'Aussie Normals': an a priori study to develop reference intervals in a healthy Australian population using the Beckman Coulter LH 750 Haematology Analyser as candidates for harmonised values.

Reference limits or intervals are important benchmarks or tools that help the clinician to distinguish between a result that is most likely to lie within a 'healthy' or diseased category. It has been suggested that a review of haematology reference intervals is long overdue. In this study we report on our findings for analytes routinely measured in a complete blood count (CBC) performed on the Beckman Coulter LH 750 analyser and an additional comparative study using the Beckman Coulter LH 750, the Sysmex XN and Abbott Sapphire. The results from the comparative study indicate that bias would not prevent harmonisation of reference intervals for these common haematology parameters. The results offered by the Aussie Normals study represent good candidates as the basis for harmonisation reference intervals.

'Aussie normals': an a priori study to develop clinical chemistry reference intervals in a healthy Australian population.

Development of reference intervals is difficult, time consuming, expensive and beyond the scope of most laboratories. The Aussie Normals study is a direct a priori study to determine reference intervals in healthy Australian adults. All volunteers completed a health and lifestyle questionnaire and exclusion was based on conditions such as pregnancy, diabetes, renal or cardiovascular disease. Up to 91 biochemical analyses were undertaken on a variety of analytical platforms using serum samples collected from 1856 volunteers. We report on our findings for 40 of these analytes and two calculated parameters performed on the Abbott ARCHITECTci8200/ci16200 analysers. Not all samples were analysed for all assays due to volume requirements or assay/instrument availability. Results with elevated interference indices and those deemed unsuitable after clinical evaluation were removed from the database. Reference intervals were partitioned based on the method of Harris and Boyd into three scenarios, combined gender, males and females and age and gender. We have performed a detailed reference interval study on a healthy Australian population considering the effects of sex, age and body mass. These reference intervals may be adapted to other manufacturer's analytical methods using method transference.

High-resolution SNP microarray investigation of copy number variations on chromosome 18 in a control cohort.

Copy number variations (CNVs) as described in the healthy population are purported to contribute significantly to genetic heterogeneity. Recent studies have described CNVs using lymphoblastoid cell lines or by application of specifically developed algorithms to interrogate previously described data. However, the full extent of CNVs remains unclear. Using high-density SNP array, we have undertaken a comprehensive investigation of chromosome 18 for CNV discovery and characterisation of distribution and association with chromosome architecture. We identified 399 CNVs, of which loss represents 98%, 58% are less than 2.5 kb in size and 71% are intergenic. Intronic deletions account for the majority of copy number changes with gene involvement. Furthermore, one-third of CNVs do not have putative breakpoints within repetitive sequences. We conclude that replicative processes, mediated either by repetitive elements or microhomology, account for the majority of CNVs in the healthy population. Genomic instability involving the formation of a non-B structure is demonstrated in one region.

Utilisation of sFLC assays - how well do we comply with guidelines?

INTRODUCTION: There is a paucity of data on the utilisation of the serum-free light chain (sFLC) and compliance with published guidelines. METHODS: Serum-free light chain assays requested at our institute for the diagnosis, prognosis and monitoring of plasma cell dyscrasias from July 2008 to March 2010 were compared with the International Myeloma Working Group (IMWG) consensus guidelines. RESULTS: In total, 1150 assays were performed (4.3 assays per patient, range 1-20). Eight hundred and forty-four (73%) of these were performed for multiple myeloma (MM), 188 (16%) for monoclonal gammopathy of undetermined significance, 72 (6%) for AL amyloidosis and 46 (4%) for smouldering myeloma. Of these, 49.6%, 22.9%, 1.4% and 69.6% of the monitoring assays were outside of IMWG recommendations, respectively. Of the 419 assays performed outside of guidelines for MM patients, 404 (96.4%) were due to monitoring of patients with a measurable M protein, while 24 (5.7%) were due to too frequent requesting (≤14 days) with 15 assays (3.6%) being noncompliant on both grounds. CONCLUSION: Utilisation of the sFLC assay shows reasonable adherence to guidelines within our centre. We propose to further optimise usage of the test with the help of administrative processes and education of clinicians.

NTproBNP concentrations in healthy children.

