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.

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.

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.

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).

'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.

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.

'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.

Longitudinal study of bone turnover after acute spinal cord injury.

Increased bone turnover is a sequel of spinal cord injury (SCI) and predisposes to a number of clinically relevant complications, including osteoporosis and fractures. There are limited data available regarding the changes in modern markers of bone metabolism after SCI. We report a 6-month longitudinal follow-up of biochemical markers of bone metabolism (free and total deoxypyridinoline, total pyridinoline, N-telopeptide, osteocalcin, and total alkaline phosphatase) and bone mineral densitometry in 30 subjects with acute SCI. Markers of bone formation showed only a minor rise, remaining within the reference range. In contrast, markers of bone resorption showed a significant rise after acute SCI, peaking around weeks 10-16, with values up to 10 times the upper limit of normal. Paired bone mineral densities (n = 11; on the average, determined 14 weeks apart) showed no change at the hip, lumbar spine, or radius, but demonstrated a decrement in the entire lower limbs. changes in biochemical markers of bone formation and resorption were comparable in patients with quadriplegia and paraplegia, except for a greater increase in quadriplegics in pyridinoline, expressed as a percentage of baseline. In conclusion, a marked increase in bone resorption and modest changes in bone formation occur after SCI, and possibly increased bone resorption occurs in quadriplegia.

Use of monoethylglycinexylidide as a liver function test in the liver transplant recipient.

The hepatic conversion of lignocaine to monoethylglycinexylidide (MEGX) has been used as a real time monitor of liver function in liver transplant recipients. Data are reported for the first 4 weeks after transplant in 50 consecutive orthotopic liver grafts in 47 adults. The MEGX concentration was significantly depressed by approximately 50% in those patients in whom there was a complicated clinical course (excluding steroid-sensitive rejection) after transplantation, compared with patients in whom major complications did not occur. The MEGX concentration in the recipients after transplant was independent of the donor MEGX concentration, but, in addition to the patient's clinical status, was strongly influenced by the recipients pretransplant biochemical profile, being inversely related to the pretransplant bilirubin concentration. MEGX concentrations < 25 micrograms/L in the first 36 hr after revascularization were predictive of greater morbidity and mortality.

Simple procedures can markedly enhance automated immunoassay performance.

Theoretically, optimal performance for an immunoassay system is achieved when both the interassay and within-run precisions are identical. Using the Ciba Corning ACS:180 automated immunoassay system, the authors made two simple changes to the operating procedures that allowed near-optimal analytic performance (as assessed with the interassay coefficient of variation determined by the protocol of the National Committee for Clinical Laboratory Standards) for four of six hormones: thyroid-stimulating hormone, luteinizing hormone, prolactin, and human chorionic gonadotropin. At low hormone concentrations, the 20% interassay coefficients of variation for the hormones assayed were as follows: free tetraiodothyronine, 1.74 pM; thyroid-stimulating hormone, .033 mIU/L; luteinizing hormone, .21 U/L; follicle-stimulating hormone, .69 U/L; prolactin, 5.03 mU/L; and human chorionic gonadotropin, 1.52 mU/L. The operational enhancements improved the analytic performance of the assay for all hormones assessed compared with the performance of previously used isotopic immunoassays.

Heterophilic antibodies remain a problem for the immunoassay laboratory.

The prevalence of heterophilic antibody interference in a modern immunochemiluminometric assay containing blocking agents was determined using thyrotropin as an illustrative example. Serum samples were obtained from 295 consecutive patients who underwent routine thyroid function testing. The following versions of the thyrotropin assay were used: protocol A (zero blocker), protocol B (routine blocker concentration), and protocol C (extra blocker). Ten patients (prevalence 3.4%) had significant levels of heterophilic antibodies (protocol A value greater than 9 SD from the protocol B value). The observed thyrotropin levels for protocols B and C were the same for all patients, consistent with the reagent blockers in routine assays adequately eliminating heterophilic antibody interference. However, seven more patients (0.03%) in series of 21,000 assessed by routine thyroid function testing had discordant results because of a concentration of heterophilic antibodies so high as to overwhelm the added blocking agents.

Pseudohyponatraemia, hypercholesterolaemia, and primary biliary cirrhosis.

A 62 year old woman with primary biliary cirrhosis was found to have a plasma sodium concentration of 115 mmol/l. Follow up showed this to be a "pseudohyponatraemia" due to a massively raised serum cholesterol concentration of 78 mmol/l. Electrophoresis of serum lipoproteins and of the lipoproteins and apolipoproteins in fractions isolated on density-gradient ultracentrifugation showed that the major portion of the serum cholesterol was being transported with lipoprotein-X. Low density lipoprotein-cholesterol concentration was also raised. Lipoprotein-X contained, in addition to albumin and apolipoprotein C, apolipoprotein E. This case is of interest because of the degree of hypercholesterolaemia, its association with pseudohyponatraemia, and the unequivocal demonstration of apolipoprotein E associated with lipoprotein-X.

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.

Urgent ("stat") testing in chemical pathology--position paper.

