REGULAR FEATURES AIMS AACB NATIONAL SCIENTIFIC ...

A

ugus

t &

Nov

ember

20

18Vo

l. 3

9 N

o. 3

& N

o. 4

AU

GU

ST

& N

OV

EM

BE

R 2

01

8 V

OL. 3

9 N

o. 3

& N

o. 4

Pa

ge

s 5

5 - 1

42

AU

ST

RA

LIA

N JO

UR

NA

L O

F M

ED

ICA

L S

CIE

NC

E

REGULAR FEATURESAIMS AACB NATIONAL SCIENTIFIC MEETING 2018

Scientific PresentationsPoster and Oral Presentations

8

STA R Max® 2 and STA Compact Max® 2 : More than just coagulation analysers, a complete Stago solution to maximise your productivity.

Con

cept

ion

agen

ceL2

R.c

om -

©20

15 D

iagn

ostic

a S

tago

- A

ll rig

hts

rese

rved

- N

on-c

ontra

ctua

l pho

tos

- 03/

2016

MaxInnovation

MaxProductivity

MaxVersatility

Max Reliability

Max

15-12024 AP Generation Max ANZ_210x297.indd 1 02/03/2016 10:28


Con

cept

ion

agen

ceL2

R.c

om -

©20

15 D

iagn

ostic

a S

tago

- A

ll rig

hts

rese

rved

- N

on-c

ontra

ctua

l pho

tos

- 03/

2016

MaxInnovation

MaxProductivity

MaxVersatility

Max Reliability

Max


Australian Journal of Medical Science August & November 2018 Vol. 39 No. 3 & No. 4 55

A u s t r a l i a n J o u r n a l o f

M e d i c a l S c i e n c e

C O N T E N T S

Instructions to authors are available on the AIMS website www.aims.org.au

Copyright: All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic, mechanical, including photocopying, recording, or by an information storage and retrieval system, without permission in writing from the AIMS. Copyright by the Australian Institute of Medical Scientists, 2018.

Disclaimer: The opinions expressed in this Journal including those of the technological and advertisement sections are not necessarily those of the Editorial Board.

August & November 2018 Vol. 39 No. 3 & No. 4AIMS National OfficeChief Executive: Mr Michael Nolan, BAppSc BA MPhil GradCertMgt FAIMS MASM Design, formatting and administration: Mrs Simona Adochiei Email: [email protected] Website: www.aims.org.au Telephone: 61 7 3876 2988 Facsimile: 61 7 3876 2999 Address: PO Box 1911 Milton Qld 4064 Australia

Editorial Board

EditorsAssoc Prof Tony Woods, BA BSc(Hons) PhD MAIMS FFSc(RCPA) School of Pharmacy and Medical Sciences University of South Australia

Board MembersDr Ross Brown, PhD MSc MBA FAIMS FFSc(RCPA) Principal Hospital Scientist Royal Prince Alfred Hospital

Ms Robyn Wells, BAppSc MAIMS General Haematology Pathology Queensland Princess Alexandra Hospital

Assoc Prof Rob Siebers, PGCertPH FNZIC FNZIMLS CBiol FSB Research Associate Professor School of Medicine and Health Sciences, University of Otago, Wellington, New Zealand Editor, NZ Journal of Medical Laboratory Science

Prof Adrian Esterman, PhD AStat DLSHTM Foundation Chair of Biostatistics School of Nursing and Midwifery University of South Australia AJMS Statistical Adviser

Dr Stuart D. Blacksell, BAppSc (MedLabSc) MPH PhD RBP FASM FACTM MAIMS Senior Researcher Mahidol-Oxford Tropical Medicine Research Unit Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

Dr Geoffrey Bosson, MSc PhD CertHSM CertTQM CertT(HE) CSci MAIMS FIBMS School of Biomedical Sciences, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom

The Australian Journal of Medical Science is the official publication of the Australian Institute of Medical Scientists.

Circulation 2000 per issue. The Journal is circulated to members in pathology laboratories, universities and research institutes throughout Australia and overseas.

Annual subscription rates are available from the AIMS National Office.

Article reprints may be organised on request from AIMS National Office.

Advertising rates are available from AIMS National Office.

Abstraction of the Australian Journal of Medical Science is through the following serial catalogue listings: Australasian Medical Index, Chemical Abstracts, and EMBASE/Excerpta Medica.

The Australian Journal of Medical Science is listed on the Register of Refereed Journals by the Australian Commonwealth Department of Education Employment and Workplace Relations.

ISSN 1038-1643

Printed by Westminster Eagle Eye Printing, PO Box 161, Paddington Qld 4064.

Design Cover and layout design by Kim Brown, 23 Denman St Exeter SA 5019 [email protected]. Cover photograph courtesy of Prof Ian Gibbins, Flinders Medical Centre, Adelaide.

AIMS AACB National Scientific Meeting 2018 Scientific Presentations 58

Poster and Oral Presentations 76

Regular FeaturesBooks for review 135

Instructions to authors 137

AIMS AJMS 131

South Pacific Congress 132

Multidisciplinary Scientific Workshop 133

APACE 134

MTS 140

Fellowship Programme 141

A D M I N I S T R A T I O N

Australian Journal of Medical Science August & November 2018 Vol. 39 No. 3 & No. 456

AIMS AACB Nat ional Scient i f ic Meet ing 2018Scientific PresentationsSpeakers index

Amor DJ ............................................................................S3Barr IG ............................................................................S10Baxter RC ........................................................................S31Buckley M .........................................................................S4Burnett L ..........................................................................S9Carpenter K ......................................................................S7Caterson ID ....................................................................S16Cheng L ......................................................................... S38Colin L ...........................................................................S37Craig ME ..........................................................................S1Dutton - Regester K ........................................................S21Dale B .............................................................................S22Fletcher N ......................................................................S25Fuller K .......................................................................... S32Fuller K ...........................................................................S39Gasiorowski R ................................................................S28Graham P ...................................................................... S42Graham P .......................................................................S44Gyedu L ..........................................................................S22Harris C ..........................................................................S15Henderson A ..................................................................S22Hoptroff J .......................................................................S22Johnston A ....................................................................S19Keane C ..........................................................................S12Khin AA ...........................................................................S22Leedman PJ ....................................................................S30Lindeman R ....................................................................S20Loh TP ............................................................................S18Lunn J ............................................................................S46Maclean F .....................................................................S23Mann G .........................................................................S35Martin J .........................................................................S36Martin R .........................................................................S11McGrow C .......................................................................S2Merlino J ........................................................................S41Micklethwaite K ............................................................S13Miranda S.......................................................................S43Newmann A ...................................................................S22Newson AJ .....................................................................S48Rozenberg R .................................................................. S14Sacks DB ....................................................................... S45Shigdar SL .....................................................................S27Shimoni O .....................................................................S34

Short Kirsty R ................................................................S47Smith S ..........................................................................S40Spring KJ .........................................................................S24Srinivasan S ......................................................................S2Stevenson WS ............................................................... S29Tchan M .......................................................................... S8Vandenbroek E ...............................................................S22Veedu RN .......................................................................S26Velickovic Z ...................................................................... S6Williams J ........................................................................S22Williams K ......................................................................S17Wilson M .........................................................................S5Winter M .......................................................................S33

Poster and Oral PresentationsAbeynayake GPN ...........................................................P10Aizawa Y ........................................................................ P11Allen D ......................................................................... P61Alvaro F ......................................................................... P25Arunachalam S ................................................................ P9Bahnisch R ..................................................................... P26Bandodkar S .................................................................. P27Chung JZY ...................................................................... P28Chung JZY .......................................................................P29Cross C ...........................................................................P31Dayanath BKTP ............................................................. P83De Leon R ...................................................................... P86Ehm M ........................................................................... P23Estoesta G ..................................................................... P32Fitzgerald B ................................................................... P47Fitzgerald B ................................................................... P48Fitzpatrick M ................................................................. P33Foyn L ............................................................................ P38Foyn L ............................................................................ P30Foyn L ............................................................................ P36Foyn L ............................................................................ P37Gay S ............................................................................. P87Ghosh R ........................................................................ P39Hall D ............................................................................. P74Herrmann M ................................................................. P12Ibrahim M ..................................................................... P60James B ......................................................................... P88Jenkins N ......................................................................... P1


Jones J ........................................................................... P22Josman N ....................................................................... P40Kanowski D ................................................................... P41Khin AA ......................................................................... P78Khoo C .......................................................................... P70Kuhn T ........................................................................... P42Lam L ............................................................................ P13Lee A ............................................................................. P79Letsatsi O ........................................................................ P2Leung CW ...................................................................... P76Lim CF ........................................................................... P43Lim CF ........................................................................... P44Liu K .............................................................................. P66Liu KC .............................................................................. P3Lo CWS .......................................................................... P14Lo CWS .......................................................................... P15Louey W ........................................................................ P67Louey W ........................................................................ P71Matthews B ................................................................... P16McEntyre CJ .................................................................. P45McLemon E ................................................................... P75McPhie D ...................................................................... P89Mitra P .......................................................................... P80Mitra P .......................................................................... P82Montgomery S ............................................................... P4Morrow C ...................................................................... P46Nascimento MP ........................................................... P18Nguyen K ....................................................................... P49Nguyen K ...................................................................... P50Novos T ......................................................................... P51Oakman C ..................................................................... P91Oostenbroek J ..................................................................P5Panapitiya N P ............................................................... P17Papasimeon V ................................................................P52Pierson-Perry J ...............................................................P24Pillay N ...........................................................................P90Pool G ............................................................................P53Pope JD ..........................................................................P54Potter JM .......................................................................P55Potter JM .......................................................................P56Punyalack W ..................................................................P92Quinlan C .......................................................................P57Quinlan C .......................................................................P58Rankin W ...................................................................... P19Rowland J .......................................................................P77

Sack MM .......................................................................P59Sahertian R ....................................................................P20Sharma S ...................................................................... P84Shepherd S .....................................................................P93Sivaneson S ....................................................................P94Sivaneson S ....................................................................P95Stranks JL .......................................................................P21Swenson R .....................................................................P62Swenson R .....................................................................P63Tan SS .............................................................................P64Thompson I ....................................................................P73Thompson S .................................................................. P65Tran C .............................................................................P72Tran D ............................................................................P68Vasiliadis C .....................................................................P85VSN Kiran Kumar Pilla VSN ............................................P69Ward G .............................................................................P6Ward G .............................................................................P7Ward G .............................................................................P8Wilschefski S ..................................................................P34Wilschefski S ................................................................. P35Woollard G .....................................................................P81

Burke DG .........................................................................O4Chiang C ..........................................................................O8Farrell C .........................................................................O16Herath Mudiyanselage AU ............................................O13Herrmann M ...................................................................O9Ibrahim M .....................................................................O11Jiang C ...........................................................................O22Jones GRD .....................................................................O17Lam CW .........................................................................O23Law Chun-Yiu ...................................................................O7Law CY ...........................................................................O15Lu ZX ................................................................................O5McGow C .......................................................................O21Rathnayake G ................................................................O19Rudd D ............................................................................O6Sikaris KA .........................................................................O1Sikaris KA .........................................................................O2Smith T ............................................................................O3Stanford P ......................................................................O18Trambas CM ..................................................................O10Vincini G ........................................................................O14Ward P ..........................................................................O12

Zakaria R ........................................................................O20


S1: Unravelling the heterogeneity of diabetes in youth – can ‘omics’ help?

Maria E Craig1Children’s Hospital at Westmead, NSW, Australia2St George Hospital, NSW, Australia3Discipline of Child and Adolescent Health, University of Sydney, Australia4School of Women’s and Children’s Health, University of New South Wales, Australia

Genome wide association and whole genome/exome sequencing studies have increased our clinical understanding of monogenic forms of diabetes that are distinct from of type 1 diabetes (T1D) and type 2 diabetes (T2D). Monogenic diabetes (MODY), which accounts for ~1–6% of paediatric diabetes cases, results from one or more defects in a single gene, which may be inherited within families as a dominant, recessive or non-Mendelian trait, or may present as a spontaneous case due to a de novo mutation. More than 40 different genetic subtypes of MODY have been identified. The diagnosis of MODY, and consideration of genetic testing, should be suspected in cases with (i) a family history of diabetes - in patients without the characteristics of T1D (ie no islet autoantibodies, low or no insulin requirements >5 years after diagnosis) or T2D (marked obesity, acanthosis nigricans); (ii) mild stable fasting hyperglycaemia which does not progress (such cases should be tested for glucokinase gene mutations (GCK-MODY) - the commonest cause of persistent, incidental hyperglycaemia in children; (iii) presence of specific features that suggest MODY subtypes, such as renal developmental disease or renal cysts (HNF1B-MODY) and macrosomia and/or neonatal hypoglycaemia (HNF4A-MODY); and (iv) patients diagnosed with diabetes < age 6 months (such cases require immediate molecular genetic testing to define their subtype of monogenic neonatal diabetes).

Establishing the correct molecular diagnosis of MODY avoids misdiagnosis as T1D or T2D; guides therapy including use of sulphonylureas; may offer more accurate prognosis of complications risk; and may enable risk prediction in a first degree relative or offspring.

Composite T1D genetic risk scores that use key gene variants associated with T1D are novel tools that can differentiate T1D from MODY and T2D, and can also be used to predict risk of future development of T1D. Increasingly, next generation sequencing is being used to investigate the role of the environment (microbiome, virome, metabolome-lipidome), as well as genetics, in the pathogenesis of T1D. The Environmental Determinants of Islet Autoimmunity (ENDIA) study (www.endia.org.au/) is an Australian prospective pregnancy cohort study that will use a systems biology approach to integrate multi-omics

analyses to explore hypotheses and mechanisms underlying the development of islet autoimmunity and T1D.

S2: Familial hypercholesterolaemia

S Srinivasan1, C Spinks2, D Sullivan3

1Institute of Endocrinology and Diabetes at The Children’s Hospital at Westmead, Australia2 Department of Medical Genomics, NSW Health Pathology, Royal Prince Alfred Hospital, Camperdown, Australia3Chemical Pathology, NSW Health Pathology, Royal Prince Alfred Hospital, Camperdown, Australia

Familial hypercholesterolaemia (FH) is the most prevalent codominant genetic cause of premature cardiovascular disease, affecting 1 in 300 Australians. FH is typified by elevated levels of low-density lipoprotein (LDL) cholesterol in the blood from birth, with a doubling of LDL in the heterozygous state and four times the norm in the rare, homozygous FH (HoFH) patients. Atherosclerosis development is accelerated and coronary artery disease (CAD) forms prematurely. Half of all untreated men with FH will suffer heart attack before 50, some as young as 20 years. The majority of patients with FH remain undiagnosed and untreated, despite the availability of safe, efficacious, and cost effective treatment that greatly reduce CAD burden. In Australia, the Dutch Lipid Clinic Network Score is the preferred clinical tool for identifying patients with FH and genetic testing is recommended to guide care for the whole family. The underlying molecular cause will be identified in the LDLR, APOB or PCSK9 genes in 80% of FH cases. Whilst an individual patient’s treatment is not solely dependant on a molecular diagnosis, there is great benefit in facilitating cascade genetic testing in family members. A multidisciplinary team incorporating paediatric and adult specialists with dieticians, nurses and genetic counsellors collaborate for the diagnosis and management of FH families. We will present the latest in diagnosis and management of FH for paediatric and adult patients in NSW.

S3: Preconception testing for fragile X, CF and SMA

DJ Amor1,2,3,4, MJ Smith1,2, T Burgess1,2,3, KL Scarff1, J Elliott1, CE Hunt1, C Barns-Jenkins1,2, C Holt1,2, K Sandoval1,2, V Siva Kumar1,2, L Ward1,2, EC Allen2,3, SV Collis2,3, S Cowie1, D Francis1,2, MB Delatycki1,2,3,4, EM Yiu2,3,4, RJ Massie2,3,4, MD Pertile1,2,3, D du Sart1,2,3, D Bruno1,2,3, AD Archibald1,2,3

1Victorian Clinical Genetics Services, Parkville 3052, Victoria, Australia2Murdoch Children’s Research Institute, Parkville 3052, Victoria, Australia3University of Melbourne, Parkville 3052, Victoria, Australia


4Royal Children’s Hospital, Parkville 3052, Victoria, Australia

Introduction

Advances in genetic testing technology have increased our capacity to identify prospective parents at risk of having a child with an inherited condition. Carrier screening has shifted from testing for a single or a small number of conditions, to much larger expanded panels. We developed the first Australian program offering simultaneous genetic carrier screening for cystic fibrosis (CF), fragile X syndrome (FXS), and spinal muscular atrophy (SMA), and have analysed the outcomes of this testing program.

