Metrological traceability of values for catalytic concentration of enzymes assigned to a calibration material.

BACKGROUND: The metrological traceability of values for the catalytic concentration of several enzymes assigned to a calibration material has been assured by following the recently published International Standard ISO 18153. METHODS: A traceable value with a measurement uncertainty was assigned for the catalytic concentration of alanine aminotransferase, creatine kinase, gamma-glutamyltransferase and lactate dehydrogenase in two materials from different sources. These are all measurable quantities, with the primary reference measurement procedure described by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and a primary calibrator giving metrological traceability to the SI unit of measurement. The metrologically traceable calibration was validated by measuring human serum samples using the primary reference measurement procedure and a routine commercial measurement procedure calibrated with the traceable materials. RESULTS: Results showed that the primary reference procedure, selected manufacturers' procedures and the end-user's routine procedure for each enzyme have the same analytical specificity. Four of eight commercial calibrators tested were commutable, whereas the others had a very small difference in absolute terms, indicating that these materials would be useful for calibration. CONCLUSION: The implementation of a reference system for enzyme measurements was demonstrated that assures the traceability of patient results to SI units.

New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects.

Consensus among clinical chemists has dictated a change in reference temperature for enzyme catalytic concentrations from 30 to 37 degrees C. Consequently, International Federation of Clinical Chemistry (IFCC) reference procedures have been redefined at the latter temperature. Acceptance in practice of these new procedures requires well-established reference values and clinical decision limits, but the establishment of reference values is complex. Therefore, as a provisional approach and to facilitate early application of the new IFCC procedures, we report our experience gained with them in the transfer of values from the consensus methods used hitherto in Germany to the new procedures. The preliminary upper reference limits were determined for catalytic activity concentrations of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) in human sera. Since enzyme measurements are almost always made on sera from non-ambulant subjects, we have used hospital patients aged 17 years and older as the subjects of our study. The catalytic activity concentrations obtained by measurements with the German consensus methods for the respective enzyme were chosen in combination with additional enzymes of similar diagnostic relevance to classify patients' samples as part of the respective reference collective. Measurements for the determination of the upper reference limits were performed manually by use of the primary reference procedures at the measurement temperature 37 degrees C according to IFCC, and also by employing mechanized measurements adapted to the reference procedures. The upper reference limits were calculated as the 97.5th percentile of the reference collectives and determined separately for women and men: ALT: 34 U/l (female) and 45 U/l (male); AST: 31 U/l (female) and 35 U/l (male); CK: 145 U/l (female) and 171 U/l (male); gamma-GT: 38 U/l (female) and 55 U/l (male); LDH: 247 U/l (female) and 248 U/l (male).

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 7. Certification of four reference materials for the determination of enzymatic activity of gamma-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase accord.

This paper is the seventh in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase. A document describing the determination of preliminary reference values is also in preparation. The certification of the catalytic activity concentrations as determined by the recently elaborated IFCC primary reference methods at 37 degrees C of four enzyme preparations, namely IRMM/IFCC 452 (gamma-glutamyltransferase), IRMM/IFCC 453 (lactate dehydrogenase 1), IRMM/IFCC 454 (alanine aminotransferase) and IRMM/IFCC 455 (creatine kinase) is described. Homogeneity data were derived from previous results. Stability was assessed using recently obtained data as well as data from previous stability studies. The collaborative study for value assignment was performed under a strict quality control scheme to ensure traceability to the primary reference method. Uncertainty of the materials was assessed in compliance with the Guide to the Expression of Uncertainty in Measurement. The certified values obtained at 37 degrees C are 1.90 microkat/l +/- 0.04 microkat/l (114.1 U/l +/- 2.4 U/l), for gamma-glutamyltransferase, 8.37 microkat/l +/- 0.12 microkat/l (502 U/l +/- 7 U/l), for lactate dehydrogenase 1, 3.09 microkat/l +/- 0.07 microkat/l (186 U/l +/- 4 U/l), for alanine aminotransferase and 1.68 microkat/l +/- 0.07 microkat/l (101 U/l +/- 4 U/l), for creatine kinase. The materials are intended for internal quality control as well as for the evaluation of test systems as required by recent European Union legislation. Furthermore, the materials can be used to transfer accuracy from a reference method to a routine procedure provided the procedures exhibit the same analytical specificity and the certified materials are commutable.