Increasingly heavy use is made of the pathology laboratory in many hospitals. This can cause problems, particularly with regard to urgent ("stat") tests, where a result is required in a short time. Many small laboratories are forced to offer a wide battery of tests, many of which may not help greatly in the acute diagnosis and/or management of the patient, and are expensive and inconvenient to perform. This paper addresses the questions as to which (chemical pathology) analytes should be available as emergency ("stat") tests, and how long should it take for results to come back.

Glutaraldehyde concentration in the sterilization of endoscopes.

Controversy over appropriate disinfection of endoscopes continues with the most commonly utilized solution in Australian hospitals, despite concern over its efficacy, being 1% glutaraldehyde (AIDAL). Review of the in-use concentrations of glutaraldehyde at Princess Alexandra Hospital has documented a significant dilutional effect in 1% glutaraldehyde by gastrointestinal endoscopes. Each hospital should determine the minimum in-use concentration acceptable for any given disinfecting solution and any given range of endoscopes. Once this level is reached, a solution change should routinely occur. Such decisions can only be made by regularly monitoring in-use concentrations of disinfectants.

Application of an immunoradiometric assay for thyrotrophin in evaluation of thyroidal and nonthyroidal disease states.

We evaluated an immunoradiometric assay for serum TSH (IRMA-TSH) and compared it with established indices of thyroid function in 208 patients with either frank thyroid disease, conditions of abnormal thyroxine protein binding, or conditions which are known to produce discordant thyroid results, viz. pregnancy or estrogen treatment and nonthyroidal illness (NTI). As expected, a wide scatter of TSH results was found in treated thyroid disease: 53 patients (53%) from both groups (initially hypo- or hyperthyroid) had TSH values less than 0.5 mU/l, but only one was considered to be clinically mildly toxic. All the pregnant, estrogen-treated and abnormal thyroxine protein-binding patients had TSH results within the reference range 0.5-6.0 mU/l, except one familial dysalbuminemic hyperthyroxinemia (FDH) patient (T4 level, 178 nmol/l) lost to follow-up with a TSH level of 8.5 mU/l, and one euthyroid, low-TBG patient being treated inappropriately with thyroxine, with a TSH level less than 0.5 mU/l. All the untreated thyrotoxic patients and 15 (26.3%) of the NTI patients had TSH results of 0.5 mU/l or lower. Because of this high incidence of low TSH levels in euthyroid NTI, we cannot recommend this IRMA-TSH as the initial test of thyroid function.

Functional and structural characterization of isolated perfused stingray liver including effects of ischaemia/reperfusion.

The morphological and functional characteristics of stingray liver were studied, including the effect of ischaemia/reperfusion. With an isolated perfused model, it was shown that the stingray liver was more resistant than the rat liver to ischaemia/reperfusion injury; this was consistent with the differing partial oxygen tensions usually present in the two species. This study confirmed that whereas stingray hepatocytes form tubules with central bile canaliculi as in other fish, the stingray liver has portal triads and a lobular architecture as in mammals. Apoptosis of hepatocytes, demonstrated in the normal liver, was only marginally enhanced by ischaemia/reperfusion. Resulting apoptotic bodies were phagocytized by macrophage-like cells in hepatocyte tubules. In contrast to rat liver, the stingray liver showed no necrosis after ischaemia-reperfusion.

Myoclonus associated with treatment with high doses of morphine: the role of supplemental drugs.

OBJECTIVE: To estimate the prevalence of important side effects in patients with malignant disease who were receiving high doses of morphine as part of their palliative treatment. DESIGN: Data on patients were collected over 12 months. SETTING: Two palliative care units in Western Australia. PATIENTS: 19 Patients with malignant disease who were receiving morphine either subcutaneously or orally as the main analgesic. 10 Patients receiving a total daily dose of morphine of at least 500 mg orally or 250 mg parenterally were enrolled in the study. The other 9 patients were enrolled after an important problem thought to be related to the morphine had been identified. All of the patients were taking drugs to supplement the treatment. INTERVENTIONS: The dose of morphine or route of administration, or both, was changed in three patients. MAIN OUTCOME MEASURE: Determination of the prevalence of side effects in the patients. Assessment of the relation of any side effects with the supplemental drugs taken by the patients. MAIN RESULTS: Plasma morphine and electrolyte concentrations were measured and a full history taken for each patient. Thirteen of the 19 patients had an important side effect; 12 of them had myoclonus and one had hyperalgesia of the skin. Plasma morphine concentrations were similar in patients with and without myoclonus, ranging from 158 to 3465 nmol/l and 39 to 2821 nmol/l respectively. Eight of the patients with side effects were taking an antipsychotic drug concurrently compared with none of those without side effects. A greater proportion of patients with side effects were taking the antinauseant drug thiethylperazine (6/13 v 2/6) and at least one non-steroidal anti-inflammatory drug (10/13 v 2/6), whereas a smaller proportion were taking a glucocorticosteroid (3/13 v 4/6). The estimated prevalence of important side effects in the total population of patients receiving palliative treatment in the two units was 2.7-3.6%. CONCLUSIONS: Myoclonus as a side effect of treatment with morphine is more likely to occur in patients taking antidepressant or antipsychotic drugs as antiemetics or as adjuvant agents or non-steroidal anti-inflammatory drugs for additional analgesia. If a patient develops myoclonus the best approach may be to change the supplemental treatment.

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.