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Pathology:
doi: 10.1097/01.PAT.0000412600.60566.8c
Abstracts

Chemical Pathology

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DIAGNOSTIC AND THERAPEUTIC APPROACHES TO ALZHEIMER'S DISEASE

Simon M. Laws1,2, Giuseppe Verdile1,2, James Doecke3,4,5, Ralph N. Martins,1,2 and the AIBL research group6

1Centre of Excellence for Alzheimer's Disease Research and Care, School of Medical Sciences, Edith Cowan University, Joondalup, WA,2Sir James McCusker Alzheimer's Disease Research Unit (Hollywood Private Hospital), Perth, WA,3The Australian E-Health Research Centre, Royal Brisbane and Women's Hospital, Herston, Qld,4CSIRO Preventative Health Flagship, Parkville, Vic,5CSIRO Mathematics and Information Sciences, Macquarie University, Sydney, NSW, Australia,6http://www.aibl.csiro.au/

Background: The definitive diagnosis of Alzheimer's disease (AD) is currently only possible following post-mortem examination of the brain for the disease's characteristic neuropathology. The successful implementation of disease interventions makes the discovery of biomarkers for early diagnosis and response to treatment paramount. Here we present results from two studies that have implications for AD diagnosis and treatment.

Methods: The Australian Imaging Biomarker and Lifestyle (AIBL) study of ageing provided the cohorts and data studied. The first study aimed to identify plasma biomarkers for the diagnosis of disease through the screening of 224 plasma analytes. The second study investigated the impact of testosterone and gonadotropins on AD biomarkers.

Results: A panel of eight biomarkers was identified that obtained sensitivity and specificity of 83% and an AUC of 87%. Testosterone and luteinizing hormone were both found to be associated with plasma and cerebral amyloid-beta levels.

Conclusions: The identification and validation of a short panel of biomarkers has significant implications for the future diagnosis, prediction and monitoring of AD. The close association of hormones with AD pathology likewise has significant implications on future treatments of the disease, with a clinical trial being the next stage in assessing the efficacy of this approach.

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METALS IN ALZHEIMER'S DISEASE

Ashley Bush

Department of Pathology, and Mental Health Research Institute, The University of Melbourne, Vic, Australia

The brain houses high concentrations of transition metals zinc, copper and iron, which it uses for specialised neurochemistry and the synthesis of heme. It is in this high flux environment that several of the culprit proteins of Alzheimer's disease (AD) aggregate, losing function and possibly becoming toxic. These include amyloid (Aβ) that aggregates outside of the cell forming plaques, and tau, which aggregates inside the neurons forming tangles. Both pathologies recruit high concentrations of metal ions. Presenilins, whose mutations cause aggressive familial AD, play a major role in metal uptake. The levels of iron in the brain rise with aging and rise even more with AD. This is caused by dissociating zinc within amyloid deposits inhibiting the iron-export activity of the Aβ precursor protein (APP), which is a major component of neuronal iron efflux. We have recently found that tau plays a major role in neuronal iron homeostasis by mediating APP trafficking. The metal-centred neuropathology of AD is the target for a new class of drugs that have shown considerable promise in clinical trials. Abnormal metal homeostasis is also reflected in the periphery in AD, and may be the basis for predictive biomarkers.

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OVERVIEW OF MATERNAL SERUM SCREENING FOR DOWN SYNDROME

Narelle Hadlow

Department of Biochemistry, Western Diagnostic Pathology, Perth, and Department of Biochemistry, Pathwest Laboratory Medicine, Queen Elizabeth II Hospital, Perth, WA, Australia

This presentation will cover the principles of Down syndrome screening and the progress of screening from the use of age, second trimester and later first trimester screening tests. Refinements of first trimester screening will be discussed, including the timing of blood and ultrasound components, accreditation of sonologists, addition of nasal bone and ductus venosus to risk assessments and progress towards measuring precision and bias in ultrasound. The benefits and disadvantages of first and second trimester screening tests will be compared. Newer developments assessing risk of neural tube defects at first trimester including loss of intracranial translucency, caudal midbrain displacement and first trimester AFP will be covered.