Negative interference of bilirubin and hemoglobin in the MEIA troponin I assay but not in the MEIA CK-MB assay.

Troponin I is a sensitive and specific marker for the diagnosis of myocardial infarction. Several commercially available immunoassays measure the concentration of troponin I in serum. The microparticle enzyme immunoassay (MEIA) for troponin I (Abbott Laboratories, Abbott Park, IL) is widely used in clinical laboratories, including our hospital laboratory. We studied the effect of bilirubin and hemolysis on the MEIA for troponin I and compared our assay with a newly available chemiluminescent assay (CLIA) for troponin I (Bayer Diagnostics, Tarrytown, NY). We also measured CK-MB concentration using the MEIA CK-MB assay. One serum pool was prepared by combining several specimens of one patient with elevated troponin I and with a diagnosis of myocardial infarction. Other serum pools were prepared by combining sera with similar troponin I values. All serum pools showed normal bilirubin concentrations and had no hemolysis. Then we supplemented aliquots of serum pools with various concentrations of bilirubin (5.0, 10.0, 15.0, and 20.0 mg/dL). After supplementation, troponin I concentrations were measured again using the MEIA and CLIA. We observed a statistically significant decrease in troponin I concentration in the presence of bilirubin with the MEIA. For example, in serum pool 1, the troponin I concentration was 16.3 (bilirubin: 0.8 mg/dL). In the presence of 5.0, 10.0, 15.0 and 20.0 mg/dL of added bilirubin, the cardiac troponin I concentrations were 13.9, 13.4, 13.3 and 13.0 ng/ml respectively. We observed similar negative interference of bilirubin in troponin I measurement by the MEIA in other pools. The troponin I value decreased slightly (not statistically significant) in one pool and did not change in two other pools in the presence of bilirubin when we measured troponin I concentration using the CLIA. Interestingly, bilirubin did not interfere with the MEIA CK-MB assay. Moderate hemolysis did not have any effect on the troponin I assay using either the MEIA or CLIA. However, gross hemolysis (hemoglobin > 40 mg/dL) interfered with both assays for troponin I.

Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB.

BACKGROUND: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum. METHODS: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography. RESULTS: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L. CONCLUSIONS: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.

Standardization of creatine kinase-MB (CK-MB) mass assays: the use of recombinant CK-MB as a reference material.

BACKGROUND: The AACC assembled a committee to identify and validate a standard creatine kinase MB isoenzyme (CK-MB) material to improve the comparability of CK-MB mass assays. METHODS: Three protocols were used. In protocol I, various CK-MB materials prepared in different matrices were screened as candidate standards. In protocol II, participating manufacturers calibrated their systems with concentrates of human heart CK-MB and then tested 20 patient samples to evaluate calibration bias. In protocol III, participating manufacturers calibrated their immunoassay systems using recombinant CK-MB2 (rCK-MB2) diluted into their respective sample diluents and measured 50 samples. RESULTS: Candidate materials showed high recovery in stripped human serum, but bias improved only from 59% to 38%. These data led to the use of human heart CK-MB diluted in each manufacturer's sample diluent. This strategy reduced bias from 31% to 15%. Because human heart CK-MB is difficult to provide, a lyophilized source of CK-MB2 was identified. rCK-MB2 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase HPLC, intrinsic protein fluorescence, circular dichroism, agarose gel electrophoresis, immunoreactivity studies, high and low temperature stability, and reconstituted stability to be equivalent to human heart CK-MB. Calibration of immunoassay systems with rCK-MB2 added into each respective manufacturer's sample diluent showed a 13% between-manufacturer bias. CONCLUSION: Lyophilized rCK-MB2 was determined suitable for use as a reference material for CK-MB mass assays.

Creatine kinase values in amyotrophic lateral sclerosis.

Serum creatine kinase (CK1) values were recorded while or soon after the diagnosis of amyotrophic lateral sclerosis (ALS) was established in 140 of 217 (65%) consecutive patients seen in the Motor Neuron Disease Clinic at this center during a 5-year period. Second creatine kinase (CK2) determinations were recorded on 48 of 140 (34%) ALS patients later in the course of the disease. The mean CK1 and CK2 values (unit, times the upper limit of normal) were 1.22 (range, 0.1 to 3.0; median, 0.80) and 1.34 (range, 0.1-9.2; median, 0.80) (P=0.11, r=0.92, mean difference=28.2%). Of the 140 ALS patients, 58 (41%) (male:female ratio: 2.9:1.0) had elevated CK1 values ranging from 1.03 to 13.0 (mean, 2.21; median, 1.67). Twelve patients (8.6%) had CK1 values greater than 3.0. Mean CK1 values were significantly greater (P=0.001) in male (mean, 1.56) versus female (mean, 0.80) ALS patients, and significantly greater (P=0.009) in limb-onset (mean, 1.34) versus bulbar-onset (mean, 0.80) disease. CK1 values correlated poorly with and were not predictive of age of onset or survival.

