Validation of NICE diagnostic guidance for rule out of myocardial infarction using high-sensitivity troponin tests.

OBJECTIVE: To validate the National Institute for Health and Care Excellence (NICE) recommended algorithms for high-sensitivity troponin (hsTn) assays in adults presenting with chest pain. METHODS: International post hoc analysis of three prospective, observational studies from tertiary hospital emergency departments. The primary endpoint was cardiac death or acute myocardial infarction (AMI) within 24 hours of presentation, and the secondary endpoint was major adverse cardiac events (MACE) at 30 days. RESULTS: 15% of patients were diagnosed with non-ST elevation myocardial infarction (MI) on admission. The hsTnI algorithm classified 2506/3128 (80.1%) of patients as 'ruled out' with 50 (2.0%) missed MI. 943/3128 (30.1%) of patients had a troponin I level below the limit of detection on admission with 2 (0.2%) missed MI. For the hsTnT algorithm, 1794/3374 (53.1%) of patients were 'ruled out' with 7 (0.4%) missed MI. 490/3374 (14.5%) of patients had a troponin T below the limit of blank on admission with no MI. MACE at 30 days occurred in 10.7% and 8.5% of patients 'ruled out' defined by the hsTnI and hsTnT algorithms, respectively. CONCLUSIONS: The NICE algorithms could identify patients with low probability of AMI within 2 hours; however, neither strategy performed as predicted by the NICE diagnostic guidance model. Additionally, the rate of MACE at 30 days was sufficiently high that the algorithms should only be used as one component of a more extensive model of risk stratification. TRIAL REGISTRATION NUMBER: ACTRN12611001069943, NCT00470587; post-results.

Analytical characteristics of the Roche highly sensitive troponin T assay and its application to a cardio-healthy population.

BACKGROUND: It is desirable that current assays for cardiac troponin (cTn) are able to meet the recommended criterion that the diagnosis and risk assessment of patients present with symptoms of myocardial infarction requires a rise and fall in cTn with at least one point above the 99th percentile of a reference population. We have evaluated the analytical characteristics of the new highly sensitive troponin T (hs-TnT) assay to see if it meets this criterion and applied it to a carefully defined, cardio-healthy Australian reference population. METHODS: An imprecision profile was determined for the Roche hs-TnT assay (Roche Diagnostics, Sydney, Australia) using multiple samples analysed on nine separate occasions. The distribution of troponin T was determined using 104 samples from a cardio-healthy population. RESULTS: The new hs-TnT assay meets the specifications of a coefficient of variation of 10% at the 99th percentile of our cardio-healthy reference population. Of the 104 samples analysed 44 showed troponin T concentrations above the manufacturer's quoted limit of detection. Age and gender differences in the median and 99th percentile troponin T concentration were observed. CONCLUSIONS: The new hs-TnT assay shows improved precision and sensitivity at very low troponin concentration. We have shown that a significant number of individuals in this cardio-healthy population had detectable circulating troponin concentration. With many apparently healthy people having detectable troponin, clinical judgement will become more important in interpreting troponin results.

Diagnostic and prognostic utility of the serum free light chain assay in patients with AL amyloidosis.

BACKGROUND: Organ dysfunction in AL amyloidosis is related to the production and deposition of amyloidogenic monoclonal light chains. These pathological light chains can now be quantified using the recently developed serum free light chain assay. METHODS: We retrospectively reviewed 31 patients with AL amyloidosis to determine the frequency of abnormal free light chain assay results at diagnosis and whether changes in the serum free light chain assay predict outcome after therapy. RESULTS: An abnormal free light chain assay was found in 30 of 31 patients (97%) at the time of diagnosis. In the subset of our patients who received treatment for AL amyloidosis, a >50% reduction of the pathological free light chain following treatment was shown to predict improved overall survival. In our series of analyses, achievement of greater magnitudes of reduction of the free light chain result did not appear to provide additional prognostic information, nor did the baseline free light chain result predict outcome. CONCLUSION: Our findings support the use of the free light chain assay in the diagnostic work-up of patients with suspected AL amyloidosis, and also as a sensitive biomarker of response to therapy.

The comparative analytical performance of four troponin I assays at low concentration.

BACKGROUND: Low troponin concentrations have been shown to be informative in the prognosis of acute coronary syndrome. We have investigated the analytical performance of four commonly used cardiac troponin I methods at concentrations approaching their analytical limit of detection. METHOD: We assayed 167 patient samples within 24 h of collection using the Beckman Coulter AccuTnI, Dade Behring Dimension, Abbott AxSYM and Bayer Centaur methods and compared their relative analytical performance. RESULTS: Of the four assays compared, the AccuTnI was observed to have greater sensitivity at low concentrations. Using the limit of detection as the threshold, the Beckman assay showed superior performance at concentrations corresponding to a 20% coefficient of variation (CV), the Dade assay had a similar performance; and at concentrations corresponding to 10% CV most assays provide similar information. CONCLUSION: The newer or recently modified assays such as the Beckman Coulter AccuTnI and Dade Behring assays are best able to identify very low concentrations of troponin.

Identification of an apolipoprotein(e) variant associated with type III hyperlipoproteinaemia in an indigenous Australian.

As a result of testing for lipid and apolipoprotein(e) (apo E) phenotype status of an indigenous Australian community, an apo E variant associated with type III hyperlipoproteinaemia has been identified. Apo E phenotype was determined by analysis of VLDL by isoelectric focusing, and genotype on DNA amplified by polymerase chain reaction, using two different restriction enzyme isotyping assays. Phenotypes and genotypes were discordant in samples from two subjects and an abnormal-sized restriction fragment was also observed in their genotyping gel patterns. DNA sequencing studies revealed this was due to a single nucleotide deletion, 3817delC, at amino acid 136 on apo E. This resulted in a new reading frame and the premature termination of the apo E protein due to a stop codon (TGA) at nucleotide 4105. The variant apo E null allele showed a recessive mode of inheritance and, in combination with the E2 allele, resulted in the type III hyperlipoproteinaemic phenotype but when inherited with the E4 allele had no marked effect on plasma lipids.

