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Abstracts: Speaker Abstracts

Please see below abstracts submitted by speakers at Focus 2017. Click the heading to jump to the relevant abstract.

Focus 2017: Speaker Abstracts

  • Metabolic and Molecular Bases for Congenital Hyperinsulinism

    Michael J Bennett (Children’s Hospital of Philadelphia, Philadephia, USA)

    Congenital hyperinsulinism (CHI) represents a group of inherited genetic disorders that result in excessive unregulated secretion of insulin by pancreatic islet beta cells. The overall frequency of CHI is about 1 in 25,000 to 1 in 50,000 live births in Western populations. The clinical and biochemical effects of excessive secretion of insulin are to induce what is often profound hypoglycaemia as a result of impaired gluconeogenesis and glycogenolysis. Circulating glucose is also directed into skeletal muscle and adipocytes compounding the hypoglycaemia. The brain is particularly dependent upon glucose as an energy source. Recurrent episodes and long exposures to hypoglycaemia can result in seizures and ultimately severe neurological damage.

    The metabolism of physiologic insulin secretion is dependent upon ATP production from a number of sources including glucose, the amino acid glutamate and pyruvate, the end-product of glycolysis. These physiologic fuels are generally elevated in the blood stream post-prandially and under normal physiological conditions coordinately regulate insulin secretion. The resulting effect is to increase cellular uptake of glucose as a glycogenic substrate when metabolic fuels are plentiful. The converse is true during periods of fasting when insulin secretion is physiologically downregulated.

    CHI may appear on imaging procedures based on 18F-fluoro-L-DOPA positon emission tomography to be either diffuse where all beta cells are abnormally functioning or focal where only certain regions of the islets are effected.

    The molecular and metabolic bases for CHI are currently being defined with a growing number of new genes being implicated in recent years. A number of transporter proteins, enzymes and metabolic regulators are required for the physiologic post-prandial secretion of insulin. Mutations in the genes that are responsible for the synthesis of these metabolic activators and regulators are typically responsible for CHI. Mutations in all three metabolic pathways (glucose, glutamate and pyruvate) have been identified that result in unregulated secretion of insulin as well as a number of mitochondrial proteins. Interestingly, many of the genetic defects result in gain of function leading to excessive ATP production.

    This presentation will discuss the genes and proteins that are required for physiologic insulin secretion and on the abnormal genes and proteins that result in CHI. The presentation will focus on recent research from my laboratory that defined a very unusual role for short-chain 3-hydroxyacyl-coenzyme a dehydrogenase (SCHAD) a protein that functions normally to generate ATP from medium and short-chain fatty acids but that results in SCHAD CHI when mutations in the HAD1 gene lead to loss of function.

    Finally, we will focus upon treatment options for CHI. Rational therapeutic intervention can be applied in certain of the genetic defects once the defect has been characterized.

  • Laboratory medicine for transgender patients

    Stephen M Gibbons (Leeds Teaching Hospitals Trust, Leeds, United Kingdom)

    Over the last twenty years there has been a positive shift in public attitudes towards the transgender community, with increasing numbers of patients being referred to gender identity clinics. Despite this there remain a number of significant challenges facing the transgender individual, not least healthcare.

    So why does this population pose such a conundrum for medical professionals? Transgender patients do not fit into our binary model of gender classification. Most trans patients are on hormone replacement therapy which is known to cause a number of side-effects, the Endocrine Society therefore recommend regular monitoring. Disappointingly, there is a severe lack of training or evidence relating to the management of transgender patients.

    The challenges to Laboratory medicine are no less daunting. Gender remains a binary classification and electronic medical records do not confer the flexibility to correctly document the gender of transgender patients. One of the most challenging issues for the laboratory is the use of appropriate reference ranges The evidence seems to suggest this will be analyte dependant, something few LIMS would be capable of managing.

    Sex hormones might be the obvious problem, but consider how you would deal with a PSA request in a women or a cervical smear in a man? These and other laboratory pitfalls will be explored with suggestions on how we as healthcare professionals can implement change to ensure that the transgender patient is not disadvantaged.

  • Harmonisation of pre-analytical factors: a European view

    Michael Cornes (Worcestershire Acute Hospitals NHS Trust, Worcester, United Kingdom)

    Over the last 25 years harmonisation has become a very important topic in laboratory medicine covering all aspects of the total testing process (TTP). There are many benefits to harmonising the TTP but at the core of these is the idea of performing the right test at the right time on the right patient in the right way. There has been a lot of progress made in the analytical part of this process of this is only one part of the TTP. A poor pre-analytical phase of the TTP results in poor sample quality and therefore potentially poor results and decision making. In recent years interest in the pre-analytical phase has grown and with it progress in harmonisation of this phase of the TTP. This progress has, in a large part, been driven by the activities of the EFLM Working Group for the Pre-analytical phase (EFLM WGPRE). Within the pre-analytical phase there are various parts that need to be standardised. The key aspects for standardisation were identified at the Pre-analytical Phase Conference and included test ordering, sample transport and storage, patient preparation, phlebotomy process (venous and capillary), how to handle unsuitable specimens and patient identification. In the case of all of these there is considerable variation across Europe and the EFLM WGPRE is devoted to providing the tools and guidance to allow laboratories across Europe to operate in a harmonised, evidence based way. This presentation aims to provide details of how the issues above are being tackled and will highlight current best practice covering aspects of demand management, reflex testing, condition specific profiles and the current situation in other aspects of the pre-analytical phase mentioned above.