Screening programs can also assess risk for other chromosomal abnormalities such as Edward, Pataus and sex chromosomal anomalies. Prediction of other adverse outcomes such as preterm birth, low birth weight and pre-eclampsia have also been studied. Practical issues in a screening program, such as interferences and confounding factors are important to recognise and will be briefly discussed.

The lecture will conclude with recognition that screening is an individual ethical choice. Pre-test information and counselling are important and the implications of the follow-up diagnostic tests should be understood.

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NON-INVASIVE PRENATAL DIAGNOSIS OF DOWN SYNDROME: A DREAM COME TRUE

Rossa W. K. Chiu

Li Ka Shing Institute of Health Sciences and Department of Chemical Pathology, The Chinese University of Hong Kong

Our group discovered the presence of cell-free fetal DNA in plasma of pregnant women. This finding offered opportunities for the development of non-invasive approaches for prenatal diagnosis. However, fetal DNA co-exists with a large background of maternal DNA and amounts to just some 10% of the total DNA in maternal plasma. Despite the technical challenges, we developed an approach based on massively parallel sequencing for the non-invasive detection of fetal Down syndrome by detecting the small increments in chromosome 21 DNA sequences in maternal plasma. We showed in a study of 750 pregnancies that Down syndrome can be accurately identified by direct analysis of maternal plasma DNA. The method could be further adapted for the prenatal detection of trisomy 18 and trisomy 13. A number of groups have since confirmed our findings. Non-invasive prenatal detection of fetal Down syndrome has since been implemented as a routine service at a number of centres around the world.

Acknowledgement: Supported by the University Grants Committee of the Government of the Hong Kong Special Administrative Region, China, under the Areas of Excellence Scheme (AoE/M-04/06), the General Research Fund Scheme of the Hong Kong Research Grants Council (CUHK463109), and an Innovation and Technology Fund (ITS/054/09).

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MASSIVELY PARALLEL SEQUENCING AS A MOLECULAR DIAGNOSTIC TOOL

Rossa W. K. Chiu

Li Ka Shing Institute of Health Sciences and Department of Chemical Pathology, The Chinese University of Hong Kong

My research focuses on the development of strategies to deliver non-invasive prenatal diagnosis of fetal genetic diseases through the analysis of circulating fetal DNA. However, circulating fetal DNA analysis poses a number of technical challenges. We recently showed that massively parallel sequencing is an ideal tool for the analysis of challenging molecular targets like circulating fetal DNA. We showed that massively parallel sequencing is sensitive to the detection of low-abundance targets even in the short fragmented form as in the case of circulating fetal DNA. Sequencing can specifically determine the genetic identity of the sequenced DNA molecules while simultaneously offers precise quantification of the relative amounts of DNA targets in the sample. We reported a strategy for assembling the fetal genome by maternal plasma DNA sequencing and developed an approach for determining fetal inheritance of both paternally-inherited and maternally-inherited genes. The cost-effectiveness of sequencing could be enhanced by using targeted approaches. We further showed that sequencing could be used to study the size profiles of fetal DNA and DNA molecules in plasma of transplantation recipients. We believe that massively parallel plasma DNA sequencing would lead to a paradigm shift in molecular diagnostics.

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EVIDENCE FOR HARMONISED REFERENCE INTERVALS

Ken Sikaris

Melbourne Pathology, Collingwood, Vic, Australia

The main purpose of a reference interval is to have something to compare a patient's initial result. Once we ask for a second test, it is probably more important to define whether that result is significantly different to the first result, as monitoring is a more sensitive way of assessing a patient's health and uses that patient as their own baseline for comparison.