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.

The lack of standardization of cardiac troponin I assay systems.

The lack of standardization of cardiac troponin I (cTnI) assay systems has meant an inability to compare absolute values between methods, and non-traceability of cTnI measurement in clinical laboratories. The present study tested whether a common calibrator for cTnI would harmonize values between different methods. Twenty fresh-frozen plasma samples and a common calibrator were analyzed in duplicate using the Dade Behring Stratus II, Chiron ACS:180, Abbott AxSYM, and Beckman Coulter Access immunoassay systems. Uncorrected cTnI values varied up to 60-fold between systems, and when correlated to the Stratus, linear regression slopes were 0.97 (ACS:180), 4.86 (AxSYM), and 0.03 (Access), verifying the differences in calibration. After correction for calibration differences by reference to the common calibrator, among-assay CV ranged from 2.7% to 55%, and was >25% for eight samples. However, the exclusion of Access results reduced the CV to 32% with only four outliers. The results show that comparable cTnI values between methods are possible by the use of a common calibrator. The lack of method harmonization for some samples may be due to non-equal antibody immunoreactivity of different plasma cTnI forms. The complete standardization of cTnI measurement requires both a secondary reference material and standardization of manufacturers' cTnI antibodies.

Plasma homocysteine levels in indigenous Australians.

OBJECTIVES: To determine plasma homocysteine levels in indigenous Australians living in urban areas, and the relationship of these levels with other risk factors in this population. DESIGN: Cross-sectional study. SUBJECTS AND SETTING: 365 urban indigenous Australian subjects, 153 men and 212 women, mean (SE) age 42 (1) years, ascertained without regard to history of atherosclerotic disease, in collaboration with community-based health centres in five indigenous communities in south-east Queensland, 1997-1998. MAIN OUTCOME MEASURES: Plasma homocysteine levels, age, sex, smoking history, metformin therapy, history of atherosclerotic vascular disease, serum creatinine level, red cell folate and serum vitamin B12 levels. RESULTS: 89 subjects (24%) had plasma homocysteine levels 15 mumol/L or above. Homocysteine levels were higher in men than in women (men: 14.4 mumol/L; 95% confidence interval [CI], 13.6-15.2; women: 11.9 mumol/L; 95% CI, 11.4-12.5) (P < 0.001); correlated with age (P < 0.001); higher in current smokers (P = 0.02); higher in subjects taking metformin therapy (P = 0.007); and higher in subjects with a history of atherosclerotic vascular disease (P < 0.001). Homocysteine levels were also correlated with serum levels of creatinine (P < 0.001), red cell folate (P < 0.001), and vitamin B12 (P < 0.001). CONCLUSIONS: These data indicate that the high plasma levels of homocysteine of Australian indigenous subjects are associated with a history of vascular disease, and correlated with, among other things, smoking, and folate and vitamin B12 nutritional deficiency. These are potentially reversible risk factors, and our data suggest that focusing public health initiatives on these issues may reduce the high prevalence of cardiovascular disease in the Australian indigenous population.

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.

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.

Measurement of Michaelis constant for human erythrocyte transketolase and thiamin diphosphate.

Human erythrocyte transketolase could be resolved from thiamin diphosphate (TDP) by acidification of the ammonium sulfate precipitate to pH 3.5, but not by other tested procedures. Resolution was 98% by chemical measurement of residual thiamin and 95% by residual enzyme activity. Reconstitution of the resolved preparation by incubation with TDP was dependent upon TDP concentration, duration, temperature, and the presence of dithiothreitol. At low TDP concentrations, 1 h was required for maximum activation; kinetic analysis then yielded an apparent Km value for TDP of 65 nM (SD 14 nM) from 100 erythrocyte lysates and similar values for reconstituted resolved preparations previously purified 400-fold and 10,000-fold. Velocity data obtained by transketolase assays in which the TDP was added to resolved preparations simultaneously with substrates yielded an apparent Km value for TDP of 2.3 microM (SD 1.6 microM) from 114 erythrocyte lysates and similar values for purified preparations. The recovery of activity following resolution and reconstitution ranged from 21 to 60% from lysates and 38 to 70% from purified preparations. Residual ammonium sulfate up to 4.9 mM decreased the apparent Km value for TDP, while a concentration of 11.3 mM increased the value in a manner competitive with TDP and with an apparent Ki value of 2.3 mM. The spectrophotometric assay of transketolase activity was greatly affected by storage of frozen solutions of the substrate ribose 5-phosphate.

High-resolution computed tomography for the comparative study of fossil and extant bone.

Special methods have been developed to use computed tomography (CT) for 3-dimensional imaging of both fossil and extant bones. Most commercial CT scanners cannot display the internal structure of fossils because the very high density is beyond the upper limits of the normal CT number scale (Hounsfield scale) of the scanners. X-ray projections from CT scans of fossils were modified by scaling the data to provide an expanded CT number scale, allowing the internal structure of highly fossilized objects to be visualized. These images were compared with state-of-the-art, high-resolution CT images of extant bone. Special image reformatting software was used to provide qualitative and quantitative 3-dimensional imaging. The recent rapid advances in CT technology have made this imaging modality the procedure of choice in much of diagnostic radiology. Use of this tool in paleoanthropology has been limited in the past by restricted access to scanners. However, new developments in CT will make this technique available to many researchers in the near future.