  • Organising Point-of-Care-Testing in the community

    Maurice O’Kane (Altnagelvin Area Hospital, Londonderry, United Kingdom)

    Point-of-care testing (POCT) is one of the fastest growing sectors of the diagnostics market. The major advantage of POCT is that the rapid availability of test results facilitates more expeditious clinical decision making and intervention. However, POCT may be more prone to error than central laboratory testing. This relates to a number of factors which include a difficult testing environment and the training, expertise and experience of POCT operators. It is self-evident that error in POCT may pose risks to patient safety. Increasingly POCT is being employed outside the hospital in a range of community settings which include health centres, unscheduled care facilities, community pharmacies and as part of ad hoc health screening projects. The range of tests undertaken may encompass those used in the diagnosis and triage of acutely ill patients, chronic disease monitoring (including patient self-measurement) and disease screening. This diversity of settings and applications poses challenges in the effective and safe implementation of POCT. Introduction of POCT in the community must include a detailed appraisal of the purpose and rationale of testing and the target patient group. As with hospital based/laboratory supported POCT there must be an appropriate governance framework that assures quality and competence of the service and which would meet the requirements for external accreditation. However, this poses particular challenges, since in the UK NHS, much community POCT takes place outside the managerial and professional governance structures of hospital Trusts in which laboratories are generally based. There will therefore be a requirement for liaison and support from laboratory professionals who can provide expertise and advice, in particular as regards  the choice of instrument, quality assurance procedures, risk assessment, training and audit. This will be resource intensive and there is a need to consider at national level how such support can be established and organised to ensure effective and safe community POCT. 

  • Metabolomic Profiling

    Dr Richard Unwin (The University of Manchester, Manchester, United Kingdom)

    Obtaining correct and accurate information regarding diagnosis, prognosis or disease course remains a challenge for the medical community. While the use of biomarkers in clinical management is pervasive, they remain in many ways a blunt instrument, providing additional knowledge to the clinician on which to make a diagnosis or to determine a course of treatment. However, it is clear that, especially in the case of complex multifactorial diseases such as cancer, cardiovascular diseases or dementia, additional indicators are required to ascertain a more accurate diagnosis, or to determine the correct or most appropriate action to take. This has led to the growing concept of ‘stratified’ or ‘personalised’ medicine, whereby a patient’s own characteristics are used to determine the best course of action for them, rather than a one-size-fits-all approach.

    However, in order for such a concept to become a success, more and better disease biomarkers are required to enable accurate stratification. One such field which is well suited to the challenge of defining patient-specific phenotype is that of metabolomics, the global study of metabolite levels.

    Metabolic profiling describes the practice of measuring large numbers of metabolites (sugars, amino acids, lipids, metabolic intermediates) simultaneously, usually using mass spectrometry, and using the pattern of metabolites to infer details about the biological status of the patient. By performing such comparisons between clinical groups (e.g. healthy versus ill, responders versus non-responders to a particular therapy) we can identify pattern which may predict diagnosis or inform us about the chances that a certain therapy will have a positive effect. Small molecules are readily accessible and are already measured in the clinical laboratory and as such this approach is, in theory, readily translatable.

    In this presentation, I will outline some of the main workflows which are used for metabolic profiling, describing the strengths and the weaknesses of this burgeoning approach to biomarker discovery and provide some key examples of how metabolic profiling of patient samples has been used to develop new panels of biomarkers to differentiate between patient strata, and to define new targets for therapeutic intervention.

  • Genotype-phenotype conundra

    Simon Heales (Great Ormond Street Hospital Laboratory Medicine, London, United Kingdom)

    Diagnostic laboratories are moving towards a combined omics approach with regards to the investigation of clinical samples. Thus patient samples may be expected to embark on parallel journeys involving genomics, proteomics, metabolomics, fluxomics etc. This with the hopeful aim of generating a comprehensive and digestible report with a clear diagnosis. In reality, we are not completely there yet. However this approach is inevitable, particularly with regards to patients with complex conditions and where there is considerable overlap of phenotype between the conditions. For disciplines such as enzymology or metabolic profiling, workload is increasing; results from genomic screens are identifying variants of unknown significance with subsequent requests for functional studies. However, the converse is also true, e.g. an abnormal metabolic profile may point strongly to a specific metabolic disease yet the clinical phenotype is atypical. The hope here is that genetic conformation provides the answer. The interplay between metabolic pathways also needs to be appreciated. Identification of an abnormal metabolite does not necessarily point towards the correct primary metabolic defect, e.g. impaired folate metabolism secondary to a mitochondrial diseases.

    Combined omics will undoubtedly lead to quicker diagnoses of very complex conditions. Additionally, reliable and specific biomarkers will continue to be identified enabling prediction of disease severity and the monitoring of responses to treatment.

