Jillian R. Tate

Cross-Reactivity of BNP, NT-proBNP, and proBNP in Commercial BNP and NT-proBNP Assays: Preliminary Observations from the IFCC Committee for Standardization of Markers of Cardiac Damage

B-type natriuretic peptide (BNP) is a 32 amino acid cardiac-synthesized hormone that reduces blood pressure and increases sodium excretion (1). Following proteolytic cleavage of proBNP, a 108-amino acid precursor, an N-terminal fragment (NT-proBNP) and BNP are released (2). Increased concentrations of BNP and NT-proBNP can be used clinically to monitor heart failure, but a lack of alignment between commercial BNP and NT-proBNP assays (3) can lead to confusion when clinicians or laboratorians compare results measured for the same analyte on different instruments. Some of this confusion arises from variable assay specificity regarding what peptides are being measured. We studied whether ( a ) BNP assays demonstrated cross-reactivity with NT-proBNP or proBNP, and ( b ) whether NT-proBNP assays demonstrated cross-reactivity with BNP or proBNP, by using 5 commercial BNP and 3 commercial NT-proBNP assays with 2 BNP, 2 NT-proBNP, and 2 proBNP materials. The NPs studied were: Peptide Institute synthetic BNP (aa 77–108), Scios human recombinant BNP (aa 77–108), HyTest human recombinant NT-proBNP (aa 1–76), Roche modified (amidated for stabilization) synthetic NT-proBNP, HyTest human recombinant proBNP (aa 1–108), and Scios glycosylated human recombinant proBNP. BNP assays evaluated were Abbott Architect, Abbott AxSYM, Bayer …

Troponin revisited 2008: assay performance

Abstract Troponin quality specifications describing the pre-analytical, analytical and post-analytical performance of cardiac troponin (cTn) assays are important for both manufacturers of cTn assays and laboratories that routinely test for cTn. Pre-analytical requirements refer not only to acceptable sample type for analysis and the stability of cTn but also to the proper handling of specimens prior to analysis to avoid pre-analytical false positive results. Analytical issues that may contribute to differences between cTn assays include analytical sensitivity and imprecision at low cTn concentration, antibody specificity and immunoreactivity of plasma cTn forms, assay specificity and the presence of falsely positive and negative interferences, and for cTnI the lack of standardised measurement, all which may impact on patient cTn results. Current second generation cTnI and fourth generation cTnT assays generally have an imprecision of around 20% coefficient of variation (CV) at the 99th percentile of the reference population, which is greater than the recommended imprecision of 10% CV. As the next generation of more analytically sensitive cTn assays are developed it can be anticipated that cTn upper reference limits will decrease by approximately 10-fold. Monitoring assay imprecision at ultra low cTn concentrations will require that the laboratory uses a quality control close to this level and a negative control to monitor baseline drift. Establishment of cTn reference ranges will require reference populations to be cardio-healthy to enable differentiation from community populations who are at increased cardiovascular risk. Close collaboration between the laboratory and local clinicians is required to ensure adequate clinical validation of more sensitive cTn assays. Clin Chem Lab Med 2008;46:1489–500.

National Academy of Clinical Biochemistry and IFCC Committee for Standardization of Markers of Cardiac Damage Laboratory Medicine Practice Guidelines: Analytical Issues for Biomarkers of Heart Failure

### A. Background In 2005, the IFCC C-SMCD recommended analytical and pre-analytical quality specifications for natriuretic peptide and their related co-metabolites assays.1 The objectives developed were intended to guide manufacturers of commercial assays and clinical laboratories that utilize these assays. The overall goal was to establish uniform criteria so that the analytical qualities and clinical performance of assays natriuretic peptide and their related co-metabolites could be evaluated objectively. As B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) become more heavily integrated into clinical practice as diagnostic and prognostic biomarkers, understanding the differences between individual assays becomes important. Further, the influence of clinical, analytical and preanalytical factors on the growing number of BNP and NT-proBNP assays commercially available begs for a better understanding of how to interpret findings of different studies predicated on BNP or NT-proBNP concentrations monitored by different assays. The Laboratory Medicine community must also work closely with the in vitro diagnostics companies to assist in defining all of the assay characteristics,1 a process that was poorly orchestrated during the developmental phase of cardiac troponin assays. When BNP or NT-proBNP assays are used as biomarkers for diagnosis, therapy decisions, and prognosis, or used in clinical trials or studies, they should be well characterized, as suggested by the list of recommendations that follow. We recommend that when designing studies that will use BNP or NT-proBNP assays, investigators …

