Standardization of high-sensitivity immunoassays for measurement of C-reactive protein; II: Two approaches for assessing commutability of a reference material.

BACKGROUND: We evaluated the commutability of a proposed reference material (PRM), with a formulation based on dilution of Certified Reference Material 470 (CRM470), for 24 high-sensitivity C-reactive protein (hsCRP) methods. We also investigated whether calibration by use of PRM was effective in harmonizing results. METHODS: A set of 40 native clinical samples was measured along with PRM and 3 dilutions of PRM. We used weighted least-squares polynomial regression (WLS/PR) to perform comparisons between all method combinations and to calculate normalized residuals for the PRM. The PRM was considered noncommutable if any of the normalized residuals for a method pair was >2. Correspondence analysis (CA) was used to explore the multidimensional relationships between methods and samples to evaluate if the PRM had properties similar to native clinical samples. Clinical sample results from the methods for which PRM was commutable were recalibrated based on the PRM results, and ANOVA was used to estimate the CVs before and after recalibration. RESULTS: After omitting data for 9 methods because of poor precision or procedural flaws, we used data from the 15 remaining methods to evaluate commutability. Using both WLS/PR and CA we found that PRM was noncommutable with 1 method. We found modest improvement in total and among-method CVs when PRM was used to harmonize the results from the 14 methods for which it was commutable. CONCLUSIONS: A PRM with a formulation based on dilution of CRM470 was commutable with native clinical samples for 14 of 15 hsCRP methods that had acceptable precision. For those methods the use of PRM may contribute to improved harmonization of results for native clinical samples.

Performance evaluation of the ADVIA Centaur anti-HBe and HBeAg assays.

BACKGROUND: Detection of HBeAg and anti-HBe is valuable for the evaluation and therapeutic management of hepatitis B infection. OBJECTIVES: To determine the clinical performance of the newly CE-approved(a) HBeAg and anti-HBe assays on the fully automated, random access ADVIA Centaur immunoassay system. STUDY DESIGN: Patient samples collected at two sites were used to compare the ADVIA Centaur assays to Abbott AxSYM assays. Consensus of discordant results was reached using Roche Elecsys assays. Additionally, two well-characterized seroconversion panels were evaluated. RESULTS: The ADVIA Centaur HBeAg assay sensitivity was 100% and specificity was 99.5%. The ADVIA Centaur anti-HBe assay sensitivity was 100% and the resolved specificity was 98.2%. Fewer samples required retesting with the ADVIA Centaur assays than with the AxSYM. In two well-characterized seroconversion panels, the ADVIA Centaur anti-HBe assay detected anti-HBe 20-25 days earlier than the AxSYM assay; the ADVIA Centaur and AxSYM HBeAg assays detected HBe reactivity on the same day. CONCLUSIONS: The ADVIA Centaur HBeAg and anti-HBe assays demonstrated good sensitivity and specificity, and thus are suitable for clinical use. Their novel algorithms require reduced retesting, suggesting these assays may be more cost effective.

Glycogen phosphorylase BB in acute coronary syndromes.

The diagnosis of myocardial damage is preferably based on measurement of the cardiac-specific troponins. However, there is an emerging need for early, specific cardiac markers. One potential candidate is the glycogen phosphorylase BB isoenzyme (GPBB). We investigated the use of a new, commercially available GPBB ELISA assay in 61 patients presenting with an acute coronary syndrome (37 acute myocardial infarction, 24 unstable angina pectoris) in comparison to established cardiac markers such as troponin T, creatine kinase isoenzyme MB (CKMB) mass, and myoglobin. Blood samples were obtained on arrival, as well as 1, 2, 3, 4, 8, 12 and 24 h later. GPBB plasma concentrations were elevated in 90.9% of patients 1 h after onset of chest pain and increased to 100% at 4-5 h. Within the first 6 h, GPBB showed the highest sensitivity (95.5-100%) and high specificity (94-96%) compared to myoglobin (85-95% sensitivity) and CKMB mass (71.4-91.3% sensitivity). As expected, troponin T showed high specificity (100%) and sensitivity >95% later in the time course (>or=3 h). In un-stable angina pectoris patients, a very high rate of elevated GPBB was observed (93.9% at 3 h) compared to myoglobin (66.7%). Cardiac troponin T and CKMB were only elevated in 33.8% and 55.0% of these patients, respectively. In conclusion, GPBB is a promising marker for the early diagnosis of acute coronary syndromes and could probably act as a marker of ischemia. However, further studies on specificity and development of a fast, automated assay are necessary before GPBB can be recommended as a routine diagnostic tool.

First WHO/IFCC International Reference Reagent for Lipoprotein(a) for Immunoassay--Lp(a) SRM 2B.