  • Big gains from big data: integrated health systems and the hospital laboratory

    Daniel Lasserson (Oxford University Hospitals NHS Trust, Oxford, United Kingdom)

    In this talk, Dan Lasserson will give an overview of how the hospital laboratory delivers a critical database for understanding the health of the population that seeks care, for understanding variation in clinician behaviour and for measuring the outcomes that result from care delivered over different healthcare settings. Using examples ranging from changing GFR estimating equations, the investigation of anaemia in primary care and the NHS England Acute Kidney Injury programme, he will describe how hospital pathology services are at the nexus of change in the NHS. The laboratory database allows us to probe variation at the level of regions and clinicians, to test the impact of changing how we make diagnoses and the impact of changing how care is delivered. It is a crucial resource to understand what the NHS achieves, how it operates and needs to be at the centre of plans to design the optimal heathcare system for the future.

  • What patients want from a pathology service: making pathology services more patient centred

    Patricia Wilkie (National Association for Patient Participation, Woking, United Kingdom)

    Patients and their relatives still lack much direct experience, direct knowledge and understanding of pathology services. Patients are aware that samples are taken and “tests” sent. But many patients still do not know precisely what the test is for, what is being measured and the meaning of results. This has become increasingly important as more patients are living with and self-managing more than one lifelong condition where lab results are important and as more patients have easier access to their medical notes both digital and paper. This paper discusses some of the implications for patients of these points and makes suggestions and recommendations for a more patient centred pathology service.

  • Pharmaceuticals on the street and how the laboratory can help

    Loretta Ford (Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, United Kingdom)

    The last 10 years has seen a dramatic change in drug use with the rise in popularity of so called legal highs, also termed new psychoactive substances (NPS).  Using the internet prescription-only medications are readily available and information can be found on abusing over-counter medication. In response toxicology services have had to move from a traditional approach of just screening for a few classic drugs of abuse such as cocaine and amphetamine and instead are introducing new technology such as mass spectrometry techniques to also screen for these different forms of abuse.

    In 2013 we introduced the first UK clinical service for routinely screening unknown drugs in patients using a Waters Xevo G2 QTof (time of flight, TOF) detector.  Capable of detecting over 1300 drugs and metabolites in a single analysis, using a series of patient cases here we demonstrate the advantage of this technique over a traditional toxicology screening service, especially for aiding clinicians in accurate diagnosis and management in real time.

  • Direct to the public testing … key issues and potential benefits for patient care

    Jonathan Berg (Clinical Biochemistry Department, SWBH NHS Trust, Birmingham, United Kingdom)

    The general public are increasingly urged to take personal responsibility for their health and within this is a rising interest in diagnostic testing. This is nothing new and patients with chronic conditions such as diabetes have for over fifty years been able to home test themselves for glucose and home-test pregnancy tests have been freely available since the 1980s. There are now direct to the public services offered either as self-tests or pharmacy delivered services for a wide range of analytes. Ten years ago we got involved with our local mental health trust to offer testing direct to the public in an alcohol awareness bus parked at locations around Birmingham. We offered a battery of tests loosely related to health issues that might arise from alcohol excess. The interest from the public in being able to have these tests done was illuminating. We discovered undiagnosed conditions such as diabetes and severe liver disease and were able to help members of the public to go and discuss their results with their GP. We asked people why they had not gone to their GP to have such tests and there was a cohort of people who did not want to be part of the healthcare system but who were interested in checking themselves out. For five years we have offered direct to the public testing for Vitamin D. This paid for service is aligned to our NHS vitamin laboratory. We incorporated a new design of blood spot card and an end-user pack which contained everything needed to take a sample and return the blood spot. We developed sensitive methods that enabled us to determine blood spot vitamin D2 and D3 to express results that are aligned to our serum vitamin D results. An important consideration is to optimise instructions and test pack components. When we had been offering the service for a year we produced a video on how to undertake the test and this was emailed to people who ordered the test. The video has been extremely helpful in ensuring the kit is used correctly and viable blood spots are produced. We send a PDF report and this includes a traffic light system to help interpret the results. Reference intervals for Vitamin D are controversial with the public but we stick to consensus values that we give out in the NHS. There are members of the public that advocate much higher suggested target Vitamin D levels but we have resisted any temptation to suggest this as appropriate. One early finding was that among the ‘worried well’ are those taking large doses of vitamin D, often supplied from internet pharmacy sites. Soon after we established our service we had to put in a system for robustly handling high results. Direct to the public services required new skills and understanding but overall this work has been beneficial not only is to those that have used the service but also to our wider role as an NHS laboratory.

  • Diabetic monitoring: developing patient-centred laboratory services

    Christopher Duff (Department of Clinical Biochemistry, University Hospitals of North Midlands NHS Trust, Stoke-on-Trent, United Kingdom)

    Around 3 million people in the UK have diabetes mellitus (DM) (prevalence 4.6%). The measurement of glycated haemoglobin (HbA1c) is the mainstay of monitoring disease control in these patients and should be performed at regular intervals. Raised levels of HbA1c are an indicator of poor diabetic control and are association with increased incidence of diabetes-related health complications and poor outcomes. Current NICE guidelines for DM (NG17, Type 1; NG28, Type 2) recommend that HbA1c levels are measured every 3-6 months. However, previous work by our group showed that a large proportion of diabetic patients do not have a HbA1c blood test according to these stipulated testing frequencies, with 20% of repeat HbA1c tests identified as ‘too soon’ and 30% of tests ‘too late’. Furthermore, we have demonstrated that suboptimal disease monitoring (testing frequency) was associated with poor diabetic control (test result), suggesting that optimising biochemical monitoring will improve control in patients with diabetes. In addition, patients who had fewer than 2 tests per year were statistically less likely to reach a target HbA1c of below 53 mmol/mol. A similar observation was made for those who have greater than 4 tests per year.

