography was also efficient for the removal of residual contaminating substances, including other enzyme catalytic activities [12,14]. Various compounds such as thioprotective agents [22,23], protease inhibitors, coenzyme such as pyridoxal phosphate [14], and albumin (bovine or human) have been added to improve the stability of enzymes during and after the purification process. The purity of a preparation may be checked by measuring the activities of potential enzyme contaminants, by polyacrylamide gel electrophoresis and by blotting transfer. When considering the purification procedures described in the BCR certification reports, their diversity is remarkable, demonstrating that several enzymes can be today partially or extensively purified as well as other proteins, provided that adapted procedures are carefully chosen. Table 2 Purification procedures for enzymes Selective precipitation Gel filtration Ion exchange chromatography Preparative isoelectric focusing Affinity chromatography
ALT, GGT, lipase . . . LD-1, amylase . . . LD-1, ALP, CK-2 . . . Lipase ACP, ALP, ALT, adenosine deaminase . . .
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6. Characterization of the purified product Molecular properties of the purified enzymes such as relative molecular mass, average isoelectric point should be compared to those of the corresponding enzyme in patients’ samples. Furthermore, catalytic properties such as the value of the optimal pH and apparent affinity constants of ECs, have also to be determined. Similarity in the catalytic properties between an EC and the corresponding enzyme in patients’ samples is a prerequisite for commutability of a material. This was well illustrated for a candidate RM of lipase and for some ECs [24,25]. As measurements may be carried out at different temperatures (30 or 37 8C), the effect of temperature on RMs and ECs must be compared to that of the corresponding enzyme catalytic activity in patients’ samples. The temperature effect is not only dependent on the enzyme, but also on the method of measurement [26]. It was demonstrated that amylase CRM (CRM476, BCR) from human pancreas has the same thermodependency in the presence of different substrates as plasma samples from patients suffering from acute pancreatitis [27]. Lessinger et al. have observed the same fact for GGT and ALP CRMs from BCR [28]. They also reported that many enzyme commercial preparations (calibrants and control materials) differed in their thermodependency in comparison with patients’ samples for three enzyme catalytic activities (amylase, ALP, GGT). Thus, these materials cannot be used as calibrators for methods carried out at 30 and at 37 8C. Also, such materials have limited value to evaluate interlaboratory coherency of results in clinical enzymology.
7. Choice of the matrix The choice of an adequate matrix is essential for stability and commutability of the materials. Because the purified enzyme is present at low mass concentration, albumin, bovine or human, is often chosen and constitutes the basis of the matrix. A buffer is also added to improve stability. Lipase is a good example that illustrates the importance of albumin (Fig. 2a and b). For this enzyme, low concentrations of bovine or human albumin are necessary for stability, but high concentrations (about 40 g / l) are generally chosen to mimic human serum, and thus to have commutable CRMs. The addition of albumin in sufficient amount is also required to give between-vial homogeneity. The nature of the buffer is also important to preserve catalytic properties and it is often a major point for ensuring commutability. Amylase CRM is a good example to illustrate this [27,29]. It has also been proposed to retain a natural matrix to prepare enzyme CRMs, for example a bovine or human serum-based matrix. A complete description of the used matrix, including buffer, effectors, stabilizers should be given in the certificate [10].
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Fig. 2. (a) Effect of temperature and albumin on the stability of human pancreatic lipase. (b) Effect of albumin concentration on the stability of human pancreatic lipase.
8. Viscosity Viscosity should be within the usual range for human sera at 37 8C i.e. 1.25 to 1.35 centipoise. This is particularly relevant for RMs and ECs used in automated procedures. Consequently, this information should be furnished to the users in the certificate because it can avoid some misuse of CRMs and ECs. However, we must recognize that information on viscosity is often lacking.
9. Stability Stability of CRMs and ECs must be clearly defined in terms of time delay and storage conditions. This information must be included in the certificate. Stability should be evaluated in liquid and solid forms (if relevant). As the temperature is
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Table 3 Estimated stability of some CRMs (stored in a solid form at 2 20 8C) GGT ALP ALT CK-2 LD-1 ACP Amylase
25 years 33 years . 50 years 10 years . 50 years . 50 years . 50 years
Estimated by accelerated degradation tests (ref. [30]).
expected to be the main factor influencing the stability of an enzyme CRM, the degradation rate is usually predicted using the Arrhenius model. For that purpose, the material is stored at different temperatures, for example at 2 20, 1 4, 1 20, 1 37 and 1 56 8C. A predicted degradation rate is derived from the Arrhenius equation [27,30]. Using this approach, the stability of enzyme CRMs is most often longer than 10 years when stored in a solid form at 2 20 8C (Table 3). Stability after reconstitution of material must also be documented. A multienzyme preparation in a frozen liquid form has been proposed as EC. Activities of this preparation were stable for at least 4 years at 2 20 8C and for 7 days at 5 8C. Apparently, it demonstrates that the preparation of a MECRM or a multienzyme calibrator is possible. However, the commutability of such a material remains to be evaluated extensively to define more precisely the interest of using this kind of material to calibrate a set of methods.