Surveys have shown that laboratories using the same analytical methods, often have very discordant reference intervals. While most laboratories do not have the resources to define their own reference intervals, when adopting someone else's the laboratory director needs to be sure of (i) the comparability of their method with that study and (ii) the comparability of their reference populations.

Getting the evidence that methods are identical, or at least highly correlated, is straightforward compared to proving the comparability of reference populations. Most assume that the reference population is simply healthy adults, and this is where their trouble will start. Firstly, a healthy child is different to a healthy adult, and the same applies for the healthy elderly and healthy pregnant women. Secondly, it is actually difficult to define ‘a healthy adult’ as, for example, over 50% of Australians are overweight or obese and a significant proportion of apparently healthy individuals have less obvious morbidities. Thirdly, as our human genomes are far more similar than different, there are few genetic differences in human homeostasis; however, there are some obvious racial differences in skin colour, height and body proportions that could affect reference intervals. Finally there are cultural differences, such as diet, that may affect our tests.

Given these potential non-analytical confounders, how can we possibly ‘harmonise’ reference intervals? The idea of harmonisation has been promoted in the European Union where countries that have undeniable differences still aim to harmonise their laws and/or regulations so that compromises made by each member are minimised. Harmonisation is not comprehensive but defined as not going further than that which is necessary. As far as reference intervals are concerned, we should aim to minimise the unjustifiable discordance that currently exists.

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STANDARDISATION OF PATHOLOGY TERMINOLOGY: HOW E-HEALTH RECORDS SHOULD WORK

Robert Flatman

Biochemistry Department, Sullivan Nicolaides Pathology, Brisbane, Qld, Australia

Attempts to progress e-health in pathology in Australia have been hindered by a lack of standardisation in key requesting and reporting parameters, such as request codes, test panels, test names, units and reference intervals. Leverage off electronic health records (e.g., development and use of decision support systems) is difficult without agreement in these areas.

The Royal College of Pathologists of Australasia (RCPA) led ‘Pathology Units and Terminology Standardisation’ (PUTS) project is showing leadership in these areas by bringing together pathologists, scientists and IT professionals to make recommendations on best practice. Working groups have been established to review request codes, recommended units, and reporting codes for seven pathology disciplines. Each group will also consider the terminology most applicable to that discipline (e.g., SNOMED-CT, LOINC, both, or possibly other) and where necessary highlight tests for which results should not be combined across pathology providers.

Work on the ambitious Personally Controlled Electronic Health record program continues, with initial operation scheduled for mid 2012. Although participation will not initially be mandatory for patients or providers, Pathology reporting will be included. The pathology industry needs to be actively engaged in this rapidly changing landscape for optimal outcomes.

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NEW APPROACH FOR THE INVESTIGATION OF LIVER DISEASES

Rossa W. K. Chiu

Li Ka Shing Institute of Health Sciences and Department of Chemical Pathology, The Chinese University of Hong Kong

We hypothesised that liver-derived mRNA, such as albumin (ALB) mRNA, would be released into human plasma as a result of liver cell death. We genotyped the ALB mRNA molecules in plasma and whole blood of liver or bone marrow transplantation recipients by RNA-single nucleotide polymorphism analysis. The plasma and whole blood ALB mRNA genotypes were compared with the DNA genotypes in the recipients and donors. Our data revealed that ALB mRNA in plasma was liver-derived, while tissue compartments other than the liver also contributed to the ALB mRNA detected in whole blood. We then used a reverse transcription quantitative real-time PCR assay to show that the plasma ALB mRNA concentrations were elevated in patients with hepatocellular carcinoma, cirrhosis or chronic hepatitis B than healthy controls. Only 21.5% of patients with liver pathologies had elevated alanine transaminase (ALT) when 73.8% of them had elevated plasma ALB mRNA concentrations. Only 49% of the hepatocellular carcinoma patients had elevated serum alpha-fetoprotein, while 91.4% had elevated plasma ALB mRNA concentrations. Liver-derived plasma mRNA may become a new class of biomarkers for the sensitive detection of liver diseases.