    The underlying cause of suboptimal testing frequency is a complex mixture of multiple patient and healthcare system factors. Having identified some of these key factors, through the use of surveys and focus groups, this talk will explore opportunities for clinical laboratories to facilitate the redesign of services to counter this important problem. Examples of current and future initiatives will be discussed, including a recently developed smartphone App to assist patients in recording their HbA1c results.

  • Variation: study it well and use the knowledge to effect

    William A Bartlett (NHS Tayside, Dundee, United Kingdom)

    Millions of quantitative test results are made available to the users of laboratory medicine services on a daily basis. The value of those data to health care systems depends upon service providers and users having an understanding of the relativity of the test result to some point of reference. That point of reference may for example be a population based reference interval, or alternatively a previous measurement performed upon the individual patient being investigated. Knowledge of between (CVG) and within subject (CVI) variability of measurands is required to enable assessment of the significance of a result in each of these contexts. It is interesting to note that whilst monitoring the course of an individual patient’s disease often relies upon assessment of the significance of change between results, it is uncommon for laboratories to routinely provide an indication of significance of any change within reports. This deficiency can be addressed through application of flags to reports based on the concept of reference change value (RCV). Newer approaches also now enable assessment of RCV across a series of results which opens up new possibilities for monitoring significance of change multiple small incremental changes in progressive conditions.

    Valid application of biological variability data requires that the provenance of the data is understood and that they are well characterised and delivered with sufficient meta-data to enable their transportability into clinical practice across health care systems. This requires standards for production, reporting and transmission of biological variation data that parallel those of conventional reference values. In the absence of such recognised standards a need has been identified to enable critical appraisal of existing data sets derived from studies published over the last 40 years, and to drive up the quality of future biological variation studies. This has led to the publication of a critical appraisal checklist by the EFLM Biological Variation Working Group (BVWG), and the formation a Task and Finish Group under the EFLM Task Force on Performance Specifications in Laboratory Medicine. The task of the latter is the delivery of a new biological variation database. The new database will enable effective transmission of objectively valid and characterised data sets to clinical users. In addition the BVWG have established a biobank of specimens delivery of new biological variation data employing contemporary analytical methods within a checklist compliant multi-centre study.

    Production of appropriately executed and powered studies of biological variation enables delivery of data with confidence limits around the estimates and with associated meta-data to enable valid clinical application across time and health care systems. A rigorous approach, that is checklist compliant, enables the resultant reference data to be used to good effect to set performance specifications for analytical systems and enable user understanding of the clinical significance of test results. Laboratory medicine services may need to develop novel approaches that exploit electronic reporting interface functionality to enable users to deliver full benefit from the knowledge attained.

  • Autopsies save lives

    Suzy Lishman (The Royal College of Pathologists, London, United Kingdom)

    Autopsies, also known as post-mortem examinations, have been performed since ancient times for a variety of reasons; from predicting the future and preparing the body for the afterlife to learning about human anatomy and disease processes.

    Most modern post-mortems in the UK are carried out at the request of the Coroner to establish the cause of death. A small number are requested by the deceased’s doctors with the consent of the next of kin to study the extent of disease or response to treatment. The number of consented post-mortems has declined significantly worldwide; less than 0.5% of deaths are investigated in this way in England.

    It is often believed that modern imaging techniques and other diagnostic tests negate the need for post-mortems, with there being nothing left to learn. Research has shown that this is not the case; post-mortems frequently reveal disease that was not suspected during life. Discrepancy rates of up to 50% are reported, with no improvement in diagnostic accuracy over the last 30 years. The decline in the number of consented post-mortems is therefore of great concern.

    The coronial post-mortem service in the UK has been described as ‘showing severe signs of strain’, with coroners struggling to find sufficient pathologists able and willing to perform post-mortems. A range of solutions is being considered, including the establishment of a national death investigation service. It is possible that future post-mortems will be less invasive, with imaging techniques being increasingly used to establish the cause of death or guide limited further investigation.

    The patient pathway does not stop the moment someone dies. Post-mortem examinations form a vital part of healthcare. A greater understanding of why and how people die benefits the family, who receive an accurate record of what happened to their loved one; the doctors who cared for the deceased, who learn to be better diagnosticians; future patients, who receive better care as a result of this learning and society as a whole, which benefits from better informed policy decisions and more appropriate investment in the prevention, diagnosis and treatment of disease.