10. Homogeneity Every material is to some extent inhomogeneous. Thus, it is necessary to evaluate inhomogeneity between units of a batch for the certified quantity. For this purpose, a method of measurement highly repeatable rather than accurate must be chosen [10]. At first glance, homogeneity seems not to be a crucial point because the filling process and lyophilisation are today well controlled. However, between-vials homogeneity must be carefully evaluated and must be taken into account to evaluate uncertainty.
11. Additional remarks The certificate which accompanies an enzyme CRM should contain all the information which is essential for correct use of the CRM i.e. the certified value with the corresponding uncertainty, and the method(s) used to determine the
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certified value(s), also details concerning the way in which the material should be stored (temperature, exposure to light), transported, the mode of reconstitution (if any) and delay before use (if relevant) must be extensively described, as well as other data concerning the CRM stability [10]. The certificate must also indicate the intended use of the material, for example to evaluate the comparability of results from laboratories using the measurement procedure retained for the certification of the material. Information on the safety of a CRM should also be provided to the user, also potential biological dangers. When commutability studies have been performed, information concerning the methods of measurement for which the CRM is commutable should also be provided. This is essential not only to promote intermethod calibration, but also to allow a correct use of this approach. The same applies to control materials when they are used for the control of trueness.
12. Description of the IFCC MECRM project The IFCC working group ‘Calibrators in Clinical Enzymology’ has a fourstep project. The first is to revise IFCC recommended methods to 37 8C; secondly, monoenzyme CRMs will be recertified at 37 8C with these methods by several laboratories. Thirdly, two single materials (ALP and AST) will be prepared and certified at 37 8C. Finally, taking into account the available information presented in the BCR certification reports, the members of IFCC working group ‘Calibrators in Clinical Enzymology’ have the project to prepare in collaboration with IRMM an MECRM containing eight purified enzymes: amylase, ALT, AST, ALP, CK, GGT, LDH, and lipase. Activity concentrations will be near to those of the single-material CRM from BCR. Also the choice concerning the sources of enzymes will be directed according to the same principles, except when significantly improved sources have been recently identified. Matrix will be constituted by buffered bovine albumin, added with some effectors. Beside purity and stability, commutability will be extensively evaluated in order to promote an intercalibration approach. Implementation of IFCC recommended methods in the laboratories participating in the certification campaign will be assessed by using single-enzyme CRMs when available.
13. Conclusions It is now possible to purify enzymes without significant alteration of their catalytic properties. Stability was shown to be satisfactory since it was evaluated to at least 10 years when stored at 2 20 8C in a solid form. However, these single enzyme CRMs are expensive and available in limited amounts. It is
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expected that in the future, a MECRM exhibiting a wide commutability will be prepared and that recommendation concerning the proper use of this material will be provided to the users to improve standardisation in clinical enzymology.
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[19] Canalias F, Visvikis A, Thioudellet C, Siest G. Stable expression of enzymatically active human pancreatic lipase in V79 cells: purification and characterization of the recombinant enzyme. Clin Chem 1994;40:1251–7. [20] Oster T, Visvikis A, Schiele F, Wellman-Rousseau M, Siest G. Gene transfer technologies for the production of enzyme and protein reference materials. Clin Chim Acta 1997;257:3–23. [21] Francis JM, Moss DW, Colinet E, Calam DH, Bullock DG. A reference preparation of human prostatic acid phosphatase: purification, characterisation and field trials. Ann Clin Biochem 1992;29:176–83. ´ [22] Lessinger JM, Tavridou A, Arzoglou P, Ferard G. Interest of using a purified, stable and commutable preparation of human pancreatic lipase in indirect assays. Anal Lett 1992;25:1453–68. ¨ [23] Mathieu M, Steghens JP, Horder M, et al. A reference preparation for creatine kinase BB. Clin Chem 1993;39:1894–8. ´ [24] Lessinger JM, Ferard G, Mignot V, Calam DH, Gaines Das R, Dourson JL. Catalytic properties and stability of lipase purified from human pancreatic juice. Clin Chim Acta 1996;251:119–29. ´ [25] Lessinger JM, Dourson JL, Ferard G. Importance of standardization of lipase assays by using appropriate calibrators. Clin Chem 1996;42:1979–83. ´ [26] Lessinger JM, Dourson JL, Ferard G. Importance of the definition of catalytic properties for the commutability of an enzyme reference material: example of lipase. Fres J Anal Chem 1998;360:494–7. [27] Moss DW, Maire I, Calam DH, et al. Reference materials in clinical enzymology: preparation, requirements and practical interests. Ann Biol Clin 1994;54:189–98. ´ [28] Lessinger JM, Ferard G, Frei J, Grafmeyer D, Labbe´ D, Maire I, Mathieu M, Schiele F, Vassault A, Vialle A. Pratique quotidienne en enzymologie clinique: du danger de l’utilisation ´ a` la temperature ´ des facteurs de conversion lies de mesure. Ann Biol Clin 1998;56:359–61. [29] Gubern G, Canalias F, Gella FJ. Determination of a-amylase activity: methods comparison and commutability study of several control materials. Clin Chem 1995;41:435–8. [30] Tydeman MS, Kirkwood TBL. Design and analysis of accelerated degradation tests for the stability of biological standards. J Biol Stand 1984;12:195–224.