Methods

Carrier screening was offered through general practice, obstetrics, fertility, and genetics settings before or in early pregnancy. Carriers were offered genetic counselling with prenatal/ preimplantation genetic diagnosis available to those at increased risk.

Results

Screening of 12,000 individuals revealed 610 carriers (5.08%; 1 in 20): 342 CF, 35 FXS, 241 SMA (8 carriers of 2 conditions), approximately 88% of whom had no family history. At least 94% of CF and SMA carriers’ partners were tested. Fifty couples (0.42%; 1 in 240) were at increased risk of having a child with one of the conditions (14 CF, 35 FXS, and 1 SMA) with 32 pregnant at the time of testing. Of these, 26 opted for prenatal diagnosis revealing 7 pregnancies affected (4 CF, 2 FXS, 1 SMA).

Conclusion

The combined affected pregnancy rate is comparable to the population risk for Down syndrome, emphasizing the need to routinely offer carrier screening. The availability of appropriate genetic counselling support and a collaborative approach between laboratory teams, genetics services, health professionals offering screening, and support organizations is essential. These results provide a platform for the development of expanded screening programs that cover a larger number of disorders.

S4: Advances in genomic testing for inherited disease

Michael Buckley

Human Genetics Society of Australasia

Purpose

Whole exome sequencing (WES) is rapidly becoming the standard of care for genetic services. I present the results of the first 500 clinical exomes performed at the Randwick Genetic Laboratory as an example of the power of this technology.

Methods

WES was performed in 204 probands with Mendelian disorders, together with family members. Ampliseq RDY exome, libraries were analysed on a Life Technologies Proton instrument. Data were analysed using an in house pipeline with variant reporting following ACMG guidelines.

Results

WES resulted in greater than 43% definitive findings with a small number of variants of uncertain significance. The diagnostic rate has increased over time, likely reflecting refinements in clinician referral practice as well as improved functionality of bioinformatics pipelines. Two families (1%) with Cantu and MoyaMoya syndromes had results which could lead to pharmacologic interventions. Thirthy-three families (16%) had de novo variants and seven (3.5%) were X-linked with significant implications for recurrence risk. Two prenatal diagnoses have been performed based on WES results. A diagnosis was made in 6/8 (75%) rapid turnaround studies, including two during pregnancy. Almost 10% of the diagnoses were unanticipated by the clinical teams and involved significant changes in diagnostic category. Four likely novel genes involved in known biological pathways were identified enabling research participation.

Conclusions

The minimum number of clinically useful outcomes was at least 20% within the 12 month period of this study. The number of secondary findings was small at about 1%. This demonstrates that WES is characterized by high levels of patient safety, clinical utility and cost effectiveness.

S5: The Australian genomics health alliance health services research project

M Wilson

Head, Department of Clinical Genetics, Western Sydney Genetics Program, Sydney Children’s Hospital Network-Westmead, NSW, 2145, Australia

The Australian Genomics Health Alliance, set up in 2014, is a national research collaboration of over 80 partner institutions including clinicians, researchers and diagnostic geneticists working together to provide evidence for the equitable, effective and sustainable delivery of genomic medicine in healthcare. In 2016 an NHMRC Genomics Targeted Call for Research grant allocated $25 million over five years for the Australian Genomics Health Alliance health services research project, which aims to demonstrate the cost effectiveness, diagnostic efficacy and impact on clinical care of genomic medicine.

The Australian Genomics project is organised under four Programs (National Diagnostic and Research Network;


National Approach to Data Federation and Analysis; Economic Analysis, Policy Implications for the Health System and Ethics; Genomics Workforce and Education), grouped across two broad clinical themes, rare diseases and cancer. Research questions for each area were determined by working groups with expert representation. For evaluation of the delivery of genomic testing it was important that this program of work occurred in a clinical setting. Testing in many cases is carried out in NATA accredited diagnostic laboratories and the research surrounding the delivery of genomic testing to patients does not disrupt clinical care pathways.

The Program One - National Diagnostic and Research Network includes a number of flagships (clinical patient cohorts) to demonstrate how clinical genomics can be applied in practice. These flagships prospectively recruit patients and provide clinical, genomic, economic and implementation data to the four programs of work. Program One encompasses other initiatives aimed at strengthening national expert diagnostic referral pathways, developing a standard national consent, establishing processes for re-evaluation of variants of uncertain significance and creating a network for functional genomics.

To illustrate the approach, this presentation will briefly summarise the outline, implementation and early impact of the Australian Genomics Acute Care Flagship, which attracted significant interest from paediatric research partners and commenced recruitment in most states in 2018.

S6: Challenges to hospital scientists manufacturing human cells

Zlatibor Velickovic

Department of Cell & Molecular Therapies, Royal Prince Alfred Hospital, Sydney, Australia

Sydney Medical School, University of Sydney, Sydney, Australia

Manufacturing of human cells is a lengthy and complex process that occurs ex vivo. It requires cycles of expansion, stimulation, treatment with factors, or genetic manipulation with viral vectors to reprogram the cells. Manufacturing of cell therapies is quite different to the traditional drug manufacturing where one product is intended for one patient and logistically this personalised medicine approach can be quite challenging. Special cleanroom facilities are designed to meet regulatory requirements for human cell manufacturing set by the code of Good Manufacturing Practice (cGMP) and mandated by the Therapeutic Goods Administration (TGA). Implementation of the Quality system is essential to ensure quality and safety of manufactured

cells. Policies and standard operating procedures need to be specifically developed to ensure that:

• strict validated manufacturing protocols are followed;

• qualified facilities and equipment are regularly maintained;

• pre-specified and approved materials are used in manufacturing;

• finished products meet pre-defined release criteria ensuring safety;

• process controls are in place to confirm critical steps in manufacture are documented;

• records are kept to provide evidence and traceability.

Department of Cell & Molecular Therapies at Royal Prince Alfred Hospital in Sydney is the leader in a GMP and PC2 facility designed and maintained to manufacture genetically modified human cells for clinical use. The facility’s specific design and operational model of laboratories will be discussed with focus on manufacturing of cell and gene therapy products to the highest level of regulatory compliance and safety.

S7: Mass spectrometry for diagnosis of inborn errors

K Carpenter

Australasian Association of Clinical Biochemistry, Sydney NSW, Australia

The development of the field of inborn errors of metabolism has mirrored advances in laboratory techniques to detect metabolites of interest in biological fluids. Traditionally using one separation technique to analyse a particular group of compounds, eg. amino acids, discovery of new disorders was slow until the middle of the last century.

The introduction of gas chromatography/mass spectrometry in the 1960s led to the identification of a whole new class of disorders, organic acidaemias. Although GC/MS has been used for other classes of compounds the next major breakthrough was the coupling of mass spectrometry with liquid chromatography, allowing the analysis of polar non-volatile compounds with minimal sample preparation.

Tandem mass spectrometry allowed the Introduction of high throughput screening for inborn errors in newborns and became the tool of choice for many quantitative analysis of many compounds of interest.

Many biochemical genetics laboratories now offer first line screening using liquid chromatography/ tandem mass spectrometry for a very wide range of conditions with rapid turnaround. LC-MS/MS is also used for diagnostic enzymology, utilising the very high sensitivity and ability to


use stable isotope labelled substrates to probe pathways with great precision.

In the future we may see mass spectrometers being used as point of care devices allowing accurate diagnosis without the need to ship samples to major centres.

S8: Inborn errors of metabolism – the adult perspective

Dr Michel Tchan

Genetic Medicine, Westmead Hospital; University of Sydney, Sydney NSW, Australia

There is now an increasing cohort of adults who are diagnosed with a genetic disorder of metabolism (also known as an inborn error of metabolism), with more than 800 patients known to the Adult Genetic Metabolic Disorders Service at Westmead. This includes those who were diagnosed in childhood, but have now graduated beyond paediatric services. Adolescence and adulthood bring new issues for these patients, including the management of pregnancy and the diseases of aging. A further group of patients are those who are diagnosed as adults, and may present with atypical symptoms or have a disease that classically presents later in life. Recognising these late-onset patients may be difficult, as their disease may be rare, and the symptoms and biochemical parameters may mimic other common diseases. As an example, an approach to making a diagnosis of an inborn error of metabolism in the setting of hyperammonemia is given. Diagnosis is important for these patients, as the inborn errors of metabolism may be amenable to treatments such as dietary modification, enzyme replacement therapy and newer approaches such as gene therapy.

S9: Whole genome analysis: how it works, and what it can do

Leslie Burnett

Genome.One, Darlinghurst Sydney NSW 2010Garvan Institute of Medical Research, Darlinghurst Sydney NSW 2010, Australia

Introduction

Extraordinary advances in sequencing technology and bioinformatics have enabled Next Generation Sequencing (NGS)/Massively Parallel Sequencing (MPS) to become available as a clinical diagnostic test. Whole Genome Analysis (WGA) is increasingly being used as a first-tier investigation, based on either Whole Exome Sequencing (WES) or Whole Genome Sequencing (WGS). The challenges of sequencing are now largely solved, and attention is turning to the new challenges of interpretation and reporting.

Methods

This presentation will provide an overview of WGA, and outline some of the approaches and applications now being used in both testing and screening. This presentation will focus on germline (inherited) disorders, although many of the issues and principles are also relevant to somatic (acquired) genetic conditions.

Results

Several example case studies will be provided to illustrate the applications of WGA for germline disorders. Examples will include cases where WGA can produce diagnoses that are otherwise unable to be made with non-genomic approaches, as well as cases where WGA may be a cost-effective first tier investigation, supplanting many conventional investigations. With knowledge and understanding of simple coding variants rapidly advancing, attention is turning to copy number variation (CNV), structural variation (SV) and non-coding variants.

Conclusion

“Everyone” agrees that WGA using WGS is going to be the end point for germline genomic testing. We are currently at the point where cost-effective testing is moving from panel-based MPS towards WGA using WES. The availability of clinically accredited reporting of CNV, and especially non-coding variants, will likely accelerate the move towards WGA using WGS.

S10: Avian influenza - why the concern?

Ian G Barr

WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, Peter Doherty Institute, 792 Elizabeth Street, Melbourne, Victoria, 3000, Australia

The year 2017 marked 20 years since the first human infections with A(H5N1) avian influenza (or “bird flu”) occurred in Hong Kong. This started a wave of zoonotic H5N1 avian viruses that rapidly spread from China to Asia to Africa and Europe/USA and continues in many countries to this day. While these viruses can be easily spread can be extremely deadly in chickens and other poultry, in man infections have been relatively rare (860 human cases since 2003) but still quite deadly (454 deaths; CFR=38%). In addition to the H5N1 outbreaks other avian influenza viruses such as H7N9 have emerged and infected many hundreds of people in Southern China (1623 people from Feb 2013 – May 2018, with 623 deaths; CFR=38%). Various control measures have been attempted including stamping out and vaccination, with limited success for H5 viruses, however, a mass poultry vaccination program in China in 2017 with a bivalent H5/H7 vaccine, seems to have succeeded in blunting subsequent human infections with H7N9. Along with these two avian influenza viruses many


more are in circulation in the avian population, some of which have infected humans. So will the next pandemic be caused by an avian influenza virus or a swine influenza virus (like the last pandemic in 2009) or will a virus merge from another source, and will it be a mild pandemic or a more severe pandemic? Time will tell, but we continue to study and speculate on the possibilities using a variety of virological and epidemiological tools as well as conducting research in an attempt to better understand and potentially mitigate the next influenza pandemic.

S11: The threat of antimicrobial resistance in animals in Australia

Dr Mark Schipp,1 Dr Robyn Martin,2

1Australian Chief Veterinary Officer2Animal Health Policy, Department of Agriculture, Canberra ACT, Australia

Antimicrobials have been a catalyst for unprecedented advances in modern medicine, including veterinary medicine. However, the revolutionary properties of antimicrobials have resulted in their widespread, and often inappropriate use. This has contributed to many bacteria developing resistance to antimicrobials, resulting in treatment complications and failures, and increased healthcare costs. Resistance to current antimicrobials is increasing faster than the development of new drugs, and effective treatments cannot keep pace. Globally, about 700,000 people die of resistant infections each year because antimicrobial drugs have become less effective at killing resistant pathogens. In animals, antimicrobial resistant infections adversely impact animal health, welfare, biosecurity and production. Another serious consequence of antimicrobial resistant infections in animals is the potential transfer of resistant bacteria to people through direct contact, environmental exposure or through foodborne transmission. With the global increase in trade and travel (including medical tourism)antimicrobial resistance presents universal challenges to the medical and veterinary professions. Addressing these issues requires not only a One Health approach with the human, animal and environmental sectors, but a wider multisectoral collaboration with other disciplines, including food safety experts, scientists, ecologists, economists and sociologists. Antimicrobial resistance is recognised as a growing global threat to both human and animal health. It has implications for food safety, food security, health security and our economic well-being. If resistant bacteria continue to spread, our inter-connected world may find itself back in the pre-antibiotic era of medicine.

S12: The evolution of immune checkpoint therapy in aggressive lymphomas.

Dr Colm Keane

Princess Alexandra Hospital, University of Queensland Diamantina Institute, Brisbane QLD, Australia

Immunotherapy has had a profound impact on the management of many cancers. Many patients with only palliative options available to them have had profound responses, and in some cases these patients may in fact be cured. The success of immune checkpoint has led to a massive development of immune based therapies. The differences between responders and non-responders have provided insights into how the immune system responds to cancers. In our laboratory, we have looked at how immune checkpoints expression within tumours may help to predict outcomes to standard chemotherapies. Our results in DLBCL and PCNSL provide hope for improving responses in these diseases, using immune checkpoint based therapy and understanding the different outcomes seen in patients. Interestingly the tumour with highest responses to immune checkpoint therapy is Hodgkin lymphoma and this talk will describe the unique mechanisms present in this disease that leads to such high response rates in patients who have failed multiple lines of therapy.

S13: Chimeric antigen receptor T-cells for cancer

Kenneth Micklethwaite

Westmead Hospital, Sydney Cellular Therapies Laboratory and Westmead Institute for Medical Research, The University of Sydney, Australia

Chimeric antigen receptor T-cells (CAR T-cells) are immune cells that have been genetically modified to enable them to detect and eliminate tumour cells. CAR T-cells specific for the B-cell antigen CD19 can cure patients with otherwise incurable leukaemia and lymphoma and are now being incorporated into standard care in the USA and Europe. CAR T-cells have potential to revolutionise the treatment of cancer, but there are significant hurdles to their widespread use including cost, logistics of production and distribution, and immediate and long term safety concerns. We have developed novel gene modification and CAR T-cell expansion methods and are testing these “homegrown” CAR T-cells in clinical trials. Early results are similar to those seen in international studies using more complex production methodologies. The local production of CAR T-cells using simplified processes will enable their broad application and hopefully replace more toxic therapies such as chemotherapy in the near future.


S14: Morphological features and treatment of inherited disorders

Gillian Rozenberg

Senior Medical Scientist, Department of Haematology, Prince of Wales Hospital, Randwick, Sydney

Two children with an inherited disorder were admitted to the Sydney Children’s Hospital which is situated on the Prince of Wales Hospital Campus. The first case is that of a nine-year-old male child that had been transferred from interstate with a diagnosis of Mucopolysaccharidosis Type VI, also known as Maroteaux-Lamy syndrome. Mucopolysaccharidosis Type VI is inherited as an autosomal recessive trait and is characterised clinically as having coarse facies, corneal clouding, joint stiffness and short stature. This child was admitted specifically for an allogeneic bone marrow transplant from a matched sibling donor.

The second case is that of a six-month-old female child admitted to the Sydney children’s Hospital with a severe microcytic hypochromic anaemia. A diagnosis of beta thalassaemia major was made on this child. Family studies were then performed on both parents; the mother and father were diagnosed as having beta thalassaemia trait.

Diagnostic tests on both these cases are discussed with an emphasis on the differential morphological diagnosis of the blood film morphology.

Treatment and cure rate of both cases will be presented by Dr Richard Mitchell, Paediatric Oncologist at the Sydney Children’s Hospital.

S15: Diagnosis and morphology of two different cases of congenital non-malignant haematological disorders

Catherine Harris

Haematologist, 4Cyte Pathology

Two children with rare congenital non-malignant haematological disorders were admitted to The Children’s Hospital Westmead. The first case was a 6-year-old girl referred from Noumea for an elective splenectomy. She had been diagnosed with Hereditary Pyropoikilocytosis (HPP) at the age of 1 in Noumea with confirmatory genetic testing performed in France. HPP is an autosomal recessive red cell membrane disorder clinically related to Hereditary Elliptocytosis (HE). HPP involves a functional defect in spectrin which results in a severe chronic haemolytic anaemia.