  • Acute kidney injury: effect of early intervention

    Andrew Lewington (St James’s University Hospital, Leeds, United Kingdom)

    There has been much interest in acute kidney injury over the last decade which has led to an increased awareness of its association with worse patient outcomes in terms of length of stay, chronic kidney disease and mortality. NHS England adopted AKI into the patient safety WorkStream and issued a level III patient safety alert mandating that all NHS trusts in England adopted the AKI warning system. There has been a gradual implementation of this over the last year which will ultimately provide big data for further analysis. In the interim many NHS trusts have AKI alert systems in place, based on rises in serum creatinine, that are linked to care bundles. It is intuitive that patients at risk of AKI, or with AKI, would benefit from earlier intervention. However the evidence is lacking at present and results are awaited from on-going studies. The presentation will describe The Health Foundation Tackling AKI Quality Improvement Programme and the interventions that have been developed across Leeds to try and improve the quality of patient care. The presentation will also describe the results from the recent NIHR funded AKI Diagnostics systematic review and economic analysis of biomarkers in the early diagnosis of AKI in critically ill patients on the intensive care unit.

  • Hyponatraemia: everyone’s problem

    Steve Ball (Manchester Academic Health Science Centre, Central Manchester University Hospitals NHS Trust, Manchester, United Kingdom)

    Hyponatraemia (serum Na+ <135 mmol/L) is common. It is associated with increased mortality and morbidity across a range of clinical contexts. The nature of the relationship of serum Na+ with clinical outcomes is unclear and there remains debate as to whether the relationship is causal.

    Recent guidance has highlighted the utility of urine Na+ concentration in the differential diagnosis of hyponatraemia. The same guidance has outlined the use of hypertonic fluids in those patients presenting with severe symptoms. Cause-directed therapy is recommended in other presentations.

    Despite this emerging consensus, there remain some key elements with which clinicians struggle. This presentation will focus on a number of these elements: an evidence-based approach to defining aetiology of the hyponatraemia; finding the balance in treating the patient rather than simply treating the serum Na+ concentration; approaches to the management of over-correction; and obstacles to implementing evidence-based practice in the management of hyponatraemia.

  • Dehydration: what is the best method of assessment?

    Lee Hooper (Norwich Medical School, Norwich, United Kingdom)

    Dehydration is a loose term for shortage of fluid, but the lack may be a shortage of fluid alone (referred to as water-loss dehydration, hypertonic, hyperosmotic or intra-cellular dehydration) or a shortage of fluid and electrolytes (salt-loss or extra-cellular dehydration or hypovolaemia). Water-loss dehydration is caused by drinking too little fluid, which leads to a concentration (and rise in osmolality) of intra- and extra-cellular body fluids.  Fluid and electrolyte loss through vomiting or diarrhoea does not raise osmolality and the loss is of extra-cellular fluid. The causes, consequences and treatment of these two types of dehydration are distinct, as are assessment methods.

    Water-loss dehydration is caused by drinking insufficient fluid and physiologically our bodies track hydration status using intra-cellular osmolality – a rise in osmolality triggers thirst (stimulating drinking) and renal concentration (reducing fluid losses).  As intra-cellular osmolality is reflected in serum and plasma osmolality, these are the key methods of assessment of fluid status. 

    Assessments of serum osmolality in older adults suggests that water-loss dehydration, not drinking enough, is very common.  The causes in older adults include reduced thirst sensation, worries about continence, physical difficulty in drinking, loss of social drinking situations and swallowing problems.  Best evidence suggests that the health consequences of water-loss dehydration for older adults include increased risk of disability and mortality, as well as increased risk of urinary tract infections, constipation and cognitive impairment. 

    Laboratory measurement of serum osmolality is labour intensive, relatively inaccurate and expensive, and the blood samples required are invasive, particularly in community settings such as residential care. We have assessed:

    • the value of simple tests and signs (such as dry mouth, urine colour and skin pinching) to assess hydration status in older adults, and
    • which osmolarity equation best predicts directly measured serum/plasma osmolality.

    A systematic review of available research and our own primary study of the diagnostic accuracy of simple signs and tests in older adults living in residential care has shown clearly that no single one-time assessment is usefully accurate in predicting hydration status.1, 2

    To assess osmolarity equations we included 5 cohorts of European older adults (595 people), of whom 19% were dehydrated (directly measured osmolality >300 mOsm/kg). Of 39 osmolarity equations, 3 had good predictive accuracy overall and in subgroups with diabetes and poor renal function. The best equation (narrower limits of agreement, low levels of differential bias and good diagnostic accuracy in receiver operating characteristic plots) was osmolarity = 1.86 × (Na+ + K+) + 1.15 × glucose + urea + 14 (all measured in mmol/L).3

    It appeared useful in people aged ≥65 years with and without diabetes, poor renal function, dehydration, in men and women, with a range of ages, health, cognitive and functional status.4

    We will discuss whether this osmolarity equation could add value to routine blood test results through screening for dehydration in older people.