Lipoprotein(a) is an important predictor of cardiovascular disease risk. The lack of internationally accepted standardization has impeded the broad application of this lipoprotein in laboratory medicine. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), through its Working Group on Lipoprotein(a) and together with research institutions and several diagnostic companies, have succeeded in developing an international reference material that is intended for the transfer of a lipoprotein(a) concentration to manufacturers' master calibrators. IFCC SRM 2B has recently been accepted by the WHO Expert Committee on Biological Standardization as the 'First WHO/IFCC International Reference Reagent for Lipoprotein(a) for Immunoassay'. The assigned unitage of 0.1071 nanomoles of lipoprotein(a) per vial is traceable to the consensus reference method for lipoprotein(a) and will enable conformity by diagnostic companies to the European Union's Directive on In vitro Diagnostic Medical Devices for the metrological traceability of calibrator materials.

European performance evaluations of the ADVIA Centaur infectious disease assays: requirements for performance evaluation according to the European directive on in vitro diagnostics.

The following article reports on newly developed ADVIA Centaur immunoassays (Bayer HealthCare LLC, Diagnostics Division; Tarrytown, NY, USA) for the detection of markers of hepatitis B infection in human serum and plasma. These fully automated assays detect antibodies to hepatitis B surface antigen (anti-HBs), total antibodies to hepatitis B core antigen (anti-HBc Total) and IgM antibodies to hepatitis B core antigen (anti-HBc IgM). The ADVIA Centaur HBV assays employ magnetic particle separation technology with direct chemiluminescence for optimal assay performance. The three ADVIA Centaur HBV assays were tested in extensive performance evaluations at two sites in Europe. Prospectively collected specimens from HBV infected subjects, blood donors, hospitalized/clinical patients and HBV vaccinees were tested under routine laboratory conditions to generate performance data. The studies resulted in the following clinical performance: overall diagnostic specificity > 99.9%, i.e. 100% for ADVIA Centaur Anti-HBs, 100% for ADVIA Centaur HBcIgM and 99.94% for ADVIA Centaur HBc Total. All of the assays showed excellent diagnostic sensitivity, as follows: 99.0% for ADVIA Centaur Anti-HBs, 98.5% for ADVIA Centaur HBcIgM and 100% for ADVIA Centaur HBc Total. Interfering substances and potential cross-reacting samples produced no effects on the results in any of the three ADVIA Centaur HBV assays. Evaluations using HBV seroconversion panels resulted in comparable or better results as compared to the reference assay(s). The performance evaluation data clearly demonstrate that the three ADVIA Centaur HBV assays are specific and sensitive automated immunoassays for the detection of antibodies to hepatitis B virus. Furthermore, these data indicate that assay performance is comparable, if not better, than the performance of currently marketed assays. Additionally, these assays have all of the advantages of being on the ADVIA Centaur immunoassay system including high throughput and full automation. Finally, these data, which had been submitted to the appropriate regulatory agencies (Notified Bodies) in Europe, have resulted in these assays now being CE marked and, thus, being readily available in the European market.

An update on laboratory productivity with infectious disease assays on the Bayer ADVIA Centaur Immunoassay System.

New biological materials and advances in robotic and computer technologies have enabled the development of automated systems designed for high-performance infectious disease immunoassays and nucleic acid amplification. The fully automated, random access Bayer ADVIA Centaur immunoassay system, offering testing for fertility, therapeutic drug monitoring, infectious disease, allergy, cardiovascular, anemia, oncology, TDMs and thyroid, has been specifically designed for use in large-volume laboratories. New immunoassay tests have been developed for the ADVIA Centaur for the hepatitis A virus, hepatitis B virus, hepatitis C virus and HIV. These assays have undergone extensive performance evaluation using samples designated in the CTS in support of obtaining the Communautés Européennes (CE) mark for European market distribution. The ADVIA Centaur Immunoassay System represents an optimal platform for infectious disease testing because of its flexibility in allowing many different assay formats and protocols with multiple incubation steps and washes coupled with its combination of magnetic particle separation and chemiluminescent detection. Additional quality features of the system design are the sample integrity verification/check, the use of disposable sample pipette tips, clot detection, the ability for sensing liquid levels, the reagent aspiration verification/check, the automatic cascade reflex testing, repeat testing, and automated reagent inventory. The ADVIA Centaur has a maximum test throughput of 240 tests per hour. Minimal hands-on time is required as a result of the large onboard capacity for reagents and supplies combined with automated maintenance and monitoring features, which streamline operations and result in a walk-away through-put of up to 840 tests.

Standardization of immunoassays for measurement of myoglobin in serum. Phase I: evaluation of candidate secondary reference materials.