INTRODUCTION: There have been limited studies generating BNP and NT-proBNP reference intervals for paediatric populations. We prospectively assessed NT-proBNP levels in a cohort of 854 healthy school children from the Lifestyle of Our Kids (LOOK) prospective longitudinal study. MATERIALS AND METHODS: NT-proBNP analysis was performed on 172 girls and 212 boys with average age 8.1 years, 183 girls and 181 boys, average age 10.1 years and 183 girls and 180 boys with average age 11.9 years. Data were stratified according to age and gender with the median, range of results and 2.5th and 97.5th percentiles calculated RESULTS: There were no significant differences between males and females at any of the 3 study ages. Significant differences were seen between the 8 and 12 year-olds, 10 and 12 year-olds and the 8 and 12 year-old boys. DISCUSSION: Our study demonstrated that NT-proBNP concentrations in healthy children progressively decline between ages 8 and 12 years. Our selection of unambiguously healthy children produced similar median but lower 97.5th percentile NT-proBNP concentrations to previously published studies.

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.

Prognostic efficacy of cardiac biomarkers for mortality in dialysis patients.

BACKGROUND: The high prevalence of cardiovascular mortality in the end-stage renal disease population is well established. The aim of this current study was to document the relative prognostic significance of established cardiac biomarkers troponin T (TnT), troponin I (TnI), B-type natriuretic peptide (BNP) and N-terminal pro-BNP (NT-pro-BNP) in this population. METHODS: A prospective cohort study of dialysis patients undertaken in a single tertiary centre in Australia. Relevant clinical and biochemical information was collected at entry and all patients followed up prospectively without any loss to follow up. End-point of interest was all-cause mortality. Statistical analysis using Cox proportional hazards was used to study relationship between competing covariates and outcome. A total of 143 patients with a mean age of 59.67 +/- 15.49 years was followed up for a median duration of 30 months. Of these patients, 89.3% were white Australians of European ancestry. Twenty-seven per cent had an established diagnosis of diabetes mellitus. The mean concentrations (+/-SD) of TnT, TnI, BNP and N-terminal peptide pro-BNP (NT-pro-BNP) were 0.08 +/- 0.04 microg/L, 0.09 +/- 0.2 microg/L, 270 +/- 117 ng/L and 1434 +/- 591 ng/L respectively. RESULTS: Twenty-eight subjects died during the period of follow up. By univariate analysis, all cardiac markers (TnT, TnI, BNP, NT-pro-BNP and C-reactive protein) were significantly associated with an increase in mortality. On Cox proportionate hazards analysis, only albumin and NT-pro-BNP showed a significant association with mortality, with hazard ratios of 0.834, 95% confidence interval (CI) 0.779-0.893, P < 0.001, and 1.585, 95%CI 1.160-20165, P = 0.004 respectively. CONCLUSION: In patients with end-stage renal failure on dialysis NT-pro-BNP provides greater prognostic information compared with TnT and TnI.

Patient-focused outcomes following detection in a hospital-based screening programme for C282Y haemochromatosis.

BACKGROUND: Haemochromatosis is a common genetic disease in populations of a northern European origin. However, there is uncertainty as to whether it is a condition that should be screened for. AIMS: To determine the proportion of persons, in a public hospital setting, who were homozygous for the C282Y mutation for hereditary haemochromatosis and the proportion of these persons who would benefit from therapeutic phlebotomy. METHODS: All persons who had blood submitted for pathology testing, had total iron-binding capacity and iron measured and transferrin saturation calculated, and where this result exceeded 40%, genotyping for the C282Y mutation was carried out. RESULTS: Of 18,779 patients screened, 887 (5.4%) were found to have transferrin saturation greater than 40%. Thirty-five of these were homozygous for the C282Y mutation. Fourteen were previously known to be affected and six of these were non-compliant with venesection. Venesection was commenced in 5 of the 21 newly diagnosed subjects. CONCLUSIONS: The proportion of detected subjects who commenced venesection was significant. Results suggest that clinical penetrance is higher in Australia than other countries and that even in the environment of a large tertiary teaching hospital, phenotypic screening identifies cases of hereditary haemochromatosis, which are likely to benefit from treatment.