The second child was a 9-week-old male admitted through the Children’s Hospital Westmead Emergency Department with fevers and a five day history of a lump to the right side

of his neck. During this admission, the diagnosis of X-linked Chronic Granulomatous Disease was made on the child. His admission was complicated by on-going anaemia requiring a transfusion. At 5 months, anti-Kx/anti-Km antibody was detected in his plasma which confirmed the diagnosis of McLeod phenotype.

Diagnostic tests and morphological features for both cases will be presented.

S16: Obesity is a chronic non-communicable disease and must be treated as such!

Ian D Caterson

Boden Institute, Charles Perkins Centre University of Sydney. NSW 2006, Australia

Obesity is highly prevalent in Australia and much of the world. It has associated complications which may be broadly considered to be metabolic, mechanical and psycho-social. The aetiology of obesity is multifactorial, and whilst there is an element of personal choice/responsibility, genetic factors and the socio-cultural environment make such choice difficult or impossible. It is therefore imperative that we understand the underlying issues and work to change them so as to make the prevention of obesity possible. Obesity is a disease (or chronic disorder) with major costs to the health system and individuals. There is a stigma attached to obesity which as well as damaging individuals also inhibits the health system and practitioners from treating it effectively.

Obesity can be managed. There is growing evidence of the effectiveness of treatment and prevention initiatives and of hard long-term outcomes of weight management. Whilst the standard approach remains attention to eating, activity, behaviour and habits, there are increasing types of effective adjunctive therapies which include CBT, use of messaging, very low energy diets, pharmacotherapy and bariatric surgery. The newer pharmacotherapy holds a lot of promise.

What is needed is for obesity to be managed as a chronic disease with the necessary multidisciplinary team and with long-term follow-up. There needs to be workforce education and a change in approach – which must be possible.

S17: Obesity and its co-morbidities

K Williams

Nepean Family Obesity Services, Nepean-Blue Mountains Local Health District; University of Sydney, Sydney NSW, Australia


The prevalence and severity of obesity has been increasing in Australia in what has been coined the ‘obesity epidemic’. Obesity is associated with many co-morbidities, which include disorders of the endocrine, respiratory, cardiovascular and gastroenterological systems. This presentation will discuss these obesity-related complications, with a focus on sleep disordered breathing and non-alcoholic fatty liver disease. It will also briefly introduce the Nepean Family Obesity Service as the first public whole of lifespan interdisciplinary clinic for managing obesity in Western Sydney.

S18: Harnessing laboratory data for better informed practice

TP Loh

Department of Laboratory Medicine, National University Hospital, Singapore

There is a saying in the research community that if the world stops performing any laboratory-based research, it will still have enough data to be analysed over the next five years. The anecdote underscores the speed and volume of data accumulation secondary to availability of high resolution and throughput laboratory methods. The use of electronic ordering system, laboratory automation and laboratory information systems have contributed to a wealth of healthcare information being generated and stored in laboratory databases. The laboratory database is a gold mine waiting to be exploited. Some examples of using laboratory database including deriving reference intervals and biological variation data for the pediatric population, which would be otherwise difficult to obtain using the conventional approach due to operational, resource and ethical considerations. Moreover, the advent of next-generation electronic medical record system (EMRS), which promises to bring together all previously silo clinical databases, have provided laboratory practitioners with unprecedented access to clinical details that can allow interesting clinical questions to be answered. The ability to draw data from the clinical diagnosis, clinical notes, medication record, radiology report and laboratory results can allow laboratory practitioners to verify the clinical performance of their laboratory tests and reference values, examine the clinical utility of laboratory requests and performed outcome-based study of laboratory tests. Moreover, access to cost data also allows the laboratory to examine the impact of the tests on the overall healthcare cost and device cost-effective diagnostic strategies for patients. All these activities help laboratory medicine add value to the healthcare system. To optimally capture these values, it is important for laboratory practitioners to foster close collaboration with our clinical colleagues

(to better understand clinical context), data scientists and biostatisticians.

S19: The role of medical laboratory science in international public health

A Johnston

Victorian Infectious Diseases Reference Laboratory, Doherty Institute, Melbourne, Vic 3000, Australia

Quality laboratory results are vital for accurate diagnosis, screening, surveillance and monitoring of serious public health conditions. This is true in Australia and around the world however a lack of appropriately skilled staff, suitable technology, and even reliable electricity can be a challenge in many international settings.

The burden of infectious diseases in low resource settings is still significant. Human immunodeficiency virus (HIV), tuberculosis (TB) and malaria remain among the top ten causes of mortality in low resource countries, along with respiratory infections, diarrhoeal diseases, ischaemic heart disease and stroke.

According to the World Health Organisation (WHO), the global incidence of HIV has declined over the past eleven years, however an estimated one million people died of HIV related illness in 2016. Tuberculosis remains a high burden disease despite a decline in new and relapsed cases. In 2016, an estimated 10.4 million people became ill with TB and there were 1.6 million deaths. WHO estimates that there were around 216 million cases of malaria globally in 2016 and malaria claimed the lives of approximately 445,000 people, mostly children under five. Laboratory diagnosis and monitoring are vital in the response to these conditions and the support of patients, contributing to accurate diagnoses and appropriate treatment options and outcomes. This presentation will describe experiences working in public health programs internationally with Médecins Sans Frontiѐres, WHO, and other non-government organisations. It will expand on the important role of the laboratory in supporting public health programs responding to HIV, TB, malaria and other public health risks. It will outline some of the challenges faced by laboratories in low resource settings.

S20: Current issues in the management of patients with thalassaemia major

R. Lindeman

NSW Health Pathology, Prince of Wales Hospital Randwick NSW 2031, Australia


Patients with thalassaemia major who have not undergone allogeneic transplantation require lifelong transfusion support. The disease pattern has changed considerably in Australia with the availability of oral chelators, improved screening of blood for infectious agents and more effective therapies for hepatitis C.

Whereas cardiac iron overload related to lifelong transfusions was previously a major contributor to early mortality, most patients are now well chelated. Adult patients nevertheless have a legacy of iron overload earlier in life, with many suffering endocrine complications including diabetes mellitus, hypoparathyroidism, hypothyroidism, pituitary insufficiency (and consequent reduced fertility) and adrenal insufficiency. Osteoporosis and its complications are common in older patients. Many patients transfused before hepatitis C screening was introduced have cirrhosis (compounded by the impact of iron overload) and are at increased risk of hepatocellular carcinoma. Despite dramatic improvements in prognosis, patients with thalassaemia major require regular and frequent hospital contact, and the impact on lifestyle and psychological consequences remain significant. Many women with thalassaemia major have now had successful pregnancies without major complications. Changing patterns of migration from countries with a high incidence of haemoglobinopathies and screening of couples in whom there is a risk of offspring with a major haemoglobinopathy have also had an impact, with increasing numbers of patients with thalassaemia intermedia, sickle cell disease and Hb H disease in Australia. The focus of the management of patients with thalassaemia major is the delivery of safe transfusions, optimising and monitoring chelation therapy, screening for and treatment of endocrine complications and infective complications of past transfusions, the management of pregnancy and the provision of psychological support for what remains a significant chronic illness. The emerging availability of modulators of erythropoiesis and gene therapy will further change decision-making and outcomes for patients with thalassaemia major.

S21: Making melanoma: how genomics is improving patient survival

K Dutton-Regester

QIMR Berghofer Medical Research Institute, Brisbane, QLD, 4006, Australia Melanoma is a cancer of melanocytes, the cells responsible for pigment production in the body. While melanoma has one of the highest cure rates of any cancer when detected early, once melanoma has metastasized the chances of survival dramatically decrease. Fortunately, the approval of numerous targeted and immune-based therapies for metastatic melanoma have had a significant impact on patient survival in the past eight years.

An important catalyst of this therapeutic success has been our significant understanding of the biology and genetics of melanoma. Genomic platforms, including next-generation sequencing, have rapidly increased our knowledge into the diverse sub-types of melanoma. This has allowed us to extract meaningful diagnostic information for use in the clinical management of patients, given us insight into the potential causes of melanoma, and have helped us identify new drug targets. Despite this, drug-resistance, patients not responding to treatment, and unintended side effects are all issues that need to be resolved by the field.

In this talk, I will provide an overview of the somatic genetics of melanoma and discuss its diagnostic relevance in context to the current landscape of therapies for late-stage disease. This will be followed by my recent research on understanding the role and potential mechanisms of transcriptional cell states in melanoma and its significance in up-front and acquired drug resistance. Lastly, I will provide an overview of my collaborative efforts with the Broad Institute of Harvard MIT to identify melanoma specific gene dependencies as novel drug targets through use of genome-wide CRISPR-knockout screens.

S22: Laboratory medicine in Myanmar – an university of South Australia student study tour perspective

L Gyedu1, A Henderson1, J Hoptroff1, C McGow1, A Neumann1, E Vandenbroek1, J Williams1, AA Khin2, B Dale1 1 School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia2 Department of Medical Technology, University of Medical Technology, Yangon, Myanmar

Introduction

In recent years the Laboratory Medicine program at the University of South Australia (UniSA) has developed substantial links with similar programs (Medical Technology) in Myanmar. To further this collaboration and expand the student learning experience, a study tour was undertaken at the University of Medical Technology (UMT), Yangon, Myanmar.

Methods

Funding was obtained from the Australian Government through the New Colombo Plan Mobility Program. A three week study tour designed to meet the objectives of this Program was developed in collaboration with UMT. Seven UniSA students commencing fourth year of the Laboratory Medicine program were approved to participate. UniSA students joined fourth year UMT Medical Technology students in lectures, tutorials and practical classes. Students visited public and private hospitals, associated pathology departments, public health and research laboratories, and observed community health visits conducted by UMT


Medical Technology students and staff. UniSA students reported their findings in oral presentations at UMT.

Results

Participation of UniSA and UMT students and staff in classes and community health visits enabled significant collaboration, knowledge development and cultural exchange. Visits to hospitals and laboratories in Yangon enabled comparison of operations with the knowledge and experience that UniSA students had from their studies and clinical placement in South Australia. Reporting of findings from these visits through oral presentations enabled critical analysis to be performed. Infrastructure and technical aspects were evaluated. Some of the challenges facing health systems and laboratory medicine in developing countries such as Myanmar were evident with substantial contrasts between public and private sectors. Differences between education systems and connection with industry in Australia and Myanmar were also appreciated.

Conclusion

The study tour provided a valuable student learning experience and broadening of understanding of the role of laboratory medicine in the Indo-Pacific region. The objectives of the New Colombo Plan Mobility Program were achieved.

S23: Histopathology – there is life in the old dog yet

F Maclean

Douglass Hanly Moir Pathology, Macquarie Park, NSW 2113, Australia

Introduction

The practice of histopathology in its current form has evolved relatively slowly in parallel with advances in both our understanding of the nature of disease and technological evolutions. In recent years the pace of change has accelerated. This change represents a challenge to the profession.

Discussion

Malignant neoplasia has plagued humans for millennia, with the oldest documented specimen of human malignancy being a 1.7 million year old case of osteosarcoma. Human understanding of disease has, however, evolved slowly. The earliest physicians embraced concepts of disease causation by the gods and goddesses, and the heavenly bodies. The Ancient Greeks lead by Hippocrates developed the humoral theory of disease, a concept that held sway for over 2000 years. Zacharias and Hans Jansen developed the first compound microscope in the 1590s. The use of the microscope in understanding disease pathogenesis was further developed through the efforts of Robert Hooke and Anton von Leeuwenhoek. Johannes Muller inaugurated a paradigm shift from one of organ-based disease to cell-based

disease, and heralded in a ‘new pathology’. Histopathology since that time has largely relied upon use of the light microscope to appreciate cellular abnormalities that underlie disease processes. In the last decades the advent of immunohistochemistry has provided useful adjunctive information to assist in diagnosis. More recently, molecular techniques have added to our disease classification systems and diagnostic accuracy. Looking forward we are on the cusp of a digital revolution, including the advent of disruptive technologies including Artificial Intelligence, that is likely to herald massive societal changes. In this presentation approaches to deal with these changes will be presented, including the expanded role of histopathology to interpret not only what is seen down the microscope but to incorporate and make sense of the explosion of diagnostic information, in light of the microscopic features.

S24: Liquid biopsy signatures in the blood: clinical and diagnostic utility for cancer therapy

KJ Spring, S Lim, J Toh, B Harris, P Ding, T Roberts, T Becker, D Adams, CM Tang, P De Souza

Liverpool Clinical School, University of Western Sydney and Ingham Institute for Applied Medical Research. Creatv MicroTech Inc, Potamac MD, and Oxford University, AustraliaSolid tumour biopsy is considered the gold standard for diagnostic review and provision of histopathologic information essential to inform cancer treatment. However, this is an invasive procedure that carries risks and limitations, such as infection, poor patient health and tumour accessibility. Liquid biopsy as a minimally invasive complement to solid tumour biopsy has great potential in developing a comprehensive and dynamic molecular understanding of a patient’s cancer to guide therapeutic options for cancer therapy. Components of liquid biopsy include circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), exosomes, extracellular vesicles (EVs) and microRNA (miRNA), all of which contain highly informative genetic information that has diagnostic and clinical utility. In this presentation, current research and different attributes of liquid biopsy will be discussed in the context of their clinical application as prognostic and predictive biomarkers to aid cancer therapy.

S25: Targeted polymeric nanomedicines as cancer therapeutics

N Fletcher,1 R Veedu,2 Z Houston, 1 Y Zhao,1 C Howard, 1 K Thurecht,1 1 Centre for Advanced Imaging (CAI), Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD, 4072, Australia


2 Centre for Comparative Genomics, Murdoch University, Perth, WA, 6150, AustraliaNanomedicine, the application of nanomaterials and biotechnology to medicine, is a rapidly expanding field of study. It comprises a multitude of approaches and many novel materials moving into clinical trials, especially in the field of oncology. Our groups approach to producing nanomedicines involves the synthesis of multifunctional hyperbranched polymers (HBPs), which are able to incorporate molecular imaging probes, therapeutics and targeting ligands within a single construct. These nanomedicines are synthesized via RAFT polymerization and are primarily composed of PEG-based monomers to enhance biocompatibility and increase circulation time. We have demonstrated successful targeting and therapeutic delivery to a range of cancer types in preclinical studies, and are also incorporating aptamers as novel targeting agents for our nanomedicines.

More recent work focuses on the production of HBPs targeted to the particularly aggressive Triple-Negative Breast Cancer (TNBC). A facile polymeric synthesis approach allowed the incorporation of multiple molecular imaging modalities as well as attachment of targeting aptamers to polymer chain ends utilizing copper-free azide-alkyne 1,3-dipolar cylcoaddition. Molecular imaging was facilitated by the incorporation of a near-infrared fluorophore for optical imaging, as well as desferoxamine (A89Zr chelator) to enable clinically relevant positron emission tomography (PET) to follow long-term polymer behaviour in vivo. This multifaceted imaging HBP was then functionalized with a locked nucleic acid aptamer RNV66 which binds to the vascular endothelial growth factors (VEGF) 165 and 121, (Marusic 2013) that are over-expressed in TNBC. The in vivo behaviour and targeting efficacy of this aptamer-functionalized construct was demonstrated in a TNBC xenograft model, with ongoing work now focusing on the incorporation of radiotherapeutics within such a targeted construct.

M. Marusic, R.N. Veedu et al, Nucleic Acids Res., 2013, 41, 9524-9536

S26: Nucleic acid aptamers: versatile molecules for targeted therapy of solid cancers & neuromuscular diseases

Rakesh N. Veedu1,2

1Centre for Comparative Genomics, Murdoch University 2Perron Institute Neurological and Translational ScienceNucleic acid aptamers [1] are short single-stranded synthetic nucleic acid ligands that can fold into complex three-dimensional shapes, forming binding pockets and clefts for specific high-affinity recognition of defined molecular targets ranging from small molecules over proteins to whole cells. These characteristics make aptamers an attractive platform for applications in

therapeutic development, drug delivery, biosensing, and nanotechnology [1]. Conventionally, aptamers are developed using a reiterative selection process over several cycles called Systematic Evolution of Ligands by EXponential enrichment (SELEX) [2]. In 2012, my group reported a rapid one-step selection methodology for developing nucleic acid aptamers [3], and by this approach aptamers can be selected in just two days unlike the traditional SELEX method. In 2015, we reported the efficacy of RNV66, an LNA-modified VEGF targeting aptamer, to inhibit breast cancer proliferation in vitro and in vivo [4]. Towards delivering drugs specifically to muscle cells, we have recently developed a novel aptamer specific to human primary muscle myoblasts by performing cell-SELEX procedures. Early this year, we have reported the development of a novel aptamer RNV95 against amyloid-beta peptides, the pathological hallmark implicated in Alzheimer’s disease. RNV95 efficiently detected low-molecular weight amyloid beta peptide aggregates in brain tissue samples of pathologically confirmed Alzheimer disease patients, and offer a great promise towards the diagnosis and therapy of Alzheimer disease [5].