BACKGROUND: Myoglobin is a low-molecular weight protein present in the cytosol of striated muscles. Its concentrations in serum can be measured by immunoassays and are used as an early indicator of myocardial necrosis. Since variability among commercial myoglobin assays exists, standardization of myoglobin assays is needed. METHODS: An international collaborative study was organized with the involvement of seven companies using 12 different automated platforms for measuring myoglobin. Five candidate secondary, i.e., matrixed, reference materials were assayed in relation to linearity, imprecision, recovery rate and commutability to demonstrate a possible identity between the materials and the usual routine serum samples. RESULTS: One lyophilized candidate material (human heart myoglobin in human serum) was selected as the most suitable secondary reference material, based on the criteria examined. Used as a calibrator a posteriori, the bias between the various myoglobin assays for a frozen human serum pool was reduced from 32% to 13%. CONCLUSION: This study provides the basis for the selection of an internationally recognized secondary reference material.

Evaluation of imprecision for cardiac troponin assays at low-range concentrations.

BACKGROUND: The European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction (MI) has recommended that an increased cardiac troponin should be defined as a measurement above the 99th percentile value of the reference group. A total imprecision (CV) at the decision limit of

The new European directive on in vitro diagnostics.

The Directive on in vitro Diagnostic Medical Devices (IVDD 98/79/EC) was officially adopted by the European Union (EU) on December 7, 1998. The IVDD aims to supplement the legal framework of the European Community, which governs the conditions for the placing on the market of medical devices, by extending the already implemented legislation to the category of in vitro diagnostic medical devices (IVDs). They consist of those devices, including reagents and reagent products, calibrator materials or instruments, as well as specimen receptacles, intended by the manufacturer for the in vitro analysis of specimens derived from the human body. This directive has introduced at the European level common regulatory requirements across Europe for the safety, quality and performance of in vitro diagnostics (IVDs), incorporating them into medical device legislation. It harmonizes the conformity assessment procedures to be applied by manufacturers before they place IVDs on the market. For certain products expressly specified in the directive, of which the most important are used for the evaluation of the safety of blood supply and patient testing, the manufacturer will have to take into account in their performance evaluation the so-called "Common Technical Specifications" (CTS). These are needed to establish the performance characteristics of the IVDs in evaluation and have the same status as harmonized standards. In the meantime, the IVD directive has been transposed into national law in all EU countries. During a transitional period ending in December 2003, manufacturers will have the option of following pre-existing national regulatory processes or taking their IVDs through the new procedures as specified in the directive. Following this, starting from 7th December 2003, adherence to the directive regulations will become mandatory, and only IVDs bearing the "Communautés Européennes (CE) mark" can then be sold in the EU.

Standardization of immunoassays for measurement of high-sensitivity C-reactive protein. Phase I: evaluation of secondary reference materials.

BACKGROUND: Inflammation contributes to the development and progression of atherosclerosis, and C-reactive protein (CRP) can be used as a marker to assess risk for cardiovascular diseases. As variability among existing high-sensitivity CRP (hsCRP) assays can lead to misclassification of patients and hamper implementation of population-based medical decision points, standardization of hsCRP assays is needed. METHODS: We evaluated five proposed secondary reference materials, including two diluted preparations of Certified Reference Material 470 (CRM470), two preparations of a serum-based material with recombinant CRP added, and one serum-based material with isolated CRP added. Twenty-one manufacturers participated in the comparison with 28 different assays. We examined imprecision, linearity, and parallelism with these materials and with fresh serum. RESULTS: All materials had similar imprecision; CVs for the undiluted materials were 2.1-3.7%. None of the materials was linear across all assays. Each had between one and three cases of nonlinearity, with one preparation of CRM470 having the fewest cases of nonlinearity. Although none of the materials was parallel across all assays, the differences in slope from fresh serum were similar across all assays. CONCLUSIONS: All materials performed similarly with regard to imprecision, linearity, and parallelism. As one preparation of CRM470 had slightly better characteristics than the other materials and because CRM470 had been certified previously as a reference material for the acute-phase reactant range, it will be used in the next phase to standardize hsCRP assays.

Reference Materials for the Standardization of the Apolipoproteins A-I and B, and Lipoprotein(a).

Measurement of the apolipoproteins A-I and B, and lipoprotein(a) enable identification of individuals at increased risk of cardiovascular disease. However, the lack of standardized methods to measure these risk markers has resulted for many years in the non-comparability of values and often a conflicting interpretation of clinical studies. Due to the collaborative efforts of the International Federation of Clinical Chemistry and Laboratory Medicine, research organizations, clinical chemistry laboratories and diagnostic companies, secondary reference materials for the apolipoproteins A-I and B, and lipoprotein(a) have been prepared and tested for their ability to harmonize test values. SP1-01 and SP3-07 WHO-IFCC reagents are now available to manufacturers for use in the value transfer of apolipoprotein A-I and B values to master calibrators, and PRM is proposed to be the secondary reference material for Lp(a). By the worldwide use of such reference materials a better traceability and standardization of measurement is being achieved in the clinical laboratories.