Practical Considerations for the Measurement of Free Light Chains in Serum

BACKGROUND A new immunoassay for free light chain measurements has been reported to be useful for the diagnosis and monitoring of monoclonal light chain diseases and nonsecretory myeloma. We describe experience with and some potential pitfalls of the assay. METHODS The assay was assessed for precision, sample type and stability, recovery, and harmonization of results between two analyzers on which the reagents are used. Free-light-chain concentrations were measured in healthy individuals (to determine biological variation), patients with monoclonal gammopathy of undetermined significance, myeloma patients after autologous stem cell transplants, and patients with renal disease. RESULTS Analytical imprecision (CV) was 6-11% for kappa and lambda free-light-chain measurement and 16% for the calculated kappa/lambda ratio. Biological variation was generally insignificant compared with analytical variation. Despite the same reagent source, values were not completely harmonized between assay systems and may produce discordant free-light-chain ratios. In some patients with clinically stable myeloma, or post transplantation, or with monoclonal gammopathy of undetermined significance, free-light-chain concentration and ratio were within the population reference interval despite the presence of monoclonal intact immunoglobulin in serum. In other patients with monoclonal gammopathy of undetermined significance, values were abnormal although there was no clinical evidence of progression to multiple myeloma. CONCLUSIONS The use of free-light-chain measurements alone cannot differentiate some groups of patients with monoclonal gammopathy from healthy individuals. As with the introduction of any new test, it is essential that more scientific data about use of this assay in different subject groups are available so that results can be interpreted with clinical certainty.

Standardisation of cardiac troponin I measurement: past and present

&NA; The laboratory measurement of cardiac troponin (cTn) concentration is a critical tool in the diagnosis of acute myocardial infarction (MI). Current cTnI assays produce different absolute troponin numbers and use different clinical cut‐off values; hence cTnI values cannot be interchanged, with consequent confusion for clinicians. A recent Australian study compared patient results for seven cTnI assays and showed that between‐method variation was approximately 2‐ to 5‐fold. A major reason for poor method agreement is the lack of a suitable common reference material for the calibration of cTnI assays by manufacturers. Purified complexed troponin material lacks adequate commutability for all assays; hence a serum‐based secondary reference material is required for cTnI with value assignment by a higher order reference measurement procedure. There is considerable debate about how best to achieve comparability of results for heterogeneous analytes such as cTnI, whether it should be via the harmonisation or the standardisation process. Whereas harmonisation depends upon consensus value assignment and uses those commercial methods which give the closest agreement at the time, standardisation comes closer to the true value through a reference measurement system that is based upon long‐term calibration traceability. The current paper describes standardisation efforts by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of cTnI (IFCC WG‐TNI) to establish a reference immunoassay measurement procedure for cTnI of a higher order than current commercial immunoassay methods and a commutable secondary reference material for cTnI to which companies can reference their calibration materials.

Standardization of troponin I measurements: an update.

Abstract Standardization of cardiac troponin I (cTnI) measurement is important because of the central role for diagnosis of myocardial infarction. In blood, cTnI is present as a heterogeneous mixture of different molecular species. The analytical problem caused by this heterogeneity may be circumvented by recognition of a unique, invariant part of the molecule that is common to all components of the mixture. Antibodies used for the development of cTnI assays should selectively recognize epitopes within this invariant part, leading to a consequential increase in the homogeneity of immunoassay reactivity. This should be associated with the use of a reference material that represents the natural and major antigen in blood after tissue release, i.e., the troponin complex. Although a primary reference material for cTnI is available, studies indicate that cTnI assays remain without harmony after recalibration using this material. To achieve closer comparability of cTnI values between assays, the use of a secondary reference material, consisting of a panel of human serum pools, is proposed for use by manufacturers to calibrate their assays. To assign true cTnI concentration values to this secondary reference material, establishment of a reference measurement procedure for cTnI is required. A practical approach to the development of a reference procedure could be to design an immunochemical assay with well-characterized specificity to the invariant part of the cTnI molecule and calibrated using the primary reference material. Clin Chem Lab Med 2008;46:1501–6.

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)

Abstract 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.