Veedu RN, 2017. Aptamers: Tools for Targeted Nanotherapy and Molecular Imaging, Pan Stanford Publishing Pte Ltd, CRC Press, Singapore.

Lipi F, Veedu RN ,2016. In vitro evolution of chemically-modified nucleic acid aptamers: Pros & cons, and comprehensive selection strategies, RNA Biology, 13(12):1232-1245.

Lauridsen LH, Veedu RN, 2012. Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA aptamer against Alpha-Bungarotoxin. PLOS ONE 7(7): e41702.

Edwards SL, Veedu RN, 2015. Targeting VEGF with LNA-stabilized G-rich oligonucleotide for efficient breast cancer inhibition. Chemical Communications 51: 9499-9502.

Chakravarthy M, Veedu RN, 2018. Development of DNA aptamers targeting low-molecular-weight amyloid-β peptide aggregates in vitro. Chemical Communications. 54, 4593-4596.

S27: Diagnostic uses of aptamers

Michelle L Bauer, Alister C Ward, Sarah L Shigdar

Centre for Molecular and Medical Research, School of Medicine, Deakin University, Geelong, VIC 3128, Australia

Immunohistochemistry (IHC) is an indispensable method for the diagnosis of malignancy when alterations in tissue morphology remain deceptively subtle. IHC can provide prognostic indications and guide personalised treatment decisions by the detection of clinically relevant markers of tumourigenicity and invasiveness. Antibodies have proven to be effective in immunohistochemistry for the diagnosis of tumours and for the detection of cell surface markers prior to commencement of immunotherapy. However, antibodies


continue to harbour issues of batch-to-batch variability and cross-reactivity that are of detriment to the accuracy and precision of clinical histopathology. Chemical antibodies, also known as aptamers, are small pieces of DNA or RNA that are generated in a similar manner to antibodies and bind in exactly the same manner as antibodies via the ‘lock and key’ mechanism. Aptamers show very high specificity to their target and, as they are chemically synthesised, they can be easily derivatised with detection moieties at one or many pre-specified locations along their sequence, depending on the application. We have developed aptamers to a number of cancer related cell surface markers and demonstrated rapid specific and sensitive staining of paraffin embedded tissues, with limited cross-reactivity and non-specific staining. We also tested their sensitivity in a chromogenic, sequential double-staining technique in paraffin-embedded tissues and were able to demonstrate the detection of cells which co-express both markers at the cell surface. Given that the majority of cells in the bulk tumour show a moderate expression of each of these markers, parallel detection of both EpCAM and CD133 enhances the likelihood that highly co-expressing cells are cancer stem cells – which represent an attractive therapeutic target. This points towards a promising future for aptamers in the fields of clinical histopathology and personalised oncology, and suggests a promising future in the diagnostic laboratory.

S28: An update on the diagnosis and classification of myeloproliferative neoplasms

R Gasiorowski

Concord Hospital; Macquarie University Hospital, Sydney NSW, AustraliaThe myeloproliferative neoplasms include essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) with the classification incorporating clinical, morphological and genetic features. Accurate diagnosis and classification of these disorders is essential to ensure optimal treatment outcomes.

Genetic testing is now a key part of the diagnostic algorithm for these patients, in particular identification of JAK2, CALR and MPL mutations. Diseases are classified according to the World Health Organization (WHO) classification, which was updated in 2016. This update incorporated four major changes.

• Reduction of the haemoglobin threshold used to diagnose PV to 165g/l for men and 160g/l for women;

• The diagnosis of prefibrotic PMF, which can present similarly to ET but has a significantly worse prognosis;

• The role of bone marrow morphology in distinguishing ET from prefibrotic PMF;

• Inclusion of CALR and MPL mutations as major diagnostic criteria for ET and PMF.

This talk will discuss the current diagnosis and classification of these diseases, with an emphasis on these 2016 changes, the importance of genetic testing and the utility of next generation sequencing (NGS) to identify other mutations such as ASXL1, EZH2, IDH1, IDH2 SRSF2, SF3B1 and TET2.

S29: Genetic investigation of AML

W.S.Stevenson

Department of Haematology and Transfusion Medicine, Royal North Shore Hospital, St Leonards, NSW 2065, AustraliaThe classification of Acute Myeloid Leukemia (AML) has evolved from bone marrow morphology alone to the increasing incorporation of genetic pathology in the WHO system reflecting a better understanding of the underlying biological basis of myeloid leukemogenesis. This additional genetic information provides a more accurate diagnosis and better prognostication for leukemia patients. This increasing use of genetic pathology in the medical laboratory has required the synthesis of different genetic testing techniques including cytogenetics, PCR and next generation sequencing with implications for test reporting. The current WHO AML classification will be outlined including changes incorporating new genetic assays. Disease stratification schema and attempts to standardize testing in Australian laboratories will be discussed with a number of case studies that illustrate these testing principles.

S30: Oligonucleotide-based therapeutics – are we there yet for liver disorders?

PJ Leedman

Laboratory for Cancer Medicine, Harry Perkins Institute of Medical Research, and The University of Western Australia, Perth, WA, 6009, Australia

Introduction

There have been major advances in oligonucleotide-based therapeutics (small interfering RNA [siRNA], microRNA [miRNA] and antisense oligonucleotides [ASO]) recently, so that it now offers the potential to treat a range of human disorders. Liver-specific siRNA targeting of PCSK9 very effectively reduced LDL cholesterol in patients already on statins (NEJM, 2017,376:1430) and ASO against Transthyretin (TTR) improved the neurological disease for patients with hereditary transthyretin amyloidosis (NEJM, 2018, 379:221). Key to these advancements has been 2nd generation chemistry


to stabilize the molecule and development of liver-specific delivery. Success with siRNA paves the way for using miRNA to treat liver disorders, eg. hepatocellular cancer (HCC), as they use the same intracellular processing machinery (Dicer, Ago2) and can be modified for liver-specific uptake. As HCC is rapidly increasing in incidence globally and has a very poor prognosis, there is massive unmet clinical need for new therapeutics.

Results

We have developed a miRNA for therapy, miR-7, which is a potent inhibitor of the EGF-receptor (EGFR) signalling pathway in HCC. miR-7 powerfully inhibits HCC growth in vitro and in vivo, downregulates phospho-Akt (P-Akt) and can overcome resistance to the only available HCC tyrosine kinase inhibitor, sorafenib. In collaboration with a US-based RNA therapeutics company, we have designed and developed 2nd generation chemistry modifications to miR-7 (miR-7 mimics) which do not require a lipid vehicle and can be targeted to the liver specifically. These novel miR-7 mimics are potent inhibitors of HCC.

Conclusions

Oligonucleotide-based therapeutics has emerged as an important new treatment paradigm, especially for human disease originating from liver dysfunction. Advances in siRNA therapeutics are providing new opportunities to apply the same technology to miRNAs for treating human cancer. Our data with miR-7 mimics suggest they may have a role as therapeutics for HCC and could be evaluated in combination with sorafenib in an early phase clinical trial.

S31: IGFBP - 3 oncogenic signaling in triple-negative breast cancer

Robert C Baxter

Kolling Institute, University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, AustraliaInsulin-like growth factor binding protein-3 (IGFBP-3) is the most abundant member of the IGFBP family, and the main transport protein for circulating IGF-1 and IGF-2. IGFBP-3 also has roles in the pericellular and intracellular environment, and influences several growth-regulatory signalling systems. In triple-negative breast cancer (TNBC), we have identified an oncogenic pathway initiated by IGFBP-3, that is now the focus of a novel targeted therapy. TNBCs are particularly aggressive forms of breast cancer, about 15% of all cases, with poor survival outcomes and no approved targeted therapies. We found that IGFBP-3, a marker of poor recurrence-free survival in basal-like TNBC, activates the lipid kinase, sphingosine kinase 1 (SphK1), which in turn transactivates EGFR, a potentially oncogenic receptor that is highly expressed in TNBC. Combined targeting of SphK1 and EGFR showed a strongly synergistic cytostatic effect on TNBC cell lines, the synergism being lost when IGFBP-3 was downregulated. In pre-clinical studies in vivo we have evaluated combining the SphK

inhibitor fingolimod (Novartis), approved as an immune modulator in multiple sclerosis, and the EGFR tyrosine kinase inhibitor gefitinib (AstraZeneca). Compared to no treatment or either single agent, the combination significantly improved survival in two xenograft models of human TNBC, and a murine TNBC model in immune-competent mice. Although fingolimod alone inhibits T cell trafficking we found that co-administration with gefitinib substantially alleviated the loss of tumour CD3+ T cells. By immunohistochemistry, tumour IGFBP-3 staining was mainly nuclear, correlated positively with Ki67 (proliferation) and negatively with cleaved caspase-3 (apoptosis), and was significantly associated with poor survival. This pre-clinical evaluation suggests that combined inhibition of oncogenic IGFBP-3 signalling through SphK1 and EGFR may be considered as a novel treatment for TNBC. Since both fingolimod and gefitinib are TGA-approved, rapid implementation is feasible once clinical safety and efficacy are demonstrated. Supported by Cancer Council NSW.

S32: “Immuno-flowfish”: imaging flow cytometry for the assessment of chromosomal abnormalities in phenotyped chronic lymphocytic leukaemia cells in suspension

Kathy Fuller1,2, Henry Hui1, Hun Chuah1,3, James Liang1,4, Hasib Siddiqi1,4, Dejan Radeski1,2,4, Wendy Erber1,3

1Translational Cancer Pathology Laboratory, School of Biomedical Sciences, The University of Western Australia, Crawley, Australia2PathWest Laboratory Medicine, Nedlands, Australia 3Department of Haematology, Royal Perth Hospital, Perth, Australia4Department of Haematology, Sir Charles Gairdner Hospital, Nedlands, Australia

Imaging flow cytometry is an automated method that enables cells and fluorescent signals to be visualized and quantified. Here we report the development and assessment of a new imaging flow cytometry method whereby fluorescent in situ hybridisation (FISH) was integrated with cell phenotyping (“immuno-flowFISH”). We assessed chronic lymphocytic leukaemia (CLL), a mature B-cell neoplasm where chromosomal abnormalities predict prognosis. Generally, for CLL, FISH is performed manually on cells in interphase on glass slides and up to 200 cells (nuclei) analysed. In this study we aimed to assess the performance of immuno-flowFISH on CLL to assess genotype. Blood mononuclear cells were phenotyped with fluorochrome-conjugated antibodies (CD3, CD5, CD19) and post-fixed. Following membrane permeabilisation and denaturation of double-stranded DNA, chromosome 12 enumeration FISH probe (CEP12) was added. CEP12 was used to identify cases with trisomy 12, a cytogenetic


abnormality of prognostic significance present in 15% of CLL. Data for up to 20,000 cells was collected on the Amnis ImageStreamX Mark II imaging flow cytometer (ISX MkII). Digital images (x60) and quantitative data (IDEAS software) were used to assess morphology and FISH probe “spots” in CLL cells identified by CD5+/CD19+/CD3- phenotype. 84% (42/ 50) cases showed the normal diploid 2 spot pattern and 16% (8/50) cases three CEP12 spots in cells with the CLL phenotype. The number of cells with 3 spots ranged from 0.1 - 45.5%. The normal B-cells (CD19+/CD5-) and T cells (CD3+/5+) in the CLL cases had normal diploid CEP12 spots. All +12 cases identified by immuno-flowFISH were independently verified by conventional FISH “on-a-slide”; there were no false positive or negative cases. This data demonstrates that automated high-throughput FISH analysis of large numbers of phenotyped whole cells in suspension is feasible, accurate and reproducible for the identification of numeric chromosomal abnormalities in neoplastic cells.

S33: Inertial microfluidic separation of circulating foetal cells: towards cell-based non-invasive testing

M. Winter, 1 M. Reza, 1 T. Hardy, 2,3 D. Zander-Fox, 3 M. Warkiani, 4 B. Thierry1

1Future Industries Institute and ARC Centre of Excellence for Convergent Bio-Nano Science, University of South Australia, Mawson Lakes, SA, 5095, Australia2 SA Pathology, Adelaide, SA, 5000, Australia 3 Monash IVF Group, Melbourne, Victoria, 3121, Australia4University of Technology Sydney, NSW, 2007, Australia

Introduction

Since its introduction in 2011, non-invasive prenatal testing (NIPT) based on circulating cell free fetal DNA has revolutionised the field of prenatal screening and has now gained broad clinical acceptance for the detection of a number of common genetic disorders. However, current technologies do not have the capability to provide information about the full range of potential genetic abnormalities. During pregnancy circulating foetal trophoblastic cells are continuously shed from the placenta into maternal blood. Although present in extremely small numbers, they provide an intact and complete foetal genome which could be utilised for non-invasive prenatal diagnosis of genetic abnormalities. However, their rarity makes isolation and analysis a tremendous challenge with cell-based NIPT being a tantalizing goal for clinical utility.

Methods

Peripheral blood samples were obtained from healthy volunteers or pregnant women (~11 weeks gestation). Samples underwent RBC lysis and were enriched using inertial microfluidics. The enriched fraction was then

processed for single cell detection, pick-up with single cell manipulation and subsequent Next Generation Sequencing.

Results

Following model studies, we have demonstrated that circulating foetal trophoblasts and syncytial nuclear aggregates can be isolated from maternal blood using inertial microfluidic technology. Subsequently, we have shown that this technology enables enriched cells to undergo sequencing which offers a multitude of screening opportunities.

Conclusion

Inertial microfluidics provides a rapid and efficient enrichment technology that can be combined with downstream analysis through semi-automated scanning, single cell manipulation and single-cell genetic analysis. The possibility to reliably isolate rare foetal trophoblastic cells circulating in maternal blood in a cost-effective fashion will enable the development of a novel translational technology platform for non-invasive prenatal genetic testing.

S34: Next-generation biomedical devices

O. Shimoni

Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney (UTS), 15 Broadway, Ultimo, NSW 2008, Australia

Introduction

Public perception of nanotechnology does not associate with a positive image due a negative connection to asbestos and its risk to a public health. Nevertheless, breakthroughs in nanotechnology and nanoscience have achieved far better thinkable health outcomes. Nanomaterials exhibit remarkable characteristics, such as high surface to volume ratio, catalytic activity, and biocompatibility making them suitable for various biomedical applications. One of the most promising potential contribution of nanotechnology is in the space of screening and diagnostic tools for early detection of diseases. Early and accurate detection of diseases exhibit several advantages, such as disease at early stage can be curable or efficiently treatable, reduction of financial burden to individuals and public health system as well as real-time health monitoring.

Results

Recent progress on development of diagnostic tool based on various properties of nanoparticles has been achieved in our Institute. In particular, I will feature our recent work on the point-of-care test for coeliac disease, biomolecular detection in dementia and mental disorders, and health monitoring throughout infectious illnesses. This talk will highlight the importance of nanoscience and nanotechnology in a future of public health.


S35: Monoclonal antibody drug treatments causing problems for the laboratory

Glenda Mann

Cabrini Pathology, Melbourne, Victoria, Australia

Monoclonal antibody drug treatments are being developed and used widely for the treatment of disease due to their enhanced efficacy and availability. However, they can cause interference with laboratory testing. Of particular interest to the Blood Bank and Biochemistry laboratories are the anti-CD38 drugs, namely Daratumumab (Darzalex), and Isatuximab. and an anti-CD47 which is now available overseas.

Because CD38 is also present on red blood cells in small amounts, it is causing major testing difficulties in the blood bank by binding to the CD38 protein on red cells and causing panagglutination, thus making all indirect antiglobulin test (IAT) screening procedures and all full crossmatches falsely positive. This means any potential underlying antibody may be masked, thus seriously compromising the ability to find appropriate blood for transfusion. The anti-CD47 drug treatment is potentially of much greater concern as not only does it interfere with antibody testing but also with blood grouping.