Acknowledgement: Supported by the Hong Kong Research Grants Council Earmarked Research Grant (CUHK463207).

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LIVER TRANSPLANTATION IN AUSTRALIA AND NEW ZEALAND

Simone Strasser

AW Morrow Gastroenterology and Liver Centre and Australian National Liver Transplant Unit, Royal Prince Alfred Hospital, Sydney, NSW, Australia

Liver transplantation (LTx) was established in Australia and New Zealand in 1985. To December 2010, 3781 LTx were performed in 3510 patients (2871 adults; 639 children). Chronic viral hepatitis, often with hepatocellular carcinoma, is the most common indication, accounting for over 40% of adult transplants. Biliary atresia is the most common indication in children. One year post-LTx survival is >90% and 5 year survival is >80%.

The greatest limitation to delivery of LTx is donor availability. Expanded criteria donors are increasingly used, including older donors, donors with hepatitis C virus (HCV)/hepatitis B virus (HBV), split livers, deceased after cardiac death (DCD) donors and live donors. Organ allocation to recipients aims to ensure equity and utility. Model for end-stage liver disease (MELD)-based allocation is utilised. Waiting list mortality is 10–15%.

Standard immunosuppressive protocols are based on calcineurin inhibitors (tacrolimus or CSA) ± corticosteroids ± azathioprine/mycophenolate mofetil. Graft loss from rejection is uncommon. Prophylaxis protocols for HBV, cytomegalovirus (CMV) and Pneumocystis carinii pneumonia fungal infections are standardised.

HCV recurrence accounts for high rates of graft failure. Histological recurrence occurs in 80%; 30% are cirrhotic by 5 years. Factors associated with severe HCV recurrence include older donors, intense immunosuppression, high HCV viral load and CMV. Currently, post LTx antiviral treatment is poorly tolerated and often ineffective, however new agents offer great promise. Hepatocellular carcinoma, primary biliary cirrhosis, primary sclerosing cholangitis and non-alcoholic steatohepatitis may recur post-LTx. Recurrent disease, infection, malignancy and cardiovascular disease remain the major causes of patient death post-LTx.

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CANCER ASSESSMENT BY PLASMA NUCLEIC ACID ANALYSIS

Rossa W. K. Chiu

Li Ka Shing Institute of Health Sciences and Department of Chemical Pathology, The Chinese University of Hong Kong

Tumour-derived nucleic acid molecules have been found in the plasma of cancer patients. Our group and others showed that the detection and quantification of circulating tumour-derived nucleic acid molecules offered value for the detection, prognostication and monitoring of cancers. A number of classes of circulating nucleic acid species have been found to be released by tumour tissues and include DNA molecules showing tumour-specific mutations or translocations, RNA transcripts, DNA methylation signatures and viral-associated sequences. A number of analytical approaches have been developed to achieve sensitive and specific detection of such tumour-specific nucleic acid markers in plasma. Strategies included the real-time polymerase chain reaction quantification of Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma, methylation-sensitive restriction enzyme based detection of hypermethylated Ras association domain family 1 A (RASSF1A) sequences in plasma of hepatocellular carcinoma patients, and digital polymerase chain reaction detection of epidermal growth factor receptor (EGFR) mutations in plasma of lung cancer patients.

Acknowledgement: Supported by the Research Grants Council of the Hong Kong Special Administrative Region, China [T12-404/11].

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NEW CONSENSUS GUIDELINES FOR THE DIAGNOSIS OF GESTATIONAL DIABETES

Alison Nankervis

Royal Melbourne and Royal Women's Hospitals, Melbourne, Vic, Australia

Australian guidelines for the diagnosis of gestational diabetes (GDM) were formulated in 1991, based largely on expert opinion. Review of these guidelines was precipitated by the publication of the Hyperglycaemia and Pregnancy Outcome Study (HAPO) and subsequent recommendations published by the International Diabetes in Pregnancy Study Groups (IADPSG).