    1. Hooper L et al. Cochrane Database Syst Rev 2015: CD009647.doi: 10.1002/14651858.CD009647.pub2
    2. Hooper L et al. Am J Clin Nutr 2016; doi:10.3945/?ajcn.115.119925
    3. Khajuria A, Krahn J. Clin Biochem 2005; 38: 514-9
    4. Hooper L et al. BMJ Open 2015; 5:e008846.doi:10.1136/bmjopen-2015-008846

  • Cortisol: how to interpret dynamic protocols

    Carol Evans (University Hospital of Wales, Cardiff, United Kingdom)

    Serum cortisol is mainly protein bound (cortisol binding globulin [80%] and albumin [10%]) and exhibits a circadian rhythm with concentrations reaching a peak in the morning and a nadir late at night. Cortisol is essential for life and deficiency can present as an Addisonian crisis, characterised by potentially fatal dehydration and salt wasting. However the initial onset is often insidious, with non-specific symptoms such as nausea and fatigue. A random serum cortisol concentration is rarely diagnostic so cortisol measurement after Synacthen stimulation is used. There is considerable variation in not only the protocol used for the test but also the ‘cortisol cut-off’ used to interpret the results. The cut-off may have been chosen for enhanced sensitivity or specificity to confirm or exclude a diagnosis. Although more often, is likely to be a compromise based on what information is actually available for the method in use. Most laboratories quantify serum cortisol by automated immunoassay. Cortisol is displaced from its binding proteins, usually by a propriety method, so that total serum cortisol can be quantified by competitive immunoassay. There are differences in bias between assays and this should be considered when choosing a cut-off. Interpreting the results of a Synacthen test is not always straight forward. Clinicians must take into consideration not only the providence of the cut-off, but also factors which can influence serum cortisol measurement for example increased or decreased cortisol binding proteins or the serum matrix, as well as the pre-test probability of disease in a given patient. Similarly, the early symptoms of cortisol excess (Cushing’s syndrome) can be non-specific. The overnight dexamethasone test is one of the recommended screening tests. This dynamic function test assesses suppression of a 9 am serum cortisol measured on the morning after late night administration of dexamethasone. In contrast to the short Synacthen test, the cut-off of 50 nmol/L is more universally employed, but with little consideration of the cortisol method bias or precision at this concentration. Suppression of the cortisol axis depends on achievement of a sufficient concentration of dexamethsone in serum. This will be affected by a number of factors, from the patient actually taking the tablet and presenting for venesection at the correct time, to factors which affect absorption and metabolism of dexamethasone. Interpreting dynamic function tests can be challenging; requiring consideration of the clinical question to be answered, factors which may influence the dynamic function test, as well as knowledge of the method used to measure serum cortisol and factors that may affect it.

  • Free thyroxine estimation by equilibrium dialysis and tandem mass spectrometry

    Alexandra Nance (Addenbrooke's Hospital, Cambridge, United Kingdom)

    Free thyroxine (fT4) status is assessed as part of investigation of function of the thyroid. Usually it is measured by immunoassay on an automated platform; however, immunoassays can be subject to interference. A fT4 concentration that does not agree with either the thyroid stimulating hormone concentration or the clinical presentation of the patient may be due to interference from, for example, heterophilic antibodies, or antibodies against the reagents or analyte. In these instances, analysis using an immunoassay from a different manufacturer is sometimes helpful in finding an answer, but not in every case.

    For patients with incongruent immunoassay results due to interference, a method which isolates the fT4 from serum proteins prior to analysis would be ideal. Two techniques have been described to achieve this: ultracentrifugation and equilibrium dialysis (ED). ED is considered to be the gold standard by Soldin, Thienpont, and other experts in the field. Previously at Addenbrooke’s we used a kit to perform equilibrium dialysis, followed by radioimmunoassay of the dialysate to measure the fT4. Methods have been developed by other laboratories in which a sample is subjected to equilibrium dialysis followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure free thyroxine. LC-MS/MS has several advantages over radioimmunoassay, including improved specificity and safety.

    We have investigated different sample extraction and analytical conditions to develop an in-house fT4 equilibrium dialysis LC-MS/MS method. This method utilises a dialysis step followed by concentration of the analyte using solid phase extraction prior to analysis by LC-MS/MS. The dialysis vessels used are commercially available and have been used previously in conjunction with a sensitive T4 radio-immunoassay (IVD Technologies, California, USA). Chromatographic separation was performed using a C18 analytical column. A Sciex 5500 triple quadrupole mass spectrometer was used in positive ionization electrospray mode. 13C (12)-labelled thyroxine was used as an internal standard.

    This method will be validated using a three-way method comparison between ED-LC-MS/MS, a one-step immunoassay (Siemens Centaur) and a two-step immunoassay (PerkinElmer DELFIA). The method comparison will use samples from patients with no known interference, and samples from patients with interference that have been received by the laboratory for investigation as part of the SAS Thyroid Service. It is hoped that, following full method validation, this ED-fT4 method will add a valuable tool to the repertoire of techniques used by the Supra-regional Assay Service laboratory to investigate unusual thyroid function test results.

  • Serum steroid panel to improve the lateralisation of primary aldosteronism during adrenal venous sampling

    David R Taylor (King's college Hospital NHS Foundation Trust, London, United Kingdom)

    Analysis of serum steroids by LC-MS/MS is increasingly replacing immunoassay, especially for those most subject to cross-reaction. However, much more is possible. By using a cocktail of deuterated internal standards, multiple steroids can be simultaneously quantified to provide a ´serum steroid panel´. This allows the incorporation of both common and rarely measured steroids in a single analysis and allows the targeting of specific adrenal diseases. One such application is the investigation of primary hyperaldosteronism (PA), which is the cause of 5-15% of hypertension. PA diagnosis is suspected in those in whom there is a raised aldosterone:renin ratio, off interfering medications. Diagnosis is confirmed by a failure of aldosterone to suppress in response to salt loading. PA is caused either by unilateral adrenal adenoma (APA) or bilateral adrenal hyperplasia (BAH). Lateralization of aldosterone over-production is addressed by adrenal venous sampling (AVS), with aldosterone:cortisol ratios from left and right adrenal veins being key. More recently, it has been suggested that ratios of aldosterone to steroids other than cortisol may be useful in identifying lateralization. Further, the steroids 18-hydroxycorticosterone and 18-hydroxycortisol have shown promise in predicting lateralization in basal samples.

