The existing interim consensus reference ranges and the future approach.

The release of the reference material for serum proteins, CRM 470/RPPHS, in 1993, has given rise to a great improvement in the between-laboratory variability of serum protein measurements worldwide. However, conversion to the new reference material has resulted in significant changes in reference values for some proteins. The establishment of new reference ranges is currently in progress; in the interim, several professional societies and diagnostic companies have agreed to use consensus reference ranges based on studies that were already undertaken.

Use of a reference material proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to evaluate analytical methods for the determination of plasma lipoprotein(a).

BACKGROUND: As part of the NIH/National Heart, Lung and Blood Institute Contract for the Standardization of Lipoprotein(a) [Lp(a)] Measurements, a study was performed in collaboration with the IFCC Working Group for the Standardization of Lp(a) Assays. The aims of the study, performed with the participation of 16 manufacturers and 6 research laboratories, were to evaluate the IFCC proposed reference material (PRM) for its ability to transfer an accuracy-based value to the immunoassay calibrators and to assess concordance in results among different methods. METHODS: Two different purified Lp(a) preparations with protein mass concentrations determined by amino acid analysis were used to calibrate the reference method. A Lp(a) value of 107 nmol/L was assigned to PRM. After uniformity of calibration was demonstrated in the 22 evaluated systems, Lp(a) was measured on 30 fresh-frozen sera covering a wide range of Lp(a) values and apolipoprotein(a) [apo(a)] sizes. RESULTS: The among-laboratory CVs for these samples (6-31%) were, in general, higher than those obtained for PRM (2.8%) and the quality-control samples (14%, 12%, and 9%, respectively), reflecting the broad range of apo(a) sizes in the 30 samples and the sensitivity of most methods to apo(a) size heterogeneity. Thus, although all of the assays were uniformly calibrated through the use of PRM, no uniformity in results was achieved for the isoform-sensitive methods. CONCLUSIONS: Linear regression analyses indicated that to various degrees, apo(a) size heterogeneity affects the outcome of the immunochemical methods used to measure Lp(a). We have also shown that the inaccuracy of Lp(a) values determined by methods sensitive to apo(a) size significantly affects the assessment of individual risk status for coronary artery disease.

Analytical and clinical evaluation of troponin I determination on dimension RXL-HM.

The evaluation of cardiac troponin I (cTnI) on the Dimension RxL-HM analyzer is presented. The one-step enzyme immunoassay is based on two cTnI specific monoclonal antibodies. Performed on a separate module of the analyzer, assay-time is 17 minutes. Using as criterion a between-run impression CV <20% the functional limit of detection was set at 0.1 microg/l. Cutoff level for minor myocardial damage of 0.1 microg/l was found. In Duchenne's dystrophy, patients showed increased cardiac Troponin T (cTnT) but no increased cTnI. In patients with a history of coronary heart disease undergoing chronic hemodialysis, cTnT and cTnI were increased. In different patients with submassive pulmonary embolism, increased cTnI was determined. In coronary artery bypass surgery without perioperative myocardial infarction, patients with extracorporeal circulation showed significantly higher cTnI at 24 h after surgery than those with minimal cardiac surgery. In patients with unstable angina, increased cTnI was found more often than on Stratus analyzer. In conclusion, the new assay is a very sensitive cTnI assay, fast and easy to perform in parallel to enzyme and substrate assays.

Standardization activities for harmonization of test results.

In the last years the search for sensitive and specific markers of renal damage and/or renal function has conducted to the development of laboratory assays for measurement of urinary proteins such as albumin, beta(2)-microglobulin, alpha(1)-microglobulin, cystatin C, etc. Furthermore, there have been new applications of already known markers based on different, reformulated methods which often rely on more advanced technologies. It is evident that such developments are connected with analytical and interpretative problems for laboratory managers and clinicians. In this situation, it is essential that international societies develop comprehensive measures for the quality management of these assays and issue uniform and carefully elaborated guidelines to ensure optimal test utilization. International activities are also directed to the development of optimized and standardized methods as well as to the production and evaluation of appropriate reference materials and, finally, to the establishment of appropriate reference ranges and cut-off values for specific analytes. The main use of reference materials is in the transfer of their accurately assigned values to the calibrators of diagnostic companies for calibration of commercially available test systems. These international standardization activities and strategies will allow a harmonized approach to disease management using a more reliable laboratory testing based on quality and value.

Use of biochemical markers in acute coronary syndromes. IFCC Scientific Division, Committee on Standardization of Markers of Cardiac Damage. International Federation of Clinical Chemistry.