HAPO was a prospective, blinded study of 23 316 women and reported a strong correlation between glycaemia at 24–28 weeks gestation and adverse maternal and neonatal outcomes. IADPSG consensus was that cut-points for oral glucose tolerance test (OGTT) plasma glucose levels with an odds ratio of 1.75 for adverse outcomes should be designated the diagnostic levels for GDM.

The new recommended diagnostic criteria for GDM are fasting venous plasma glucose (VPG) of ≥5.1 mmol/L; or 1-hour VPG ≥10.1 mmol/L; or 2-hour VPG ≥8.5 mmol/L, based on an OGTT routinely performed at 24–28 weeks gestation. Glucose challenge tests are no longer part of the diagnostic algorithm. It is anticipated that adoption of these criteria will increase the prevalence of GDM to 12–13%, with significant implications for workload management.

Recommendations for early screening for DM in pregnancy are being considered. The consensus view is that there should be universal screening at the first pregnancy visit with method and interpretation of testing not yet defined.

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ANTIPHOSPHOLIPID ANTIBODIES AND PREGNANCY

Stephen Adelstein

Department of Clinical Immunology, Royal Prince Alfred Hospital, Camperdown and Sydney Medical School, University of Sydney, NSW, Australia

Acquired thrombophilia is commonly associated with detection of autoantibodies against phospholipid-protein complexes that include lupus anticoagulant, anticardiolipin antibodies, anti-prothrombin and anti-beta-2 glycoprotein I antibodies. The antiphospholipid syndrome is present when these autoantibodies are found in association with clinical manifestations that include venous or arterial thromboembolism or pregnancy morbidity, such as recurrent spontaneous miscarriages, fetal deaths, second or early third trimester fetal deaths or fetal growth retardation.

Because the diagnosis of antiphospholipid syndrome is based on laboratory data, optimal performance and interpretation of the tests is essential. However, heterogeneity of the autoantibodies and absence of gold standard assays makes analysis of laboratory results a challenge for pathologists and clinicians alike. As a further complication to rational use and interpretation of the tests, the causal relationship of aPLs and thrombosis is difficult to establish as they are present in normal, asymptomatic individuals, with thrombotic events occurring only in approximately 30% of patients with antiphospholipid antibodies. Obstetric complications are also difficult to correlate meaningfully with test results because of heterogeneity in study populations, and changes in anti-coagulant management and the threshold for its use in everyday practice over time.

Characterisation of the molecular basis of the pathogenic mechanisms involved in the syndrome will hopefully provide a more rational basis for definition of the syndrome, the use of diagnostic tests and management in the future.

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BIOMARKERS OF LIVER FIBROSIS: EVIDENCE FOR THE CLINICAL UTILITY OF THE ELF TEST IN HEPATIC DISEASE

Katherine M. Soreng

Siemens Healthcare, USA

Chronic liver disease (CLD) is on the increase in many countries, driven by factors such as obesity, alcohol abuse, and infectious diseases such as viral hepatitis B and C. While liver biopsy remains the primary standard of care in the diagnosis of liver fibrosis or cirrhosis, limitations such as sample quality and challenges to obtaining repeat biopsies constrain utility. Recent advances in liver disease testing include minimally invasive blood tests to assess levels of serum biomarkers associated with the fibrotic process. The Enhanced Liver Fibrosis (ELF) assay from Siemens incorporates three direct markers of liver fibrosis in a unique algorithm that has been shown to have both diagnostic and prognostic performance. This presentation will review some of the recent published evidence supporting a role for biomarkers of liver fibrosis in the diagnosis and management of CLD patients.

© 2012 Royal College of Pathologists of Australasia

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