Various documented suggested solutions have been put forward to help alleviate the anti-CD38 problem. These include enzyme techniques, eg DTT, papain, ficin, trypsin, etc. However, they are not without their limitations and also may be somewhat impractical in Australia owing to changes with in vitro diagnostic regulations. This does not negate the need to send the samples for genotyping and give genotyped matched blood when possible. Potential solutions for negating anti-CD47 interference will also be discussed.

Our laboratory uses a commercial automated prepapainised panel, and testing has shown that a good proportion of underlying clinically important antibodies may still be detected. However, as it is not an IAT method and is performed on saline cards, it cannot be used as a complete solution.

We have also tested Daratumumab patients’ plasma spiked with known antibodies, against other Daratumumab patients’ genotyped red cells as a mini panel, thus showing that Daratumumab patients’ red cells have lost their CD38 protein but other antigen systems remain intact. All of our direct agglutination tests (DATs) have been negative, and titres of anti-CD38 varied depending on the cells chosen.

These antibody treatments can also cause interference with protein electrophoresis and flow cytometry.

With the rise in development of these monoclonal drugs, it can be assured that there will remain a challenge for testing in the laboratory.

S36: Using genotyping to select blood for patients on immunotherapy, such as Daratumumab

Jacqui Martin

Australian Red Cross Blood Service, Brisbane, QLD, Australia

Anti-CD38 therapies, such as Daratumumab and Isatuximab are now being widely used in Australia for the treatment of Multiple Myeloma. These anti-CD38 therapies interfere with routine immunohaematology testing, because CD38 is expressed on red cells. Patients treated with anti-CD38 often have pan agglutination in the antibody screen, which may be accompanied by a positive Direct Antiglobulin Test (DAT). This group of patients often receive blood transfusion prior to and during treatment. The positive DAT and/or recent transfusions mean the patient’s own phenotype cannot be determined by serology.

The interference with routine immunohaematology testing presents a challenge in ensuring there are no underlying allo antibodies and makes serological cross matching impossible. To manage these challenges it is recommended to provide phenotype matched red cells for transfusion to reduce the risk of alloimmunisation. Unlike traditional serology, molecular genotyping is not affected by transfused cells or a positive DAT. Genomic DNA is isolated from the patient’s leukocytes (from EDTA whole blood sample) allowing us to predict the phenotypes in these patients where this was previously not possible.

The Guideline Chart “Considerations for Pre-Transfusion Immunohaematology Testing in Patients Receiving Anti-CD38 Monoclonal Antibody Therapy” published on the ANZSBT website in May 2018 recommends considering phenotype / genotype matched blood for Rh (C, c, D, E, e), K, Jka, Jkb, Fya, Fyb, S and s, particularly where long-term transfusion support is anticipated. The Blood Service has genotyped 164 patients receiving anti-CD38 therapy and is endeavouring to provide antigen matched blood where transfusion is required.

The number of orders received by the Blood Service for phenotyped red cells has increased from approximately 6000 to 8000 each month in the past 18 months (a 33% increase). This increase in demand for phenotyped red cells requires careful management to ensure that we have enough of the appropriate phenotyped red cells to support all patients, where antigen matching is appropriate.


S37: A quantitative approach to primary prevention of Alzheimer’s disease: progress in blood-based screens and other methods to detect persons at risk

Colin L Masters MD

The Florey Institute, University of Melbourne, AustraliaThe etiology of Alzheimer’s disease (AD) is best understood through the deposition of Aβ-amyloid (Aβ). There are two basic forms of AD. The common (>95%) form is sporadic, and is caused by the failure to clear Aβ (mean age at onset 80 years). The rare (< 5%) autosomal dominant familial form is caused by the over-production of Aβ42,also on a background of failure to clear (mean age at onset 45 years). In both forms, the kinetics of Aβ accumulation are similar, taking about 30 years to accumulate a total of approximately 7mg of Aβ. Thus we estimate that sporadic AD starts about the age of 50 years and the autosomal dominant form starts about 15 years of age. The advent of validated biomarkers (PET/CSF Aβ and tau) now provides us with unprecedented opportunities for preclinical diagnosis, enabling the development of primary and secondary prevention strategies. Predictive algorithms utilizing age, biomarkers, polygenic and vascular risk scores are now being developed from longitudinal cohort studies to estimate times of onset and rates of cognitive decline.

From the time of the first identification of the C-terminus and the neuronal origin of the Aβ we have endeavoured to develop a peripheral blood-based biomarker of AD. Recently, an high performance assay for Aβ42 in plasma which correlates with PET/CSF Aβ was described. Another long sought-after goal has been to identify genetic and environmental determinants of Aβ deposition and rates of cognitive decline. Genetic loci which interact with sleep, diet and physical activity may provide the first clues. Applications of biomarker screens (blood, CSF, PET) to subjects who are about to cross the lower cutpoint threshold will define a population who may be suitable for primary prevention clinical trials.

S38: Serum and brain miRNA exosomal biomarkers associated with neurodegenerative diseases

Lesley Cheng1, Laura J. Vella2, James D Doecke3,4, Robyn Sharples1, Mitch Shambrook1, Laura J. Ellett5, Colin L Masters2, Malcolm Horne2, Vicki A. Lawson5, Kevin J Barnham2,3 & Andrew F. Hill1

1The Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.2The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia.3CSIRO Digital Productivity Flagship/The Australian E-Health Research Centre, Herston, Queensland, Australia

4CSIRO Food and Nutrition Flagship, Melbourne, VIC, Australia 5Department of Pathology, University of Melbourne, Melbourne, Victoria, Australia

Introduction

Alzheimer’s disease (AD) affects more than 55 million people worldwide and is expected to double every 20 years in the absence of disease-modifying drugs. Therapeutic strategies aimed at limiting neurodegeneration require methods to diagnose the disease in preclinical patients. Several blood-based tests have been explored to detect AD however, evidence is required to determine whether blood sampling is an appropriate specimen to diagnose brain diseases. Previously we isolated serum exosomes from AD patients which displayed an abnormal composition of 16 specific microRNA (miRNA) biomarkers compared to controls.

Methods

To provide evidence that our serum exosomal miRNA biomarkers are suitable for the detection of a brain condition, we also profiled exosomes isolated from post-mortem human AD (n = 8) and control (n = 8) brain tissues. Exosomes were extensively characterised to meet the minimal experiments requirements set out by The International Society for Extracellular Vesicles to be defined as exosomes and small RNA profiling was performed by next-generation sequencing.

Results

Brain derived exosomes (BDEs) were found to contain a unique profile of small RNA, including miRNA, compared to whole tissue. Furthermore, all 16 serum biomarkers, identified in our previous study, were detected in BDEs including a panel of BDE specific miRNA that target genes involved in AD pathology. Small RNA profiling was also performed in serum exosomes and BDEs isolated from Parkinson’s disease (PD) subjects and a mice model of Prion disease.

Conclusion

This work has identified a highly specific panel of miRNA that is both present in the brain and blood of AD and PD patients. The miRNA candidates can be used to develop a blood-based diagnostic test highly relevant to a brain disease, equivalent to non-invasive brain biopsy, and further studied to understand AD pathology and other neurodegenerative diseases to identify therapeutic targets.

S39: “Immuno-flowfish”: imaging flow cytometry for MRD monitoring in phenotyped leukaemia cells

Kathy Fuller1,2, Henry Hui1, Wendy Erber1,2


1Translational Cancer Pathology Laboratory, School of Biomedical Sciences, University of Western Australia, Crawley, Australia2PathWest Laboratory Medicine, Nedlands, Australia Image-based flow cytometry combines high resolution digital images with the quantitative fluorescence information gained from a standard flow cytometer, in a single platform. The imaging functionality allows the localisation of FISH probe signals (“spots”) to be localised within the (stained) nucleus of cells, a feature that cannot be achieved by standard flow cytometry. Here we present the development and assessment of a new imaging flow cytometry method whereby fluorescent in situ hybridisation (FISH) is integrated with cell phenotyping (“immuno-flowFISH”). We have assessed this method on normal lymphocytes and chronic lymphocytic leukaemia (CLL). We have successfully detected numeric (+12) and structural (del(17p)) chromosomal abnormalities in CLL cells identified by phenotype using fluorochrome-conjugated CD3, CD5 and CD19 antibodies. The method requires initial antibody labelling followed by post-fixation. Following membrane permeabilisation and denaturation of double-stranded DNA, the relevant probe is added. Data for up to 100,000 cells is collected on the Amnis ImageStreamX Mark II imaging flow cytometer. Digital images (x60) and quantitative data (IDEAS software) are used to assess cell morphology, phenotype and FISH probe binding. Trisomy 12 and del(17p) can be successfully detected in phenotyped CLL cells (CD5+/CD19+/CD3-) with a limit of detection of at least 0.13%. Automated high-throughput FISH analysis can be performed to assess the chromosomal (numeric and structural) in large numbers of phenotyped normal and neoplastic cells in suspension. This new method enables FISH probe signals to be analysed in a large number of cells in suspension at high throughput, providing accurate analysis of chromosomal abnormalities to low detection levels. It is therefore suitable for minimal residual disease analysis, in contrast to traditional slide-based FISH.

S40: MRD in a clinical flow lab: a practical guide

S Smith

Clinical Flow Cytometry Laboratory, ICPMR, Westmead Hospital, Sydney NSW, AustraliaAssessment of Minimal Residual Disease (MRD) by flow cytometry has become an integral component in the treatment of many haematological malignancies. These are currently the most resource intensive assays for a clinical Flow Cytometry laboratory. Furthermore, the assay techniques and analysis strategies are different from those currently used for diagnostic assays in the majority of Australasian laboratories. The implementation and maintenance of these assays would have a significant impact on any clinical laboratory.

This presentation discusses practical considerations when performing MRD assays in an Australian clinical flow cytometry laboratory. It will include assay technique, panel development and selection, analysis and reporting. A laboratory needs a balance between standardisation and flexibility to achieve a consistent and sustainable workflow. Standardised panels and analysis protocols are essential for identifying a neoplastic population over different time points, whether weeks or years apart. However there must be flexibility to adapt to any presentation Leukaemia Associated Immunophenotype (LAIP), and to evolving treatments that block antigen targets. Participation of clinicians in the review of results is vital to maintain confidence in the service and ensure that MRD by flow cytometry continues to contribute to patient care.

S41: Antimicrobial resistance - a threat to public health

John Merlino 1, 2

1Department of Infectious Diseases and Immunology, Sydney Medical School, University of Sydney, Sydney, NSW, Australia

2Department of Microbiology and Infectious Diseases, Concord Hospital, NSW Health Pathology, Sydney, NSW, Australia

Antimicrobial resistance is a complex issue and is a threat to public health globally. While emerging and re-emerging diseases have captured news headlines with outbreaks such SARS, bird flu, Ebola and Zika virus around the world, leading health experts tells us that there is a more serious threat to public health – antimicrobial resistance (AMR). This is not a new phenomenon. We have been aware of AMR since the beginning of antibiotic usage in therapeutics medicine. When AMR organisms known as ‘superbugs’ emerge and spread in society they become a major public health issue. AMR is an increasingly serious threat to global public health that requires action across all government sectors and society.

S42: RCPAQAP update

P Graham1, D. Holzhauser1 R. J. Massie2, GRD Jones3

1The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP), St Leonards, NSW, Australia; 2Royal Children’s Hospital, Melbourne, VIC, Australia; 3SydPath, St Vincent’s Pathology, Darlinghurst, NSW, AustraliaThe RCPAQAP Update session will feature new programs in development for 2019 as well as a preview of the new survey report review functionality in myQAP.


John Massie is a Paediatric Respiratory Physician and member of the RCPAQAP AACB Sweat Testing Advisory Committee. He will present on the recently published sweat test guidelines with emphasis on the Australasian context. He will also present on the new therapies that restore CFTR function and the role of sweat chloride testing as a biomarker of drug activity and adherence.

Graham Jones who is the Chair of the RCPAQAP Chemical Pathology Advisory Committee will provide an overview of the new Body Fluids program as well as the latest Liquid Serum Chemistry results.

S43: Coagulation results and consequent treatment of chronic liver disease patients

S Miranda

ICON Cancer Care, Townsville QLD, AustraliaStandard coagulation tests, including PT and INR are frequently used to assess haemostasis. In liver disease, an elevated INR is used to reflect an impairment in the synthetic function of the liver and is incorporated into disease prognostic scores. However, impaired protein synthesis affects both pro-coagulant and anticoagulant factors important in haemostasis. The PT/INR are useful in diagnosing deficiency of procoagulant proteins, but are insensitive at detecting decreased levels of the anticoagulants. Traditional dogma based on incorrect interpretation of high INR is that individuals with liver disease have a bleeding tendency and are “auto-anticoagulated”. However, there is growing consensus that despite laboratory tests suggesting a bleeding tendency, haemostasis in liver disease is rebalanced with a bias towards a pro-thrombotic state.

Peri-procedural transfusional support with fresh frozen plasma and other blood products are often employed with the aim of reducing perceived bleeding risk, in spite of a growing body of literature showing that prophylactic plasma transfusion based on baseline INR does not reduce bleeding in patients, and may in fact cause harm. Against the use of plasma infusions stands the knowledge that the resulting volume expansion contributes to increase portal vein hypertension, aggravates decompensation and increases the risk of bleeding and rebleeding from oesophageal varices. Of addition concern, epidemiological studies have suggested that patients with CLD have to greatest risk of TRALI. In the setting of liver disease with high INR, thromboestography (TEG/ROTEM) offers a new and alternative global assessment of haemostasis including coagulation factor function, platelet contribution to clot formation as well as fibrinolysis. Real-time viscoelastic testing has proven a robust tool guiding blood product transfusion during liver transplantation and may prove useful for other invasive procedures in patients with chronic liver disease.

S44: RCPAQAP Chemical pathology clinical cases

P Graham, N Wijeratne, J. Smith, P Coates, GRD Jones

Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) Patient Comments Advisory Committee

Members of the RCPAQAP Patient Comments Advisory Committee will present cases taken from recent Patient Report Comments surveys.

This unique program is both educational and challenging. Patient details, related clinical notes and pathology results are sent to Participants to respond as if they were being asked by a clinician to interpret and advise.

The Committee reviews all the submissions from Participants and adjudicates on comments which are ranked from recommended to misleading. Additional commentary is provided with each report with references for further reading.

The program is recognised by the RCPA as a component of the continuing professional development requirement for all practicing pathologists.

S45: Assessment of haemoglobin A1c in patients with haemoglobin variants

DB Sacks

National Institutes of Health, Bethesda, MD 20892, USAThe prevalence of diabetes is increasing rapidly worldwide. There are estimated to be 425 million people in the world with diabetes currently and by 2045 this number is expected to reach 629 million. Glycated haemoglobin, most commonly measured as haemoglobin A1c (HbA1c), has an integral role in the management of patients with diabetes. Large prospective clinical studies have clearly documented that HbA1c is both an indicator of long-term glycemic control and predicts risk for the development of microvascular complications. These attributes, combined with low intra-individual variability and the lack of influence of food ingestion (a fasting sample is not necessary), make HbA1c appealing for the diagnosis of diabetes. The recent adoption of HbA1c by several influential clinical organizations for the screening and diagnosis of diabetes increases the need for further understanding the reliability of HbA1c results. Conditions that change erythrocyte lifespan (e.g., hemolytic anemia, certain haemoglobin variants and blood transfusion) alter HbA1c concentrations. Published reports indicate that some diabetic individuals have HbA1c values that are lower or higher than expected based on their clinical presentation and blood glucose results. The measurement of HbA1c in individuals with haemoglobin variants that may influence the glycation of haemoglobin or interfere in HbA1c assays will be discussed.

S46: Norovirus and cruise ships

J Lunn


School of Biotechnology and Biomolecular Sciences, UNSW, Sydney NSW, Australia

Acute gastroenteritis (AGE) is one of the most common diseases of humans. Despite the improved safety in food handling and prevention strategies, AGE remains the leading cause of mortality among children under the age of five and is estimated to cause 1.4 million deaths annually worldwide. Between 2007 to 2016 there has been a 320% increase in the number of cruise ships entering Sydney harbour with 320 in 2016. Subsequent outbreaks on board are not unusual and are facilitated through person-to-person transmission, insufficient cleaning of contaminated surfaces and infected passengers refusing to be isolated. Furthermore, norovirus has a low infectious dose and is more resistant to commonly used disinfectants than other viruses. Norovirus is now notorious for causing gastroenteritis outbreaks on cruise ships all over the world, affecting hundreds of passengers in one setting. In the context of cruise ships, we examined the recent trends in emerging norovirus, its ability to cause outbreaks and discussed the factors that influence its spread on board.