Elevated serum cardiac troponin I in older patients with hip fracture: incidence and prognostic significance.

INTRODUCTION: Cardiovascular complications are the main causes of morbidity and mortality in patients with osteoporotic hip fracture (HF). The aim of this prospective study was to evaluate the incidence and prognostic significance of elevated cardiac troponin I (cTnI) in the early peri-operative period in older patients with HF. MATERIALS AND METHODS: A blind evaluation of myocardial injury as detected by cTnI elevation in 238 consecutive older patients with low-trauma HF (mean age 81.9 +/- 7.8 (SD) years; 72% females). Data on demographic and clinical characteristics, in-hospital mortality, hospital length of stay and discharge destination were collected prospectively. Serum cTnI level was analysed from blood collected routinely in the first 72 h of hospital admission. RESULTS: Sixty-nine (29%) patients had elevated cTnI (>0.06 microg/l) but myocardial injury was clinically recognised in only 23 (33%) and only 24 (34.8%) had a history of coronary artery disease (CAD). Patients with elevated cTnI were significantly older, more often had American Society of Anaesthesiologist status score >or=3, a history of CAD or stroke and more often were current smokers than the patients without cTnI elevation. In multivariate regression analysis only age was an independent predictor of cTnI elevation. Patients with cTnI release were twice as likely to have a length of stay >or=20 days (P = 0.047) and 2.7 times more likely to be discharged to a long-term residential care facility (RCF) (P = 0.013). cTnI level >or=1 microg/l was a strong independent predictor of all-cause mortality with 98.3% specificity and 89.1% negative predictive value. CONCLUSION: Peri-operative myocardial injury is common in older HF patients but is frequently unrecognised clinically. Elevated blood cTnI level is an independent predictor of prolonged length of hospital stay (>or=20 days), need for long-term RCF and mortality (if cTnI >or=1 microg/l).

Cushing's syndrome in a clinic population with diabetes.

BACKGROUND: A recent report has suggested that occult Cushing's syndrome (CS) may be present in a significant number of patients with type 2 diabetes mellitus. The aim of this study was to determine whether any patients in our clinic population with diabetes had this condition. METHODS: One hundred and seventy-one consecutive overweight attendees at the diabetic clinic were enrolled in a study to assess the presence of occult CS. The initial screen was with the 1 mg overnight dexamethasone suppression test and follow-up testing, where indicated was with a 24 h collection for urine-free cortisol. RESULTS: Thirty-one of 171 patients had a positive result from the overnight dexamethasone suppression test. Follow-up testing with 24 h urine-free cortisol reduced the number of patients with positive results to 3. Two of these were shown to have alcoholic pseudo-CS. The third patient has had several high urine-free cortisol results, in the presence of normal scans of pituitary and adrenals. He has no stigmata of CS and is being observed. CONCLUSION: Based on the results of our study, there would appear to be little value in screening type 2 diabetics for CS, in the absence of clinical suspicion.

Effects of hypoxia/reperfusion injury on drug disposition in the rat isolated perfused liver.

1. Ischaemia-reperfusion injury is known to be associated with a range of functional and structural alterations in the liver. However, the effect of this injury on drug disposition is not well understood. The present study was designed to examine the effects of hypoxia/reperfusion on the disposition of glutamate and propranolol in the rat isolated perfused liver. Both glutamate and propranolol are mainly metabolised in the pericentral region of the liver. 2. Hypoxia/reperfusion was established using the slow flow-reflow method of perfusion in both anterograde and retrograde perfusion. Glutamate metabolism was measured by the recovery of [(14)C]-glutamic acid and [(14)C]-labelled metabolites in a single pass in both anterograde and retrograde perfusion in the presence of a steady state concentration of unlabelled glutamic acid. Propranolol disposition, mean transit time and normalized variance were assessed from the outflow concentration-time profile of unchanged [(3)H]-propranolol determined after a bolus injection of [(3)H]-propranolol using HPLC and liquid scintillation counting. 3. Hypoxia/reperfusion of livers did not affect oxygen consumption, but caused significant changes in enzyme release, lignocaine hepatic availability and bile flow. 4. Hypoxia/reperfusion did not affect the hepatic metabolism of glutamate to carbon dioxide or the hepatic extraction of propranolol. Small but significant changes were evident in the distribution parameters of mean transit time and vascular disposition for the hypoxic-ischaemic liver. 5. It is concluded that reperfusion injury induced by slow flow-reflow perfusion did not influence the extraction of glutamate or propranolol, but may have affected pericentral morphology and solute distribution.