This paper presents evidence and suggestions from the IFCC Committee on "Standardization of Markers of Cardiac Damage" (C-SMCD) on the use of biochemical markers for the triage diagnosis of acute coronary syndromes. There is general agreement that both 'early' and 'definitive' biochemical markers of myocardial damage are necessary and that these assays must be available with a turnaround time of 1 h or less. Currently, myoglobin is the marker that most effectively fits the role as an 'early' marker, whereas 'definitive' markers are cardiac troponins. Since the sensitivity of the initial electrocardiogram is only 50% for detecting myocardial infarction, the use of biochemical markers may significantly contribute to the early diagnosis and become relevant when the electrocardiogram is not diagnostic. In addition, new sensitive biochemical markers, particularly the cardiac troponins, are presently the best to detect the presence of minor myocardial cell damage. With regard to this, two decision limits are probably needed for the optimal use of troponins: a low abnormal value suggesting the presence of myocardial damage and a higher value suggesting the diagnosis of myocardial infarction according to traditionally used criteria. Properly designed studies should be performed to establish limits for each commercially available troponin assay. Finally, it is recognized that there is no need for the use of any biochemical marker when the clinical diagnosis is unequivocal, other than for diagnosing reinfarction, estimating the infarct size, and monitoring thrombolytic therapy.

Proposals from IFCC Committee on Standardization of Markers of Cardiac Damage (C-SMCD): recommendations on use of biochemical markers of cardiac damage in acute coronary syndromes.

This paper presents evidence and suggestions from the IFCC C-SMCD on the use of biochemical markers for the triage diagnosis of acute coronary syndromes. There is general agreement that both 'early' and 'definitive' biochemical markers are necessary and that these assays must be available with a turnaround time of 1 h or less. Currently, myoglobin is the marker that most effectively fits the role as an 'early' marker, whereas 'definitive' markers are cardiac troponins. Since the sensitivity of the initial electrocardiogram is only 50% for detecting myocardial infarction, the use of biochemical markers may significantly contribute to the early diagnosis. New sensitive biochemical markers, particularly the cardiac troponins, are presently the best criterion to detect the presence of small myocardial cell damage. Two decision limits are probably needed for the optimum use of troponins: a low abnormal value suggesting the presence of myocardial damage and a higher value suggesting the diagnosis of myocardial infarction. Additional studies should be performed to establish limits for each commercially available assay. Finally, it is recognized that there is no need for the use of any biochemical marker when the clinical diagnosis is unequivocal, other than for diagnosing reinfarction, estimating the infarct size, and monitoring thrombolytic therapy.

Proposals from the IFCC Committee on Standardization of Markers of Cardiac Damage (C-SMCD): strategies and concepts on standardization of cardiac marker assays.

The search for sensitive and specific biochemical markers of cardiac damage has resulted in development of methods for the measurement of cardiac markers such as myoglobin, CK-MB mass and the cardiac troponins (cardiac troponin I and cardiac troponin T). There have been new clinical applications of already known markers based on improved, reformulated methods which often depend on advanced technologies. These developments are connected with analytical and interpretative challenges for the laboratory manager and for the clinician. In this situation, it is essential that international professional societies develop comprehensive and carefully elaborated guidelines for the quality management and use of these measurements and their results. Several professional associations have formed committees working on different issues regarding the measurement of biochemical markers of cardiac damage. Recognizing the huge interest in this field and substantial diagnostic relevance of these markers, the IFCC has established the Committee on "Standardization of Markers of Cardiac Damage" (C-SMCD) in 1997 inviting the already operating American and European groups to designate some of their members into the new committee. The task of the IFCC C-SMCD is to coordinate the different activities of the national groups, with preparation of international documents and recommendations under the auspices of IFCC and to initiate various standardization activities. The establishment of consensus/reference methods as well as development of primary and secondary matrix reference materials for markers of cardiac damage are extremely important in order to achieve comparability of test results, thus leading to an effective patient care.

International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Standardization Project for the Measurement of Lipoprotein(a). Phase 2: selection and properties of a proposed secondary reference material for lipoprotein(a).

The International Federation of Clinical Chemistry and Laboratory Medicine Working Group for the Standardization of Lipoprotein(a) Assays has initiated a project to select a secondary reference material for lipoprotein(a) that can standardize the measurement of this lipoprotein. Most of the analytical problems with lipoprotein(a) assays are due to apolipoprotein(a) kringle 4 type 2 reactive antibodies and values being expressed in mg/l mass units rather than as nmol/l of apolipoprotein(a) particles. In Phase 2, four manufactured materials were compared for analytical performance, commutability properties and method harmonization in 27 lipoprotein(a) test systems. Results of precision and linearity testing were comparable for all materials whereas testing for the harmonization effect resulted in an among-assay coefficient of variation for corrected lipoprotein(a) values of between 11% and 22%. The material that gave maximum harmonization achieved a variation of < 8% for 18 immunonephelometric and immunoturbidimetric assay systems. It can be hypothesized that this residual variation in part takes into account the inaccuracy of lipoprotein(a) measurement due to apolipoprotein(a) size polymorphism. On the basis of acceptable analytical performance, maximal harmonization effect and documented stability, a lyophilized material has been selected as the common calibrator for lipoprotein(a) to be used in a value transfer procedure by diagnostic companies.