Of the norovirus-associated cruise ship outbreaks investigated in Sydney, the pandemic norovirus (GII.4 Sydney 2012) was found to be responsible for 66.7% (n=8/12). In late 2016, the emergence of five new noroviruses were observed and the lack of herd immunity to these viruses led to a sudden increase of institutional gastroenteritis and cruise ship outbreaks in both Australia and New Zealand.

The constant evolution of norovirus allows the creation of new strains capable of escaping herd immunity. These emerging viruses often appear early on cruise ships thus active surveillance is vital to protect ship’s passengers and prevent outbreaks and can even inform the general population of what norovirus strains to expect.

S47: Chronic medical conditions and severe influenza virus infections

Kirsty R. ShortSchool of Chemistry and Molecular Biosciences, University of Queensland, Australia

The current obesity and type 2 diabetes epidemics represent two of the biggest health crises of the 21st century. Obesity and type 2 diabetes confer susceptibility to numerous different complications, including severe influenza virus infections. Specifically, both conditions significantly increase the risk of being hospitalised with, and dying from, influenza. The mechanisms that underscore this susceptibility remain poorly defined. Our research group uses a series of in vitro and in vivo models to identify how chronic metabolic conditions increase the severity of influenza. In the context of diabetes, our data suggest that diabetic fluctuations in blood glucose levels increase the severity of influenza virus infections by inducing an aberrant pro-inflammatory response. In contrast,

in obesity we observe a systemic immunosuppressive response which is then associated with increased respiratory and extra-respiratory viral replication. Our current research seeks to use this in-depth mechanistic information to develop new therapeutic options to protect the growing number of people living with obesity and diabetes. Given that 2017 was one of Australia’s worst influenza seasons on record, this research is both highly timely and pertinent.

S48: The end of targeted testing? Should patients routinely be given more information?

AJ Newson

The University of Sydney, NSW, Australia

Introduction

As diagnostic and prognostic methods in clinical medicine and public health continue to improve, the amounts of information they can give rise to also increases. Technologies such as next generation DNA sequencing provide the ability to look at more points in the genome with greater depth than previously. This raises the question of what to do with the extra information that is sometimes found, or is deliberately looked for. In policy, research and clinical practice, there is an increasing tendency to search for and report extra information beyond that which informed the initial indication for testing. This gives rise to the question of whether, as a general presumption, such testing should be targeted or broad.

Methods

This paper will address this question using methods of theoretical bioethics. It will proceed by way of critically analysing concepts and relevant examples to come to an ethical position (making an argument) on the possible future routine provision of expanded information in medical testing.

Results

An argument will be presented that patients should not routinely be given more information than that which is requested by then, or that which addresses the core clinical question driving testing. This is based on two lines of argumentation: (1) over-emphasis on the role of information within patient autonomy; and (2) possible overdiagnosis. The implications of this position for current service provision will be considered; and counter-arguments will be assessed and rebutted.

Conclusion

The end of targeted testing is not yet upon us. With currently available technologies, it should not yet be routine to provide patients more information rather than less. While new testing technologies can provide richer and finer grained information than was previously available, we should not use them to drive technology-led practice. Rather the key rationale prompting the test should be upheld and promoted.


P1: Evaluation of the Abbott alinity ci system

N Jenkins, E Leung, K Nguyen, S Burgess, V Deane, J D‘Agostino, HG Schneider

Alfred Pathology Service, Melbourne, Victoria, 3004, Australia

Introduction

The recently released Abbott Alinity ci is a multi-module integrated clinical chemistry and immunoassay system.

The Alinity c-series can process samples using photometric and potentiometric Methods. The Alinity i-series uses chemiluminescent microparticle immunoassay (CMIA).

The aim of this study was to evaluate an extensive menu of routine diagnostic assays.

Assays evaluated include UEC, LFT, Calcium, Phosphate, Magnesium, Glucose, Creatine Kinase, high sensitivity Troponin-I, TFT, CRP, Cholesterol, Triglyceride, HDL, Lactate, and β-HCG.

These assays were compared against the established Abbott Architect ci16200. Correlation and precision studies were performed in this preliminary evaluation.

Methods

Fresh and frozen de-identified patient samples (n=40) representing the linear range of the evaluated assays were analysed on both the Abbott Architect ci16200 and the Abbott Alinity ci system. Intra – and inter-assay precision on the Alinity was evaluated using quality control material as well as patient pool samples. Sample testing was performed according to manufacturer’s kit inserts.

A commercial statistics program (Analyse-it) was used to evaluate the data.

Results

Passing Bablok analysis showed acceptable correlation between the Abbott Architect ci16200 and the Abbott Alinity ci, with slopes for individual analytes ranging between 0.91 to 1.05.

Correlation coefficients were between 0.98 to 0.99.

Intra and inter precision CVs were all below 5%.

Conclusions

The assays evaluated on the Abbott Alinity ci analyser demonstrated good precision. Method comparison between the Abbott Alinity ci system and the Abbott Architect ci16200 showed acceptable agreement.

P2: Method evaluation for arsenic speciation using HPLC-ICPMS

O Letsatsi , M Galdamez, M Baxter, R Flatman, G Ward, L Price, D Kanowski

Sullivan Nicolaides Pathology, Bowen Hills, Brisbane, QLD. 4006, Australia

Introduction

Patients with detectable total arsenic require speciation to determine an organic or inorganic origin, as inorganic arsenic is carcinogenic. The aim of this study was to establish and evaluate a reliable arsenic speciation method, using HPLC coupled with ICPMS.

Methods

The method uses a 1260 InfinityII HPLC, 7900 ICP MS and G3288-80000 anion exchange resin column in an adaptation of an Agilent Technologies procedure (1). The 16 minute isocratic HPLC run uses 2 mmol/L pH 11.0 phosphate buffer, and Germanium and Yttrium internal standards rather than Indium specified in the original Agilent method. Calibration standards and spiked urine samples were prepared using Dimethylarsinic acid (DMA), Arsenite (As(III)), Monomethylarsonic acid (MMA) and Arsenate (As(V) standards obtained from Spex (www.spexcertiprep.com) and Arsenobetaine (AB) from Sigma-Aldrich. Urine quality control material was obtained from Recipe (PM Separations, Australia).

Results

The method allowed separation, identification and quantification of AB, DMA, As(III), MMA and As(V). Calibration curves showed linearity up to 50 ug/L. Limit of quantitation at 20% CV of AB, DMA, As(III), MMA and As(V) was 1.0 ug/L. Between run imprecision at levels ranging from 2.5-43.4 ug/L was less than 16% CV and within run imprecision was less than 4.7% CV. Retention times of AB, DMA, As(III), MMA and As(V) were approximately 2.8, 3.8, 4.4, 7.9 and 12.5 minutes respectively.

Discussion

This study did not investigate minor species of organic arsenic, which can be found in some patient urine samples after ingestion of seafood.

The method described showed excellent sensitivity, linearity and separation of arsenic species. The five main species of arsenic were reliably separated at a rate of 16 mins per injection.

Reference

Agilent 7700/7500 Series ICP-MS, As Speciation Analysis Handbook: Agilent Techologies,Inc.2009,2010

P3: A fully automated LC-MS/MS method with enzyme hydrolysis of codeine and morphine glucuronide in human urine

KC Liu1, H Luu2, H Nguyen1,W Louey1, ZX Lu1,K Sikaris1

1 Melbourne Pathology, Collingwood, Victoria, 3066, Australia2 Shimadzu, Rowville, Victoria, 3178, Australia


Introduction

LCMS is the golden standard for confirmatory testing for non-negative immunoassay screening in drug of abuse. Typically requires two-three hours by multi step manual preparation. Which lead to reduce in turnaround time and potential sample preparation error. Unlike most of the liquid handling system which prepare samples in 96-well plate, the fully modular Shimadzu CLAM-2000 system automated sample preparation seamlessly from reagent and sample dispensation, vortex mixing, suction filtration for sample clean up, incubation for enzyme hydrolysis follow by automatic transfer sample vial to autosampler for LC separation and MS confirmation and quantitation.

Methods

Certified blank urine was spiked with three glucuronides (Codeine-6-G, Morphine-3-G Morphine-6-G) concertation at 500, 1000, 3000, 10000µg/L. Fully automated sample preparation (CLAM-2000) consists of mixing buffer solution with the deuterated internal standards, filtration follow by incubation at control temperature with b-glucuronidase. Chromatographic separation achieved by reverse phase chromatography with gradient elution 0.1% formic acid in water and methanol mobile phase. Analysis was performed on a Shimadzu 8060 by ESI. We evaluated the recovery of codeine and morphine form their respective glucuronides. Within run precision was evaluated by comparing our QC at 240, 360 µg/L ±20% cutoff of AS4308:2008, Accuracy was assessed by Austox quality assurance program with z-score.

Result

Our method is linear between 20-2000µg/L with R2 = 0.9999. Recovery for Codeine-6-G is 82-90%, Morphine-3-G is 93-108% and Morphine-6-G is 100-105%. Within run precision at 240,360 µg/L CVs are 3.3% and 3.8% for codeine, 2.3% and 3.2% for morphine at respective concentration. The z-score for Codeine is – 0.60 to 0.48 and morphine is between – 0.21 to 0.98 in spike and authentic urine from Austox.

Conclusions

We developed a fully automated sample preparation and analysis solution for drug of abuse in urine. The CLAM-2000 eliminates the need for sample preparation, increase efficiency, highly precise and accurate.

P4: Oestradiol measurement in IVF. Comparison of four immunoassay platforms with Liquid chromatography – tandem mass spectrometry (LC-MS/MS)

S Montgomery, K Young, M Henman, D Kanowski, G Ward, L Price.

Biochemistry Dept, Sullivan Nicolaides Pathology, Bowen Hills, Qld 4006, Australia

Introduction

Measurement of oestradiol (E2) is important in the investigation of female reproductive function as well as checking the response to ovarian stimulation in IVF. Drug interferences (eg. Fulvestrant) in immunoassay as well as poor sensitivity in post-menopausal women and children has required the development of sensitive and specific LC-MS/MS methods to meet this clinical need. However, LC-MS/MS is not currently practical when large sample numbers are being processed and is probably not required in the majority of patients having IVF therapy. To address the need in the IVF setting E2 levels were measured in IVF samples by 4 immunoassay platforms and correlated with LC-MS/MS to determine which immunoassay provided a “best fit” to LC-MS/MS.

Methods

Serum samples from 50 patients undergoing IVF treatment cycles were assayed by the following immunoassay platforms: Abbott Architect; Roche E602; Beckman Access and Siemens Immulite. The results from each of these immunoassays were then correlated with our in-house LC-MS/MS assay. Results

The Oestradiol concentration range tested was between 300 and 19000 pmol/L. Passing-Bablok regression comparisons and Pearson’s r2 values (LC-MS/MS vs Immunoassay) were y = 0.9693x + 19.55 and r2 = 0.9940 (Abbott Architect); y = 1.018x + 66.98 and r2 = 0.9584 (Roche E602); y = 0.8905x + 58.9 and r2 = 0.9662 (Beckman Access); and y = 0.9147x – 25.77 and r2 = 0.9880 (Siemens Immulite).

Conclusions

The correlation statistics indicate that the Abbott Architect had better agreement with LC-MS/MS within the measured oestradiol concentration range than the other three immunoassay platforms tested. Our study demonstrates that immunoassay may be dependably used for the IVF patient population with correlation statistics very similar to an LC-MS/MS method.

P5: Evaluation of the BD Vacutainer® Barricor™ Collection Tube in a STAT Lab

J Oostenbroek1, R Flatman2

1Sullivan Nicolaides Pathology, Greenslopes Private Hospital, QLD. 4120, Australia2Sullivan Nicolaides Pathology, 24 Hurworth Street, Bowen Hills, QLD. 4006, Australia

Introduction

Fast turnaround times (TAT) are highly sought after in critical care environments. The BD Vacutainer® Barricor™


Collection Tube is designed to offer faster TATs and a very clean plasma sample. We evaluated the Barricor against conventional BD serum separator tubes (“SST”) in a busy private hospital and stat laboratory environment.

Methods

A number of 115 parallel Barricor/SST collections were compared for:

• Ease of collection;

• Clotting time;

• Quality of sample separation post centrifugation;

• Result comparability for routine biochemistry analytes.

Samples were centrifuged in a Drucker Apex 6 DASH high speed centrifuge for 3 minutes at 5500rpm (4000g). Electrolyte and liver function test panels were performed on 45 Barricor and SST collections (analysis on Roche Cobas 6000), with result differences compared against RCPAQAP allowable limits.

Results

Collectors reported no issues with Barricor collections. Barricor tubes were noted to be visually clear post centrifugation, meeting or exceeding the clarity of SSTs in all cases. Pre-analytical processing times were significantly shorter for Barricor (0min clotting time, 3 mins centrifugation) versus SST (30mins clotting time, 10 mins centrifugation).

Nine assays showed differences above QAP allowable limits: TP (69% of patients), K (40%), LDH (29%), chloride and bicarbonate (9%), calcium (4%), ALT, AST & albumin (2%).

Three assays with > 10% variation versus QAP limits:Assay Name Range of differences

(Barricor versus SST)

Average difference

(Barricor versus SST)

Total Protein +0.2 – +10.9 g/L +5.3 g/L

Potassium -0.8 – +0.3 mmol/L -0.2 mmol/L

LDH -88 – +72 U/L -5 U/L

Conclusions

The Barricor tubes significantly improved TATs, while providing visually clean plasma samples. No issues were

noted by collection staff. A high speed centrifuge is required for the shortened 3min separation time. Some result differences were noted versus SST, however these were in line with expectations for lithium heparin plasma versus serum.

P6: Analytical evaluation of the Abbott alinity chemistry and immunoassay platform

G Ward1, K Rowland1, J Smith1, J Nicolson1, N Coonan1, R Flatman1, A Liu2, D Clifford2, N Gasparillo2, N Taylor2, D Kanowski1, L Price1

1Sullivan Nicolaides Pathology, Brisbane QLD 4006, Australia2Douglas Hanly Moir Pathology, Sydney NSW 2113, Australia

Introduction

Abbott Diagnostics have recently introduced next generation chemistry and immunoassay platforms – Abbott “Alinity” c and i modules. The reagents and analytical principles are largely unchanged from previous Abbott platforms, however hardware and software have had considerable changes. The aim of this study was to evaluate analytical performance of the c (chemistry) module and i (immunoassay) modules.

Methods

A full analytical evaluation was performed at two Sonic Healthcare laboratories (DHM Sydney and SNP Brisbane), including linearity, LOB/LOD/LOQ, patient comparisons (n=120) with existing platforms (Abbott c16000 and i2000), and assay imprecision. U-cortisol was compared against HPLC and extraction immunoassay, and Vitamin D against Diasorin XL. The evaluated assay menu consisted of 26 general chemistry analytes (ISE, photometric, and immunoturbidimetric) and seven immunoassays (chemiluminescent immunoassay). The majority of analytes were in a serum matrix, however some urine analytes were studied.

Results

A majority of analytes performed as well, or better, than existing Abbott c16000 and i2000 platforms, with 2-3% interassay CV for chemistry, and <5% interassay CV for most immunoassays. Assay LOD and linearity met performance criteria. Direct U-cortisol agreed very well with HPLC and extracted urine cortisol results (slope 1.05, intercept 3.77, r2 0.999).

Conclusions

Analytical performance of the Alinity chemistry and immunoassay platforms was generally equivalent, or in some cases slightly superior, to current Abbott platforms (c16000 chemistry and i2000 immunoassay).

P7: Abbott PSA reagent change evaluation


G Ward, B Teis, K Rowland, J Smith, J Nicolson, R Flatman, D Kanowski, L Price.

Sullivan Nicolaides Pathology, Brisbane QLD 4006, Australia

Introduction

In December 2017 Abbott Diagnostics implemented a new internal measurement standard (IMS) for Total PSA measurement. Abbott Product information indicated that there would be a down shift in results with reagents using the new IMS. The Abbott comparison study between reagents calibrated against either the old or new IMS used 117 patient samples of which ~15 samples had a PSA < 1ug/L with limited numbers of samples <10 ug/L. The aim of this study was to further investigate the correlation between the reagents with larger numbers of patient samples with PSA <10 ug/L; and determine the LoQ with the new IMS reagent preparation over multiple Abbott i2000 immunoassay platforms.