Analytical evaluation of the CK-MB mass assay on the Technicon Immuno 1 system.

OBJECTIVES: To evaluate the performance of the Technicon Immuno 1 CK-MB mass assay. DESIGN AND METHODS: The precision was measured using the BioRad Liquichek CK-MB controls and the Technicon Immuno 1 SETpoint CK-MB calibrators. The linearity was verified by diluting 9 patient serum samples containing high CK-MB concentrations, and the minimum detectable concentration determined by repetitive analysis of the zero calibrator. The assay was compared with the Johnson & Johnson Vitros CK-MB activity assay using 111 patient serum samples. The reference range was determined using serum samples from 100 healthy adult volunteers. RESULTS: The assay shows within-run CVs varying from 1.5 to 7.5% and between-day CVs from 1.5 to 4.8% for CK-MB concentrations ranging from 3.9 to 99.0 micrograms/L. The linearity study gave correlation coefficients greater than 0.9961. There was a very good correlation between mass and activity assays (r = 0.985). The reference values were estimated at 0.0-4.2 micrograms/L and the minimum detectable concentration at 0.5 micrograms/L. CONCLUSIONS: The evaluation shows that the Technicon Immuno 1 CK-MB mass assay has the analytical characteristics suitable for its inclusion in the modern clinical repertoire.

Clinical laboratory investigation of the Sanofi ACCESS CK-MB procedure and comparison to electrophoresis and Abbott IMx.

This evaluation was undertaken to verify the application protocol for the CK-MB assay on the ACCESS Immunoassay Analyzer (Sanofi Diagnostics Pasteur, Chaska, MN). The results show that the ACCESS CK-MB assay total imprecision was 6.8% to 9.1%. Analytical linearity of the ACCESS CK-MB assay was excellent in the range of < 1-214 micrograms/L. A comparison of the ACCESS CK-MB assay with the IMx (Abbott Laboratories, Abbott Park, IL) method shows good correlation r = 0.990 (n = 108). Linear regression analysis yielded Y = 1.36X-0.3, Sx/y = 7.2. ACCESS CK-MB values also correlated well with CK-MB by electrophoresis with r = 0.968 (n = 132). The linear regression equation for this comparison was Y = 1.08X + 1.4, Sx/y = 14.1. The expected non-myocardial infarction range of CK-MB determined by the ACCESS system was 1.3-9.4 micrograms/L (mean = 4.0, n = 58). The ACCESS CK-MB assay would appear to be rapid, precise and clinically useful.

A reference preparation of creatine kinase BB isoenzyme.

Creatine kinase (CK; EC 2.7.3.2) catalytic activity in serum is widely measured in clinical chemistry practice and provides information for diagnosis and follow-up in many pathological conditions affecting heart, muscle, and brain. Depending on the organ involved, the predominant CK isoenzyme in serum varies. However, routine methods measure total CK catalytic activity, and standardized methods for doing so have been recommended by the International Federation of Clinical Chemistry and by several national scientific societies. Many commercial kits for those methods are now available. With use of a reference material for CK, commercial reagents can be compared with standardized methods, improving confidence in the results. Here we present a reference preparation of CK consisting of the BB isoenzyme purified from human placentae. We describe the procedure of purification and the properties of the lyophilized preparation of CK-BB, which has been certified by the Community Bureau of Reference of the Commission of the European Communities under the designation CRM 299. The preparation can be used to calibrate assays of the catalytic activity of CK-MM and CK-MB, as well as CK-BB.

Creatine kinase and fibrillation potentials in patients with late sequelae of polio.