Relevance of markers of hemostasis activation in obstetrics/gynecology and pediatrics.

Pregnancy and puerperium are considered to be hypercoagulable states with increased incidence of thromboembolic events. During normal pregnancy, changes in the hemostatic mechanism involve increased stasis and increased coagulation factors and/or decreased levels of anticoagulant proteins such as protein C and protein S as well as enhanced thrombin generation and decreased fibrinolytic activity. The physiological or pathophysiological activation of hemostasis during pregnancy results in the generation of the so-called activation markers which increase, reflecting hypercoagulability and therefore representing an imbalance in the hemostatic system. The most interesting markers of hemostasis activation and, thus, of thrombin generation are: thrombin-antithrombin III complex (TAT), antithrombin III itself, prothrombin fragment 1+2 (F 1+2), fibrin monomer (soluble fibrin) and D-Dimer (which indicates also an increased fibrinolytic activity). Together with fibrinogen levels and platelet counts, the activation markers are useful tools in different pathological situations in pregnancy to predict and monitor the severity of the condition. Recently, a higher incidence of factor V Leiden mutation has been demonstrated in selected populations in whom thrombotic events developed during pregnancy and puerperium. Therefore, the combination of APC resistance/FV Leiden mutation and pregnancy may predict a high risk for thromboembolic phenomena. In newborns, the activation markers are elevated immediately after birth and decline to near adult levels during the first 24 h of life. During infections the activation markers are increased showing the same behavior as in the mature adult system. In neonates and children, the same etiologies can be responsible for acquired and inherited pathological hypercoagulable states as in the adult.

International Federation of Clinical Chemistry standardization project for the measurement of lipoprotein(a). Phase I. Evaluation of the analytical performance of lipoprotein(a) assay systems and commercial calibrators.

A secondary reference material for lipoprotein(a) is required to standardize the measurement of lipoprotein(a) in clinical laboratories worldwide. Towards this aim, the International Federation of Clinical Chemistry Working Group for the Standardization of Lipoprotein(a) Assays has initiated a standardization project involving a total of 33 diagnostic company and clinical chemistry laboratories from 12 countries. In Phase 1, the analytical performance of 40 lipoprotein(a) assay systems was evaluated by testing sera and manufactured lipoprotein(a) calibrator materials for precision, linearity, and parallelism. Twenty test systems were nonoptimized according to the results for a pooled serum, which tested nonlinear in 16 systems and imprecise in 4. Acceptable analytical properties and harmonization of lipoprotein(a) values were shown by some commercial calibrators, suggesting their possible use as reference materials. This study highlights the problems that currently occur for lipoprotein(a) measurement in existing assay systems.

ProC Global: the first functional screening assay for the complete protein C pathway.

In clinical practice, venous thromboembolic complications are much more frequent than bleeding disorders. In fact, disturbances within the protein C pathway due to coagulation factor V (FV) Leiden mutation and deficiency of protein C or protein S are the most frequent abnormalities in hereditary thrombophilia. Furthermore, acquired dysfunctions of the protein C system may predispose the single individual to an increased thrombotic risk. A routine-suited screening assay that would allow the monitoring of the proper interplay of factors in the protein C pathway could add an important factor to the basic coagulation profile. This consists of the prothrombin time and of the activated partial thromboplastin time, which currently allow only a screening for increased risk for bleeding but not for venous thromboembolism. A new functional screening test for the protein C system such as the presented ProC Global should therefore facilitate detection of FV Leiden as well as deficiency of protein C and protein S. The results of the present evaluation indicate that ProC Global is highly sensitive to activated protein C resistance/FV Leiden (100%) and protein C deficiency (90%) and sensitive to protein S deficiency (63%). Furthermore, the assay gives a quantitative measure of the net potential of the protein C pathway in relation to the intrinsic procoagulant system. The use of this assay for a prospective assessment of thromboembolic risk is the subject of current studies.

Quality management and international standardization programs for protein immunoassays.