The relationship between dosing of alosetron and discontinuation patterns reported by patients participating in a follow-up programme.

BACKGROUND: Alosetron was reintroduced for treatment of irritable bowel syndrome with a risk management programme in November 2002. Recommended starting dosage was 1 mg/day for 4 weeks. If symptoms remained uncontrolled, dosage could be changed to 2 mg/day. AIM: To describe alosetron dosages and associated patient characteristics from the Lotronex follow-up survey programme. METHODS: Patients reported dosages of alosetron at start and regular follow-up intervals. Analyses were limited to patients with the potential to have at least 1 year of follow-up (enrolled between 9 December 2002 and 31 December 2003). RESULTS: At baseline, 75% of 2817 respondents reported starting on 1 mg/day, 17% on 2 mg/day and 8% on other doses. Adherence to recommended starting dosage did not vary by status at end of follow-up, previous alosetron experience or age. At last reported dose, 50% of respondents were using 1 mg/day; 29% were using 2 mg/day. Discontinuation was not related to baseline doses. Longer times to discontinuation were associated with previous use, symptoms for more than 6 months and dose change throughout follow-up. CONCLUSIONS: High adherence to recommended dosing at baseline and follow-up suggests that the risk management programme is encouraging safe use of alosetron, including adherence to dosing recommendations.

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.

pH-regulated secretion of a glyceraldehyde-3-phosphate dehydrogenase from Streptococcus gordonii FSS2: purification, characterization, and cloning of the gene encoding this enzyme.

Streptococcus gordonii and other viridans streptococci (VS) are primary etiologic agents of infective endocarditis, despite being part of the normal oral microflora. Recently, a surface-bound glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been found on the cells of all tested streptococcal species, where it has been implicated as a virulence factor. In contrast, we observed that a soluble extracellular GAPDH was the major secreted protein from S. gordonii FSS2, an endocarditis strain. The biochemical properties and gene sequence of S. gordonii GAPDH are almost identical to those of other streptococcal GAPDHs. Growth at defined pHs showed that secretion of GAPDH is regulated by environmental pH. GAPDH was primarily surface-associated at growth pH 6.5 and shifted to > 90% secreted at growth pH 7.5. Others have identified S. gordonii promoters that are up-regulated by a pH shift similar to that experienced by organisms entering the blood stream (neutral) from the oral cavity (slightly acid). Analysis of our results suggests that secretion of GAPDH may be a similar adaptation by S. gordonii.

Automated measurement of unsaturated iron binding capacity is an effective screening strategy for C282Y homozygous haemochromatosis.

BACKGROUND: C282Y hereditary haemochromatosis is an appropriate condition for population screening. Transferrin saturation, the best screening test to date, is relatively expensive, labour intensive, and cannot be automated. Unsaturated iron binding capacity is a surrogate marker of transferrin saturation and its measurement can be automated. AIMS: To evaluate a screening strategy for C282Y hereditary haemochromatosis in a tertiary hospital environment based on unsaturated iron binding capacity as the initial screening test. METHODS: Measurement of unsaturated iron binding capacity was adapted to the main laboratory analyser. An unsaturated iron binding capacity of less than 30 micromol/l was identified as an appropriate decision point and 5182 consecutive subjects were screened over 28 consecutive days. RESULTS: Of those screened, 697 had an unsaturated iron binding capacity less than 30 micromol/l. Of these, transferrin saturation was greater than 40% in 294. A total of 227 were able to be genotyped for the C282Y mutation. Nine subjects homozygous for C282Y were identified. Based on full cost recovery, affected persons were identified at a cost of Aus$2268.77 per case (approximately US$1496). CONCLUSION: Automated measurement of unsaturated iron binding capacity enables a cost effective, large scale population screening programme for C282Y hereditary haemochromatosis to be developed.