The incidence of an elevated creatine kinase (CK) in a group of polio patients with delayed weakness (15/29) did not differ from polio patients without delayed weakness (9/31) or others with amyotrophic lateral sclerosis (ALS; 10/21). Mean CK in polio patients without delayed weakness (151 IU/L) was lower than the CK in those with delayed weakness (270 IU/L) or ALS (224 IU/L) (P less than 0.05). An elevated CK in polio patients with delayed weakness did not correlate with new or residual weakness. These findings suggest that muscle overuse is either not important or inadequately measured by CK. Widely distributed fibrillations were associated with an elevated CK for all polio patients combined (P less than 0.01). Fibrillations occurred in more muscles of polio patients with delayed weakness (P less than 0.01) and implies that late denervation may play a role in the development of new weakness in some polio patients.

Quantifying the MB isoenzyme of creatine kinase with the Abbott "IMx" immunoassay analyzer.

In this two-step automated assay of the MB isoenzyme of creatine kinase (CK-MB), developed for the Abbott "IMx" immunoassay analyzer, monoclonal anti-CK-MB antibody immobilized onto latex microparticles and polyclonal anti-CK-MM antibody coupled to alkaline phosphatase are used. Within-run CVs ranged from 3.9% to 9.0%, between-run CVs from 0.0% to 5.6%, and the sensitivity was 0.2 microgram/L. Twenty-four results can be obtained in about 37 min. Analytical recovery of CK-MB added to human serum or plasma ranged from 89% to 109%. Icteric, lipemic, or hemolyzed samples did not interfere with CK-MB recovery. Cross-reactivity with CK-MM and CK-BB was 0.012% and 0.001%, respectively. The normal reference interval was 0-5 micrograms/L. IMx CK-MB results correlated well with CK-MB enzyme activity as determined by electrophoresis (n = 203; r = 0.97; slope = 0.90; y-intercept = -4.29) and with commercial immunoassays. We think that this assay will be useful for confirmation of acute myocardial infarction, both in critical-care units and in the clinical laboratory.

Differential diagnosis of patients with abnormal serum creatine kinase isoenzymes.

For the diagnosis of myocardial injury, particularly AMI, CK-MB has become the gold standard. Changing CK-MB activities in serially collected blood from patients with suggestive signs and symptoms of AMI is almost pathognomonic for infarction. Nevertheless, an increased CK-MB cannot be equated with AMI owing to the many other types of inflammatory, traumatic, and miscellaneous forms of injury to the heart and the trace activities of CK-MB in skeletal muscle. Other enzyme tests for AMI are less efficient. In order of decreasing efficiency, the tests are CK-MB, CK, LD1 greater than LD2 or LD1/LD2 greater than 0.76, AST and LD; the latter two tests are not cost effective and add little or nothing when results for CK-MB, CK, and LD isoenzymes are available. The value of the isoforms of CK-MM and CK-MB remains to be established. Early evidence suggests that they could be helpful in the diagnosis of AMI; however, owing to the greater technical difficulties in performing these tests, their use is necessarily more restricted. Enzyme testing on admission and then every 12 hours for 2 days is sufficient and effective in making the initial diagnosis. In patients presenting early after an attack, CK and CK-MB are often normal. Decisions on AMI cannot be made on blood tests collected in the emergency department. Clot-lysing agents like streptokinase, urokinase, and tPA have changed the therapy of AMI dramatically. Enzyme tests clearly separate patients with and without successful therapeutic or spontaneous reperfusion. With successful reperfusion, the uniform finding has been a "washout" phenomenon with significantly earlier peaking times for CK and CK-MB. The isoforms of CK and myoglobin give the earliest peaks after successful reperfusion. With faster turnaround times for these tests, they may become important tools in patient management.

Production and certification of secondary enzyme reference materials (ERMs). Part 1: Preparation of the sera and some of their properties.

We produced three batches of a human-serum-based enzyme reference material (ERM) enriched with human aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), creatine kinase (EC 2.7.3.2), and lactate dehydrogenase (EC 1.1.1.27). The added enzymes were not exhaustively purified; thus the final ERMs contained some enzymes as contaminants, of which only glutamate dehydrogenase activity might interfere. The stability during storage and after reconstitution was good. The commutability of the four enzymes in the three ERM batches was also good, except when German or Scandinavian methods for aminotransferases were involved. The temperature-conversion factors for the ERMs were equivalent to those for patients' sera. Reactivation after reconstitution was complete within 5 min and was independent of the temperature of the reconstitution fluid. We believe that these secondary ERMs will aid in the transfer of accuracy between well-defined reference methods and daily working methods so that clinical enzymology results will become more comparable from laboratory to laboratory.