Methods

All samples were assayed by Abbott Architect PSA using reagents calibrated against either the new or old IMS. The number of samples tested for each group of PSA levels was as follows: PSA<0.2 ug/L (n=136); PSA<1.0 ug/L (n=185); PSA 1.0-9.9 ug/L (n=136). Limit of quantitation (LoQ) studies were performed by 6 separate Abbott i2000, on five patient pools (PSA <0.007 ug/L to 0.1 ug/L) over 5 days.

Results

The following equations were obtained by Passing-Bablok analysis:

• PSA (<0.2ug/L) Old IMS = 1.02New IMS (n=136)

• PSA (<1.0ug/L) Old IMS = 1.03New IMS (n=185)

• PSA (1.0-9.9 ug/L) Old IMS = 1.07New IMS (n136)

At PSA 0.006 ug/L the inter-assay CV on 6 analysers ranged from 6.7%-11.3%.

Conclusions

The studies confirmed a small but clinically insignificant decrease in PSA levels <10 ug/L.

The LoQ of 0.006 ug/L is consistent with our previous studies using the Old IMS and lower than that stated by the manufacturer. Thus there will be no changes to clinical interpretation. Manufacturers and laboratories should ensure an acceptable number of samples over a range of clinically important levels are assayed to test changes in assay standardization.

P8: Measurement of serum androstenedione. Comparison of three immunoassay Methods pre and post organic extraction with LC-MS/MS

S Stoner, K Young, B Teis, B McWhinney, D Kanowski, G

Ward, L Price.

Biochemistry Dept, Sullivan Nicolaides Pathology, Bowen Hills, Qld 4006, Australia

Introduction

Clinical applications of serum androstenedione measurement include: assessment of hirsuitism and virulization; assessment of catheter placement in adrenal venous sampling in primary aldosteronism; and prediction of IVF fertilization rates in insulin resistant women. Differences between LC-MS/MS and immunoassay Methods have been documented for testosterone with less information available for androstenedione. Organic extraction prior to immunoassay can improve the accuracy of immunoassays. The aim of this study was to assess androstenedione results from immunoassays pre – and post-extraction of serum samples with an LC-MS/MS method.

Methods

Androstenedione was measured in approximately 100 patient samples by the following: Waters LC-MS/MS (Pathology Queensland); Diasorin XL; IBL RIA; and Siemens Immulite 2000. The immunoassay samples were assayed before and after extraction in ethyl acetate:hexane (3:2 by volume). Extracted samples (organic fraction of 150 uL serum in 1.5 mL solvent) were evaporated and reconstituted in Diasorin Endocrinology Diluent prior to immunoassay.

Results

Direct and post extraction immunoassay results were higher than those measured by LC-MS/MS. Extraction procedures interfered with the Diasorin XL assay. Post extraction (PE) results were lower for the RIA and Immulite. Correlation between all assays was poor. Selected Passing-Bablok equations were as follows: Immulite = 2.55LC-MS/MS – 1.7 (n=84; r2 0.601); Diasorin = 1.82LC-MS/MS – 0.3 (n=96; r2 0.812); RIA = 1.98LC-MS/MS + 1.0 (n=98; r2 0.567); Immulite PE = 2.35LC-MS/MS – 0.39 (n=41; r2 0.776); RIA PE = 1.23LC-MS/MS – 0.87 (n=96; r2 0.711).

Discussion

Significant improvement is required in direct androstenedione immunoassays in order to achieve results comparable with LC-MS/MS. Diasorin XL immunoassay correlated best with LC-MS/MS although results were ~1.8 times higher. Extraction reduced RIA results to levels 1.23 times higher than LC-MS/MS indicating that removal of potential interfering proteins and/or polar steroids improved the assay. Correlation between the Diasorin XL and RIA(PE) with LC-MS/MS may be improved by standardization or harmonization.


P9: Review of factor XIII screen versus assay: Results from the RCPAQAP

S Arunachalam1, Bonar R1, F Estepa1, E Duncan2 1RCPAQAP, Sydney, Australia2SA Pathology, Australia

Introduction

Factor XIII (FXIII), initially known as fibrin stabilising factor, is involved in cross-linking of fibrin in clot formation. In the absence of FXIII the clot becomes unstable and is susceptible to lysis by plasmin. Although deficiency in this plasma protein is rare, clinical manifestations of this condition can be life-threatening. Currently, clot lysis screens are used when FXIII deficiency is suspected and assays to determine levels of FXIII. The Royal College of Pathologists Australasia Quality Assurance Programs (RCPAQAP) provides external proficiency testing for FXIII. This review aims to summarise results of a three-year period for the relevance of screening and quantitation of FXIII.

Methods

The FXIII proficiency program contains four samples per year. Survey data from 2015 to 2017 (12 samples), were analysed with FXIII levels ranging from <1% to 110%. Survey materials consisted of a combination of patient and commercial FXIII deficient samples.

Results

Three of the twelve survey samples had FXIII levels of 1 to 2 %. Only (21/34) 62% of participants using a screening test correctly interpreted a severe deficiency in these samples. FXIII assay results for participants using the Liatest Methods had a median FXIII of 1.4%, whereas, the chromogenic method had a median FXIII of 5.5%. It was noted that 59% of chromogenic method users did not use a ‘blank’.

The remaining nine samples had FXIII of ≥4%. Screen results had (99/109) 90% of participants correctly reporting FXIII deficiency to be excluded. FXIII assay by both Methods produced results closer to the target and correctly excluded FXIII deficiency.

Conclusions

The FXIII assay results showed better sensitivity to detect a severe deficiency compared to lysis tests. FXIII lysis tests presented low sensitivity which could cause a FXIII deficiency diagnosis to be missed. Therefore, even if the screen results indicate exclusion of FXIII deficiency, a FXIII assay is essential to assess bleeding risk.

P10: Serum cystatin C as a marker of early nephropathy in Sri Lankan patients with type II diabetes mellitus

GPN Abeynayake, MM Gunatillaka

National Hospital of Sri Lanka, Colombo, 01000, Sri Lanka

Introduction

Diabetic nephropathy is a significant cause of end-stage renal disease and therefore its early detection is essential. The effectiveness of cystatin C in detecting diabetic nephropathy in Sri Lankan patients with type 2 diabetes remains uncertain as many studies have been conducted in western populations.

Methods

An observational, cross sectional study was conducted at the diabetic clinic in National Hospital of Sri Lanka. We recruited 140 patients consecutively excluding those with malignancy, thyroid disease, blood pressure > 130/80 mmHg, chronic kidney disease, steroid therapy, liver disease and acute illness. Blood for serum creatinine, cystatin C and 2nd void urine for creatinine and albumin were obtained on a subsequent date and those were analyzed using Kone 60i automated analyzer. Patients’ blood pressure was measured twice before sample collection to confirm normal blood pressure. Patients were grouped according to urine albumin : creatinine ratio (ACR) reported in mg/mmol as follows; < 3, normoalbuminuric (Group 1; 97 patients), and 3 to 30, microalbuminuric (Group 2; 35 patients). Eight patients with ACR > 30 were excluded. Receiver characteristic operating curves (ROC) were generated to assess diagnostic efficiency of serum creatinine and cystatin C to predict diabetic nephropathy.

Results

There was a significant increase (p < 0.01) in both serum cystatin C (0.864 ± 0.256 mg/L Vs 0.656 ± 0.180 mg/L) and creatinine (74 ± 16.2 µmol/L Vs 59 ± 13.5 µmol/L) in group 2 compared to group 1. ROC analysis showed that cystatin C had a higher area under the curve (0.826; 95% confidence interval from 0.728 to 0.92) compared to creatinine (0.779; 95% confidence interval from 0.688 to 0.869) in detecting diabetic nephropathy when microalbuminuria was considered as the gold standard.

Conclusions

Serum cystatin C is an effective biochemical marker for detection of early diabetic nephropathy.

P11: A case with non-detectable placental growth factor-1 (PlGF-1) in first trimester testing

Y. Aizawa1, P. Williams1,2, V. Tasevski3, J. Hyett4, J. Sherfan1

1Royal Prince Alfred Hospital, NSW Health Pathology Central Sydney, Australia2Charles Perkins Center, Faculty of Medicine and Health, University of Sydney, Australia3Royal North Shore Hospital, Northern NSW Health Pathology, Australia4Royal Prince Alfred Hospital, Sydney Local Health District, Australia


First trimester testing for aneuploidy at approximately 12 weeks of a 33 year old female (Parity:1; Gravida:3; Terminations:1-omphalocele) gave results on the Kryptor Compact Plus of: PAPPA – 3.662 IU/L, fBhCG – 82.8 IU/L, PLGF-1 <3.6 ng/L (LOD 3.6 ng/L). The low PlGF prompted us to perform comparative measurements on the Roche Cobas 8000 (LoD 3 ng/L) and Perkin Elmer Delfia Xpress (LoD 1.9 ng/L) instruments. The results were 20.8 ng/L and 15.8 ng/L on the Cobas and Delfia respectively. Upon further investigations into the possible cause of the undetectable PlGF-1 result, ruling out sampling errors and sample integrity, it was noted that the Kryptor method is specific to free PlGF-1 isoform, whereas the Cobas and Delfia detected total PlGF. The Kryptor package insert states cross reactivity with PlGF 2 of 13% vs 28% and 30% for the Cobas and Delfia respectively.

This led us to make the assumption that the patient has a predominant form of the 2, 3 or 4 isoform undetectable by the Kryptor method. However, proving this requires further testing.

Despite the undetectable PlGF-1 result, this did not affect the patient’s risk of aneuploidy (1:2798 for Trisomy 21). It did, however, increase her risk of developing preeclampsia (PE) before 34 weeks to 1:22.

PlGF is a useful marker in the detection of aneuploidy in pregnant women when combined with fBhCG and PAPP-A, but low levels of maternal PlGF in the first trimester has been implicated in impaired placentation, consistent with the prediction of high risk PE.

This is the first case of a patient that we have measured with undetectable levels of PlGF-1. Laboratories should be aware there is a lack of standardisation with commercially available PlGF assays and understanding the assays’ cross reactivity with other isoforms will help to make informed patient result interpretation.

P12: LC-MS/MS based 25(OH)D status in a large central european outpatient cohort – gender and age specific differences

M Herrmann,1,2, S Giuliani,1,2, V Barbieri,3, AM Di Pierro,2

1Medical University of Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Graz, Austria2Laboratory of Clinical Pathology, Hospital of Bolzano, Italy3Obstmarkt 31, 39100 Bolzano, Italy

Background

Developed countries have a high prevalence of vitamin D deficiency. In previous studies 25-hydroxy vitamin D [25(OH)D] was predominantly measured by immunoassays, which are known for their variable

analytical performance. The present study assessed serum 25(OH)D in a very large European outpatient cohort by liquid-chromatography-tandem-mass-spectrometry (LC-MS/MS).

Material and Methods

Between 2015 and 2016 were extracted 74,235 serum 25(OH)D results generated under routine conditions from the laboratory information system of the Department of Clinical Pathology at Bolzano Hospital (Italy). In 3,801 cases parathyroid hormone (PTH) was requested in parallel. Serum 25(OH)D was measured by a NIST-972 aligned commercial LC-MS/MS method. The distribution of serum 25(OH)D concentrations in males and females of different age groups, the prevalence of 25(OH)D2 and seasonal variability were studied.

Results

Statistics state that 25(OH)D testing was requested three times more often in females than in males. Average 25(OH)D concentration in the entire cohort was 68.6 nmol/L (7.5-1880 nmol/L). Females had a 7 nmol/L higher average 25(OH)D concentration than males, which increased significantly with age. 37.9% and 28.3% of males and females, respectively, had a deficient 25(OH)D concentration of <50 nmol/L. 620 samples (0.84%) had measureable amounts of 25(OH)D2. In samples with a normal PTH 25(OH)D was 9 nmol/L higher than in the entire cohort. Seasonal variation ranged between 20-30% and was most pronounced in young individuals. 25(OH)D2 remained constant throughout the year.

Conclusions

Vitamin D status in South Tyrol is rather good. Higher 25(OH)D concentrations in individuals with normal PTH suggest a functional deficit in a substantial number of subjects. Seasonal variation of serum 25(OH)D is a relevant aspect in young and middle-aged adults, but becomes less relevant in elderly subjects. Serum 25(OH)D2 is of minor practical importance in South Tyrol

P13: Prescription records of patients with high urinary total metanephrines without a diagnosis of phaeochromocytoma or paraganglioma

L Lam, G Woollard, C Kyle

Department of Chemical Pathology, LabPlus, Auckland City Hospital, New Zealand

Introduction

Total 24 hour urinary metanephrines is a first-line screening test used in the detection of paraganglioma and phaeochromocytoma (PPGL). Anti-hypertensives and other medications (e.g. Tricyclics) are a well-recognized cause of false positive urine metanephrines. We examined


the prescription records of a cohort of patients suspected of PPGL with high urinary total metanephrines to characterize the extent of pharmaceutical effect on urinary metanephrines.

Methods

Upper decile of urine metanephrines reported over a one year period in 2015 was examined. Patient dispensary records were accessed via the electronic database (TestSafe), which connects all pharmacies and laboratories in the Auckland Region. Patients from other regions and those with PPGLs were excluded. Records were examined to determine whether: ACE inhibitors, spironolactone, alpha-blockers, beta-blockers, calcium channel blockers, diuretics, tricylics, SSRI/SNRI, monoamine oxidase inhibitors, antipsychotics, methyldopa and L-DOPA were being prescribed at the time of testing.

Results

Sixty of 258 patients (23%) had a likely non-pharamaceutical cause of high urinary metanephrines. Excretion of normetanephrine was positively associated with the number of above medications prescribed (R=0.22; P<0.05). By contrast, this association was not observed for metanephrine or methoxytyramine. Patients without meidcation had similar urinary metanephrine levels compared to patients being prescribed any single medication in isolation.

Conclusions

Patients with higher urinary metanephrines are of much greater interest as potentially having PPGL. Three quarters of these pateints are being prescribed one or more susceptible medications. These affect normetaneprhine far more than metanephrine and 3-methoxytyramine. The increase in urinary normetanephrine increases with the number of medications.

P14: Quantitation of plasma angiotensin II by liquid chromatography mass spectrometry

CWS Lo, TKC Tsui, RCW Ma, MHM Chan, CS Ho

Department of Chemical Pathology, Prince of Wales Hospital, Hong Kong

Introduction

Quantitation of plasma angiotensin II (Ang II), the active mediator of the renin-angiotensin system (RAS), is challenging due to its low physiological concentrations. We report a validated liquid chromatography mass spectrometry (LCMS) method to overcome this challenge.

Methods

Ang II was extracted from EDTA plasma by an offline solid phase extraction procedure with a Waters MAX µElution

plate. LCMS quantitation was performed on a Waters TQS system monitoring the 3+ ions of the peptide. Analytical performance of the LCMS method was validated according to CLSI guidelines. Stability of Ang II was studied at different sampling conditions including different temperatures and the presence of protease inhibitors. Local reference interval was established from 145 healthy normotensive subjects (57%female, 21-60 years old). Plasma renin activity (PRA) and aldosterone concentrations were also measured in these reference samples using LCMS Methods. Ang II concentrations were also measured in hypertensive patients on different medications

Results

The Ang II LCMS method did not have significant matrix interference and carryover. The measurable range was 3.3-700pmol/L. Between batch precision coefficient of variation was <7% over the Ang II concentrations of 8.6-110pmol/L. There was no significant difference in Ang II concentrations when collected at room temperature with and without protease inhibitors. The stability was up to at least 1 month when stored at -70oC. The reference intervals, independent of sex, decreased with increasing age. There were significant correlations between AngII, PRA and aldosterone. Ang II concentrations were higher in patients on Ang II receptor blockers than those on ACEI and beta blockers.

Conclusions

Performance of the present LCMS method is suitable to quantitate Ang II to study the RAS system. Ang II collected at room temperature into EDTA bottles is stable at -70 oC for up to 29 days.

P15: Plasma aldosterone /angiotensin II ratio for screening primary aldosteronism

CWS Lo, TKC Tsui, RCW Ma, MHM Chan, CS Ho

Department of Chemical Pathology, Prince of Wales Hospital, Hong Kong

Introduction

The aldosterone/renin activity ratio (ARR) has been used for screening primary aldosteronism (PA). However, the measurement of plasma renin activity (PRA) is tedious requiring sample incubation for hours at two temperatures. Plasma angiotensin II (AII), the active mediator of the renin-angiotensin system, correlates with PRA. The turnaround time for measuring AII by liquid chromatography mass spectrometry (LCMS) is faster than that of PRA. Therefore, aldosterone/AII ratio (AAIIR) may be an alternative for screening PA.