    In this study an LC-MS/MS method for simultaneous quantification of 16 steroids in serum (pregnenolone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone, aldosterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, 18-hydroxycortisol, 21-deoxycortisol, cortisol, cortisone, testosterone, progesterone, androstenedione and DHEA) was developed and validated. The utility of the method in predicting lateralization from both basal and AVS samples (15 PA patients) was then assessed.

    Steroids were extracted from serum following addition of deuterated internal standards by protein precipitation, followed by liquid-liquid extraction using ethyl acetate. Steroids were then dried down under nitrogen, reconstituted in water:methanol and steroids resolved using a reverse phase C18 column. Steroids were detected using a triple quadrupole mass spectrometer operated in positive electrospray ionisation mode. Calibration curves were constructed from 8 standards prepared in charcoal-stripped serum at concentrations bracketing expected physiological and pathological ranges. Lower limit of quantitation ranged from 0.36 nmol/L for 11-deoxycortisol to 4 nmol/L for pregnenolone. Linearity over the chosen ranges and inter and intra-assay CVs of <10% were achieved for all analytes. In the clinical samples, adrenal to peripheral venous ratios were larger for 11-deoxycortisol, androstenedione and 17-hydroxyprogesterone than for cortisol, making these steroids useful in confirming aldosterone lateralisation. LC-MS/MS measurement yielded larger aldosterone:cortisol ratios than immunoassay. In peripheral samples, DHEA, corticosterone and cortisol were all higher in BAH than in APA (all p<0.05). In these samples, 18-hydroxycortisol tended to be higher in APA than BAH, whilst 18-hydroxycorticosterone was higher in BAH than APA, although neither association reached statistical significance.

    In conclusion, serum steroid panelling by LC-MS offers a promising diagnostic aid for primary hyperaldosteronism in both basal and AVS samples, by combining the measurement of steroid hormones and their precursors in a single analysis.

  • Tazocin pharmacokinetics in an intensive care unit dialysed cohort

    Harriet Allison (Ninewells Hospital, Dundee, United Kingdom)

    Patients admitted to the intensive care unit (ICU) can be extremely vulnerable to infection. Sepsis alone is the leading cause of mortality in non-cardiac ICUs. Therefore, effective antimicrobial therapy is critical for improving outcomes in these patients.However, the unique physiology typically encountered in critically ill patients which is often coupled with the requirement for renal replacement therapy can dramatically distort antibiotic pharmacokinetics.

    Sustained Low Efficiency Dialysis (SLED) has been introduced as the main method of renal replacement therapy within Ninewells Hospital ICU. This is a hybrid technique using intermittent haemodialysis equipment along with reduced blood and dialysate flow rates and is performed over eight hours. Tazocin is a combination antibiotic used to treat hospital acquired pneumonia and septicaemia and contains the beta-lactam antibiotic, piperacillin and the beta-lactamase inhibitor, tazobactam. This antibiotic is administered intravenously as a bolus dose of 4.5 g every eight hours. For β-lactam antibiotics, the characteristic that best links drug exposure with the antibacterial effect is the fraction of the dosing interval during which the free drug concentrations are above the bacterial minimum inhibitory concentration.

  • Test evaluation (Joint with AACB)

    Phillip Monaghan (University of Manchester, Manchester, United Kingdom)

    The Working Group for Test Evaluation (WG-TE) is a joint collaboration between the EFLM and AACB (Australasian Association of Clinical Biochemistry). Membership of this WG-TE represents collaboration between experts in evidence-based laboratory medicine, evidence-based diagnosis and epidemiology, and IVD industrial partner R&D.

    Key deliverables for this Working Group are to provide general guidance on the process of test evaluation studies and criteria, depending on the purpose or intended clinical application of new tests within clinical pathways. To this end, the WG-TE has published a number of articles addressing the current challenges of test evaluation, providing a cyclical test evaluation framework and a practical checklist tool for unmet clinical needs assessment for new tests. The WG-TE has also recently hosted the first training course on developing medical tests that improve patient outcomes, in Leiden, Netherlands attended by over 50 delegates from 20 different countries; a huge success. 

    Contributing to the WG-TE activities as a young scientist member has been a fascinating experience and I have really valued the opportunity to work on this important aspect of laboratory medicine. My presentation will cover 1) what I have learnt, achieved and contributed to whilst on the WG-TE, 2) my experience of working closely with European colleagues, 3) major outputs from the WG-TE, and 4) advice to prospective young scientists.

  • How should rare inherited metabolic diseases be optimally managed?