The huge number of new immunoassays for the measurement of specific proteins/tumor markers commercially available at present has made necessary comprehensive measures for the quality management of such laboratory tests. Very important for the comparability of test results are international standardization programs for the establishment of consensus/reference methods as well as for the development of suitable reference materials. Such programs are presently conducted by various international organizations and professional societies. Important characteristics of such reference materials are molecular composition, purity, matrix, additives, storage conditions, stability. Internationally accepted reference materials which have been prepared, calibrated and certified will contribute to the harmonization of results with different test systems, improving their quality and clinical use.

Quality management and standardization programs in hemostaseology.

Total quality management (TQM) of laboratory services is connected with a comprehensive system of quality assurance, which integrates quality development, quality maintenance, and quality improvement. TQM concentrates not only on analytic performance and organizational issues, including specimen collection, reporting, and interpretation of results, but focuses also on the benefits to society related to the use of specific laboratory tests in prevention, early detection, and therapy monitoring, as well as on outcome measures. A prerequisite to TQM are international and national standardization programs for the establishment of optimized and standardized methods, as well as for the development and evaluation of suitable reference materials. Reference materials that have been or are being prepared, calibrated, and certified by international and national reference institutions will certainly contribute to the harmonization of results, with different test systems in hemostaseology, improving their quality and value.

Consensus of a group of professional societies and diagnostic companies on guidelines for interim reference ranges for 14 proteins in serum based on the standardization against the IFCC/BCR/CAP Reference Material (CRM 470). International Federation of Clinical Chemistry. Community Bureau of Reference of the Commission of the European Communities. College of American Pathologists.

The release in 1993 of a new reference material for serum proteins, CRM 470/RPPHS 5 has given rise to a great improvement in the between-laboratory variability of serum protein measurements worldwide. Conversion to the new reference material results in significant changes in reference values for some proteins. The establishment of new reference ranges will take a considerable time, and in the interim several professional societies and diagnostic companies have agreed to use consensus reference ranges based on studies already undertaken.

New international reference preparation for proteins in human serum (RPPHS).

Quality-control surveys in recent years, in various parts of the world, have shown poor between-laboratory agreement for measurements of plasma proteins. Despite the existence of international reference materials distributed by the World Health Organization, standards produced by diagnostics manufacturers and professional organizations differ significantly in their ascribed values. The reasons for this are complex but include poor availability of the primary materials, confusion about their use, and the fact that their turbidity on reconstitution precludes their use in modern optical immunoassays. This unfortunate situation led to an important initiative to produce sufficient quantities of a widely available, optically clear secondary reference material for plasma proteins that could be used worldwide by manufacturers, professional organizations, and laboratories. Here we present an overview on how the laboratory community, including manufacturers, clinical laboratories, professional societies, and regulators, has reached what we consider is a successful conclusion to a difficult problem.

International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B.

To minimize differences in apolipoprotein measurements among laboratories and methods, a standardization program involving common suitable reference material is needed. The Committee on Apolipoproteins of the International Federation of Clinical Chemistry initiated a collaborative study for the standardization of test systems for measuring apolipoproteins (apo) A-I and B, with 25 company laboratories and three research laboratories involved in apolipoprotein analysis to: (a) evaluate calibration differences among the test systems; (b) evaluate whether comparability of the data can be achieved with the use of frozen serum pools to recalibrate the different systems; and (c) evaluate and select suitable candidate reference material. We used 26 test systems for apo A-I and 28 for apo B. Relatively modest differences were found in calibration for apo A-I, but very wide differences were observed for apo B methods. After uniform calibration, the overall among-laboratory CV for apo B decreased from 19% to 6%. Three lyophilized serum preparations for apo A-I and three liquid-stabilized serum preparations for apo B were selected for further evaluation as candidate international reference materials.

Serum levels of alpha-1 microglobulin and beta-2 microglobulin in bone marrow transplant recipients treated with cyclosporin A.

The levels of alpha-1 microglobulin (alpha 1m) and beta-2 microglobulin (beta 2m) in serum were estimated in 77 bone marrow transplant recipients. In comparison to pre-transplant levels, the highest levels of alpha 1m and beta 2m were found during impairment of renal function, i.e., during cyclosporin-induced nephrotoxicity and during treatment with other nephrotoxic drugs (P less than 0.001). The alpha 1m levels were less elevated during infections and acute graft-versus-host disease (P less than 0.01), while beta 2m levels were markedly elevated during the same conditions (P less than 0.001). The linear correlations between serum creatinine and alpha 1m and creatinine and beta 2m were r = 0.7 and 0.8, respectively (P less than 0.001). The overall correlation between alpha 1m and beta 2m was 0.4 (P less than 0.001). It is concluded that alpha 1m might be a complement to serum creatinine levels in monitoring renal function after bone marrow transplantation.