Discordance with 3 Cardiac Troponin I and T Assays: Implications for the 99th Percentile Cutoff

BACKGROUND We compared the 99th percentile reference intervals with 3 modern cardiac troponin assays in a single cohort and tested the hypothesis that the same individuals will be identified as above the cutoff and that differences will be explained by analytical imprecision. METHODS Blood was collected from 2005 apparently healthy blood donors. Cardiac troponin was measured with Abbott Architect STAT high sensitive troponin I, Beckman Coulter Access AccuTnI+3, and Roche Elecsys troponin T highly sensitive assays. RESULTS The 99th percentile cutoff limits were as follows: Abbott cardiac troponin I (cTnI) 28.9 ng/L; Beckman Coulter cTnI 31.3 ng/L; and Roche cardiac troponin T (cTnT) 15.9 ng/L. Correlation among the assays was poor: Abbott cTnI vs Beckman Coulter cTnI, R(2) = 0.18; Abbott cTnI vs Roche cTnT, R(2) = 0.04; and Beckman Coulter cTnI vs Roche cTnT R(2) = 0.01. Of the results above the cutoff 50% to 70% were unique to individual assays, with only 4 out of 20 individuals above the cutoff for all 3 assays. The observed differences among assays were larger than predicted from analytical imprecision. CONCLUSIONS The 99th percentile cutoff values were in agreement with those reported elsewhere. The poor correlation and concordance amongst the assays were notable. The differences found could not be explained by analytical imprecision and indicate the presence of inaccuracy (bias) that is unique to sample and assay combinations. Based on these findings we recommend less emphasis on the cutoff value and greater emphasis on δ values in the diagnosis of myocardial infarction.

Recommendations for standardized reporting of protein electrophoresis in Australia and New Zealand

Background Although protein electrophoresis of serum (SPEP) and urine (UPEP) specimens is a well-established laboratory technique, the reporting of results using this important method varies considerably between laboratories. The Australasian Association of Clinical Biochemists recognized a need to adopt a standardized approach to reporting SPEP and UPEP by clinical laboratories. Methods A Working Party considered available data including published literature and clinical studies, together with expert opinion in order to establish optimal reporting practices. A position paper was produced, which was subsequently revised through a consensus process involving scientists and pathologists with expertise in the field throughout Australia and New Zealand. Results Recommendations for standardized reporting of protein electrophoresis have been produced. These cover analytical requirements: detection systems; serum protein and albumin quantification; fractionation into alpha-1, alpha-2, beta and gamma fractions; paraprotein quantification; urine Bence Jones protein quantification; paraprotein characterization; and laboratory performance, expertise and staffing. The recommendations also include general interpretive commenting and commenting for specimens with paraproteins and small bands together with illustrative examples of reports. Conclusions Recommendations are provided for standardized reporting of protein electrophoresis in Australia and New Zealand. It is expected that such standardized reporting formats will reduce both variation between laboratories and the risk of misinterpretation of results.

Point: Put Simply, Standardization of Cardiac Troponin I Is Complicated

The analysis of heterogeneous protein analytes is very complicated to standardize, but these measurements must be viewed as fundamental to the practice of clinical chemistry. The clinical importance of selected protein measurements such as hemoglobin A1c (Hb A1c),5 thyroid-stimulating hormone, and cardiac troponin I (cTnI) is underscored by their routine use in the diagnosis, prognosis, monitoring, and management of disease, and their incorporation into professional guidelines. Standardization is difficult for proteins because there are few reference measurement procedures for these analytes, few primary (pure substance) reference materials (RMs) have been developed, and some of the RMs that are available can be used only for assay calibration with restrictions. In addition, few secondary (matrix-based) RMs with assigned values are available. In fact, the reference measurement system for the majority of clinically relevant proteins fall into the relatively weak Category 4, according to International Organization for Standardization (ISO) document 17511 (1). Furthermore, protein reference measurements sometimes appear to belong in a higher category but are in fact subject to analytical artifacts and prone to bias. By the criteria of the recently published “roadmap for harmonization” (2), standardization and harmonization of measurement would be regarded as high priority and mission critical for many proteins in laboratory medicine. cTnI is one such protein because its measurement represents the cornerstone for the diagnosis, prognosis, and management of patients with suspected and confirmed acute coronary syndromes (3, 4). Standardization is based on the concept of metrological traceability, as described, for instance, in the ISO 17511 document (1). According to ISO 17511, standardization-of-measurement results for a substance require the metrological traceability chain outlined in Fig. 1A. This chain begins with a primary reference measurement procedure, which assigns quantity values to a primary RM. Primary RMs are used to assign values …