    Michael Badminton (Cardiff University School of Medicine and University Hospital of Wales, Cardiff, United Kingdom)

    Rare diseases are conditions with a prevalence of less than 1 in 2000 and although individually rare, affect an estimated 3 million people in the UK. The majority of rare diseases have a genetic component and present in childhood, with inborn errors of metabolism forming an important subset. Being adult onset, the acute porphyrias have tended to be diagnosed and managed through distinct clinical and diagnostic services based in Clinical Biochemistry or Haematology Departments. As a consequence centres providing a clinical service for acute porphyrias applied for and received funding for a highly specialised clinical service (the National Acute Porphyria Service or NAPS) in 2012.

    The principle behind a specialist service is to help patients with a rare disease receive high quality clinical care, preferably close to where they live. A service specification which defines the objectives and expected outcomes for the patient cohort should be developed with input from patient groups. For patients with active acute porphyria NAPS provides immediate clinical support and advice to local secondary care clinicians to assist in the treatment of acute attacks in the patient’s local hospital. Collaborative care is encouraged because the drug treatment (haem arginate) is funded and provided by NAPS. Patients can then be managed through defined care pathways to ensure they receive appropriate aftercare, genetic counselling and family screening, access to prevention therapies and other support services such as pain management and clinical psychology. This may include specialist centre outpatient visits, outreach clinics in satellite centres, as well as telephone or “Skype” consultations where appropriate. Regular contact and support from an experienced specialist nurse is also critical to ensuring that healthcare needs are being met locally. Patients requiring regular intravenous treatments can receive these at home through commissioning of a Homecare service with an appropriate private healthcare company. Liaison with general healthcare professionals supports the management of other more common clinical conditions and situations (e.g. pregnancy) in patients with rare disease. Of particular importance in acute porphyria patients is advice on which medications are safe to administer in collaboration with the UK Porphyria Medications Information Service (UKPMIS).

    National Rare Diseases plans, developed following a directive from the European Union, also highlight the need to empower patients through close collaboration with patient support groups, improve access to diagnostic testing to allow early intervention, co-ordinate appropriate care, and foster research into rare diseases with the aim of improving treatment options for patients. Networking of specialist services at both national and international levels, which encourage sharing of expertise in order to improve standards of care for patients is being fostered and the European Porphyria Network and the British and Irish Porphyria Network have been very active in support of patients. Ongoing implementation of many of these ideals may however prove challenging in an under resourced NHS, particularly given the relatively high cost of many novel treatments.

  • Alkaptonuria: evolution of a service

    Lakshminarayan Ranganath (Royal Liverpool University Hospital, Liverpool, United Kingdom)

    Alkaptonuria (AKU) is an iconic autosomal recessive inherited disorder of the phenylalanine/tyrosine metabolism characterised by deficiency of homogentisate dioxygenase leading to increased circulating and urinary homogentisic acid (HGA). Oxidative conversion of HGA to a melanin-like pigment is body tissues is responsible for the multisystem disease dominated by premature spondyloarthropathy. In 2003 there was no National Health Service available for rare disease AKU in the UK. Since AKU has a prevalence of 1 in 250,000, patients were isolated and unable to obtain satisfactory healthcare at a local level. A consultant chemical pathologist in Royal Liverpool University Hospital (RLUH) and a patient with AKU established the AKU Society, a charitable patient society in 2003, to support and advice patients. Around this time a clinical trial of nitisinone in AKU in the USA reported an inconclusive outcome despite remarkable metabolic efficacy. To pursue the possibility of treating AKU patients with nitisinone, further work was needed and led by RLUH. However, since AKU patients were needed to pioneer nitisinone treatment in AKU, a national survey of primary care physicians was carried and this identified 81 UK, 450 European and 1000 patients worldwide. At the time, systematic assessment of patients with this crippling disease was lacking, and it was felt that this lack might have been a reason for the inconclusive clinical trial. For these reasons the RLUH was established as a National Centre for AKU (NAC) and funded by the NHS Highly Specialised Services, employing off-label use of nitisinone. The patients identified have been able to access the NAC and able to access nitisinone free of charge and access a multidisciplinary team of experts. The NAC utilises a novel outcome measure, the AKU Severity Score Index, pioneered by RLUH. Established in 2012, the NAC now has more than 70 patients that have attended for assessment and care. Outcome measures of AKU studied in the NAC show a promising improvement in patients following off label use of unlicensed nitisinone. A range of therapies other than nitisinone are offered in the NAC and their use is personalized to patients. Credible developments in AKU at the RLUH have also led to the European Union providing a Framework Programme 7 grant of 6 million euro in a project called DevelopAKUre in 2012. The first of 3 studies has identified the optimal dose of nitisinone in AKU for the first time. Since nitisinone is expensive and inhibits the enzyme p-hydroxyphenlypyruvate dioxygenase causing tyrosinaemia, a second cross-sectional study is also being carried out to clarify the natural history to determine the best time in the life cycle to begin nitisinone in AKU. The third study is a 4 year nitisinone outcomes study that is expected to report of non-metabolic efficacy of nitisinone in AKU in 2019. These studies in the NAC, DevelopAKUre and others have already advanced our knowledge of AKU, providing novel metabolic insights and improved healthcare in AKU. The continued evolution of the NAC will bring advances in AKU to all patients with AKU worldwide.

Updated 24 March 2017

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