Method

Plasma samples were collected from normotensive controls (n=135, 56.3% female, age 22-59 years) and hypertensive patients who underwent screening for PA (n=155, 45% female, age 21-74 years). Plasma aldosterone (PALD), PRA and AII were measured by LCMS Methods; ARR and AAIIR were calculated. Hypertensive patient records were reviewed. PA status were determined by the endocrinologists based on oral salt loading or saline infusion plus imaging results. Statistical analyses were performed on MedCalc software.

Results

In this cohort of hypertensives, 35 patients were diagnosed with PA. Both PRA and AII correlated significantly with PALD in normotensives and non-PA patients, but not in PA patients. ARR and AAIIR correlated significantly in controls and patients. Both ratios in patients were higher than those in controls. Receiver operating characteristic analyses showed optimal PA screening cutoff values for ARR and AAIIR were 577 and 60, respectively. At these cutoffs, specificities were 85% for both ratios; and sensitivities were comparable at 89% and 91% for ARR and AAIIR, respectively. There was no significant difference in the AUC between these two ratios. The likelihood ratios for positive and negative test results for AAIIR were 6 and 0.1, respectively, an acceptable test to rule out PA.

Conclusions

AAIIR has similar diagnostic efficiency as ARR for screening PA. AAIIR has the advantage of a faster turnaround time than that of ARR.

P16: Insulin-like growth factor-1 analysis using liquid chromatography tandem mass spectrometry: comparison between MRM and MRM3 acquisition modes

B Matthews, B Hiley, C White and AR Horvath.

NSW Health Pathology, Prince of Wales Hospital, Randwick, NSW 2031, Australia

Introduction

The Endocrine Society recommends IGF-1 as the first line test for the clinical assessment of GH disorders. IGF-1 immunoassays have high analytical variation and lack specificity. Therefore alternative Methods by liquid chromatography mass spectrometry (LC-MS/MS) have been developed. To improve specificity, we compared multiple reaction monitoring (MRM) and MRM3 acquisitions that utilise secondary fragmentations in a linear ion trap assays for IGF-1.

Methods

Abundant serum proteins were precipitated using acidified acetonitrile followed by endoproteolytic digestion with trypsin. LC-MS/MS was employed to assay

the IGF-1 specific peptide GFYFNKPTGYGSSSR. The MRM assay targeted the Y13 and Y12 transition ions. The MRM3 further fragmented these ions targeting the Y9 ion.

Results

The average extraction efficiency was 96.15%. An eight point linear calibration curve, between 10 to 800 ng/mL, provided sufficient range to diagnose GH deficiency and excess. The 10 ng/mL LLOQ was statistically validated with an acceptable CV of 12% for the MRM method, though much higher variability was observed in the MRM3 assay (CV 34%). At higher calibrator concentrations (25 ng/mL and above) the analytical variability of the MRM3 method was more acceptable (CV < 10%). The MRM and MRM3 assays were compared using two batches of different patient pooled samples with 10 replicates in each. The first showed a high correlation between the two Methods (MRM: average IGF-1=110.4 ng/mL, CV 6.8%; MRM3: average IGF-1=109.6 ng/mL, CV 6.9%; T-test P=0.66) while the second showed significant variation (MRM: average IGF-1=92.2 ng/mL, CV 3.9%; MRM3 average IGF-1=112.7 ng/mL, CV 6%; T-test P <0.001).

Conclusions

Correlation with a clinically validated IGF-1 method or analysis of certified reference material is required to determine the more accurate method. Under the conditions tested, the MRM assay provided a more analytically robust measure, essential for clinical diagnosis of GH disorders.

P17: Distribution of HbA1c and its association with lipid parameters among an apparently healthy Sri Lankan adult population

Panapitiya N P, Ginige L, Lankananda B D, Jainulabdeen F F, Munasinghe D, Navodika L H D, Jayasekara L P C P, Aththanayake A M I S, de Silva T S T M, Katulnada P, Katulanda G W

Medical Research Institute, Colombo, Sri Lanka

Introduction

Diabetes is amongst the leading causes for mortality and morbidity in South Asia. Since dyslipidaemia is a well known risk factor for diabetes, we aimed at determining the lowest levels of different lipids which significantly associate with diabetes.

Methods

A descriptive cross sectional study was conducted among 422 voluntary, non pregnant, healthy(not diagnosed to have Diabetes/ Hypertension/ Hyperlipidaemia) adults (>18 years). Fasting blood samples were obtained following informed written consent and tested for HbA1c, lipid profile, Apolipoproteins A1 (ApoA1) and B (ApoB)


using fully automated assays. The results were interpreted against ADA cut-off levels for HbA1c and NCEPATPIII for lipids. Chi square, Odds ratio (OR) with 95% confidence interval (CI) and t test analysis were performed using SPSS 23.

Results

Our population had a female predominance of 62.9 %. Mean age of females and males were 40.5 years (+/-12.2) and 41.3 years (+/-11.8) respectively. 14.7 % had HbA1c ≥ 48 mmol/mol. Mean HbA1c increased with increasing age and was significantly different between males (5.8 mmol/mol +/ – 1.2) and females (5.6 mmol/mol +/-0.7) (p < 0.001).

Percentages of participants who exceeded desirable levels for Total Cholesterol (TC) ≥ 5.8 mmol/L, Low Density Lipoprotein (LDL-C) ≥ 3.4 mmol/L, non High Density Lipoprotein (nHDL-C) ≥ 3.4 mmol/L, Apo B ≥ 1.2 g/L, Triglycerides (TG) ≥ 1.7 mmol/L and High Density Lipoprotein (HDL-C) < 1.03 mmol/L were 66.2 %, 62.6 %, 85.6 %, 70.7 %, 31.2 % and 22.2 % respectively.

Considering 48 mmol/mol of HbA1c as the cutoff level, LDL-C = 3.1 mmol/L (p = 0.01; OR = 2.5 (1.15 – 5.47 ), ApoB = 1.2 g/L (p = 0.03; OR = 2.1 (1.06 – 4.20), TG = 1.1 mmol/L (p = 0.02, OR = 2 (1.07 – 3.91) were the lowest levels at which the significant associations detected. TC, nHDL-C and HDL-C did not show significant association with HbA1c at 48 mmol/mol.

Conclusions

Majority of our population had elevated TC and LDL-C levels. Diabetes and hypertriglyceridemia were observed in the minority. Even mild elevations of TG/LDL-C/ApoB cause 2 folds increase in risk for diabetes.

P18: Comparison of thyroid function tests in capillary and venous paired blood samples

MP Nascimento¹ C Yu¹, E Moore¹, C McDonald¹, A Simpson¹, ²

¹ACT Pathology, The Canberra Hospital,

²ANU Medical School, Garran, ACT 2605, Australia

Introduction

Hypothyroidism is an endocrine disorder which has significant associated morbidity particularly in paediatrics. Patients require ongoing follow-up to ascertain adequate hormone replacement. Venepuncture in this age group is often difficult and fingerprick testing could be an appropriate alternative. Despite widespread use of capillary dried blood spot for thyroid disease screening in neonates, there is limited data comparing thyroid function tests performed on venous and capillary blood. Our study compared concentrations of TSH, fT4 and fT3 in paired serum samples collected over a period of three days.

Methods

Paired capillary and venous blood were collected from 14 healthy adult volunteers (11 females, 3 males). Paediatric and standard serum separation tubes were used for capillary and venous samples respectively. Specimens were analysed on the same day of collection on the Abbott Architect ci16200. Two pairs of samples were checked for haemolysis using analyser indices. In three samples with volumes below 270 uL, TSH assay was performed using onboard dilution of 50 uL of serum with 200 uL of saline solution.

Results

Venous ranges of TSH (0.369 – 2.311 mIU/L) and fT4 (12.07 – 22.11 pmol/L) demonstrated strong correlation with paired capillary samples with R² values of 0.992 and 0.964 respectively. The relationship for fT3 (3.67-4.99 pmol/L) was less strong with R2=0.700. Haemolytic indices ranged from 0.02 to 0.05 for venous and 0.26 to 0.33 for capillary samples.

Discussion

Fingerprick blood collection for measurement of TSH and fT4 offers an alternative in populations with difficult venous access, such as paediatrics. Our result demonstrated acceptable haemolytic indices with both sample types.

P19: Clinically significant inter-assay discordance in serum prolactin in Australia

Sunita MC De Sousa,1–3 Mohamed Saleem,1,3,4 Wayne Rankin,1,3,4 David J Torpy,1,3

1Endocrine and Metabolic Unit, Royal Adelaide Hospital, Adelaide, Australia;2Department of Genetics and Molecular Pathology, Centre for Cancer Biology, an SA Pathology and University of South Australia Alliance, Adelaide, Australia;3School of Medicine, University of Adelaide, Adelaide, Australia; 4Chemical Pathology Division, SA Pathology, Adelaide, Australia.

The magnitude of prolactin elevation guides the differential diagnosis of hyperprolactinaemia and parallels prolactinoma diameter. Severe hyperprolactinaemia (>10-fold ULN) is generally due to macroprolactinoma or pregnancy. Causes of mild hyperprolactinaemia (<4-fold ULN) include microprolactinomas, dopamine interference, primary hypothyroidism, polycystic ovary syndrome, prolactin co-secretion in acromegaly or Cushing’s disease, and physiological changes (e.g. stress).

We observed eight patients with 28–166% higher prolactin by the Roche versus Siemens assays during routine clinical practice. Some patients had cause for true hyperprolactinaemia but there were no clinical


changes in any case to explain the higher Roche assay levels. For example, a woman with schizophrenia had hyperprolactinaemia at 7-fold ULN by Roche, prompting investigation for prolactinoma. Pituitary MRI was normal and repeat prolactin by Siemens assay was only 2.5-fold ULN, consistent with her longstanding antipsychotic use.

Hence, we measured serum prolactin in both assays using split clinical samples (n=40) across a range of serum prolactin (5–5051 mIU/L). This revealed that serum prolactin was approximately 50% higher by the Roche compared to Siemens assay, despite similar reference ranges. Review of the original Roche data showed no technical error in reference interval calculation: the reason for discordance thus remains unexplained. We speculate that the recent divergence in measurements may relate to either progressive positive bias with successive reagent lot numbers, or antibody deterioration.

Our findings of prolactin inter-assay discordance emphasise the importance of verifying reference intervals and performing bias checks over time. Endocrinologists should be aware of the potential for prolactin overestimation and the utility of repeat testing on different platforms. In mild hyperprolactinaemia by the Roche platform, with normoprolactinaemia by other platforms, patients may be spared from unnecessary endocrine reviews and MRI studies. In true hyperprolactinaemia, separating patients with mild versus severe hyperprolactinaemia will narrow the diagnostic possibilities.

P20: Quantitation and separation of 17β-estradiol, 17-estradiol and a contaminant with the same nominal mass in an LC-MS/MS clinical assay

R Sahertian, SL Ramsay, AR Horvath

NSW Health Pathology, Department of Clinical Chemistry & Endocrinology, Randwick, NSW, 2031, Australia

Introduction

The most biologically active estrogen is the endogenous 17β-estradiol. High-sensitivity estradiol assays are needed for certain patient populations, including males, children, postmenopausal women and patients on aromatase inhibitors for breast cancer. Estradiol concentrations are particularly low in postmenopausal women (typically <100 pmol/L). Hormone replacement therapies for post-menopausal women often are estradiol-based. 17-β estradiol has higher relative binding affinity to estrogen receptors ER/β than the 17- estradiol epimer (Zhu BT et al 2006) and therefore requires adequate chromatographic separation from each other. Equivalent immunoassays are interfered by sex hormone binding globulin and human serum albumin (Iqbal MJ, et al 1983) where LC-MS/MS methods have obvious advantages. In spite of higher analytical specificity and sensitivity, in our LC-MS/MS assay we have observed an infrequent contaminant of same

nominal mass and similar fragmentation to 17-β estradiol which, if undetected, could lead to confounding results.

Methods

Our LC-MS/MS method measures 17β-estradiol in serum (250μL) by multiple reaction monitoring (MRMs); analytical range 5-5000 pmol/L. The gradient was extended to enable sufficient separation of both β and epimers. MRM triggered EPI (enhanced product ion) scan was utilised to monitor full product ion scans for all molecular species with nominal mass of m/z 271. Additional MRMs were added to the method that were observed to be significant for both epimers and the contaminant.

Results

The and β epimers of estradiol were baseline separated chromatographically. A contaminant with nominal mass was observed in several patients and was not sufficiently separable from the later eluting epimer, however it’s product ion scan was sufficiently different from both epimers of estradiol although strikingly similar.

Conclusions

With the extended gradient method described, both epimers were quantified and the contaminant was differentiated based on retention time and ion ratios, to provide reliable estradiol results.

P21: Use of urine c-peptide to creatinine ratio to assess graft function in islet transplant recipients

Stranks JL1, Rankin W2,3,4, Coates P2,3,4, Coates PT4,5,6.1 Diabetes and Endocrine Unit, Lyell McEwin Hospital2 Chemical Pathology Directorate, SA Pathology3 Endocrine and Metabolic Unit, Royal Adelaide Hospital4 Discipline of Medicine, University of Adelaide5 Central Northern Adelaide Renal and Transplantation Services6 Centre for Clinical and Experimental Transplantation

Introduction

Islet transplant recipients have traditionally required mixed meal testing with serial serum c-peptide levels to assess ongoing graft function. While this has been considered the gold standard for assessing islet function, this approach has limitations in terms of accessibility, cost, and the need for a day admission at an Endocrine testing unit and therefore issues with distance for patients living in a rural location. We present the results of six previous islet transplant recipients who underwent paired mixed meal tests and a urine c-peptide-creatinine ratio (UCPCR) for comparative graft function evaluation.

Methods

After an overnight fast, recipients drank a standardised Resource nutrient shake (combination carbohydrate/fat)


with serial c-peptide (Roche E601), insulin (Advia Centaur) and glucose measurements at 0, 15, 30, 60, 90 and 120 minutes as per the standard mixed meal test protocol. In addition, the patients produced a urine sample for UCPCR testing at 120 minutes post drink. All patients were asked to void at the start of the test to avoid contamination from any overnight endogenous insulin production.

Results

Of the six patients, one patient was unable to produce a urine sample at 120 minutes so was excluded from the analysis. Of the others, 90 minute serum c-peptide (scp90) levels and UCPCR correlated well when predicting poor graft function (three of the five patients), using the previously quoted ratio cutoff of <0.34 nmol/mmol. For the 2 insulin independent patients, their scp90 and UCPCR ratio were more variable but still within quoted parameters for moderate–good graft function.

Discussion

The UCPCR test provides a potentially more convenient and accessible alternative to mixed meal testing. Further assessment is required to test reproducibility in patients with good islet graft function and evaluate if home post-meal urine testing would be similarly useful, as seen at one other centre.

P22: Performance Evaluation of the Atellica CH Enzymatic Hemoglobin A1c Assay*

Jones J, Robinson C, Gisiora J, Dai J.

Siemens Healthcare Diagnostics Inc., Newark, DE, U.S.

Background

According to the WHO, 422 million adults were living with diabetes in 2014, and an estimated 1.6 million deaths directly related to diabetes occurred in 2015. Early diagnosis is important for diabetes care. One measurement of glycemic state is glycated haemoglobin (HbA1c). A new enzymatic HbA1c Assay (A1c_E) has been developed* for the Atellica® Solution CH Analyser. In the pretreatment step, the erythrocytes are lysed and the haemoglobin oxidised to methaemoglobin with sodium nitrite. In the first step of the reaction, the N-terminal fructosyl dipeptide fragment is cleaved from the haemoglobin beta chain with a protease. Concurrently, methaemoglobin is converted into azidemethaemoglobin with sodium azide, and the total haemoglobin concentration is determined by measuring the absorbance at 478/694 nm. In the second step, fructosyl peptide oxidase is added to react with the fructosyl dipeptide generating hydrogen peroxide which reacts with the chromagen in the presence of peroxidase developing a colour measured at 658/805 nm.

Methods

Assay linearity and precision was evaluated using CLSI protocol EP06-A and EP15-A3. Two levels of commercial

controls and four whole blood pools ranging from ~25.7 to ~107.7 mmol/mol HbA1c were tested. Each sample was assayed five times per run, two runs per day, for five days. A method comparison study (n = 40 samples) was conducted between the Atellica CH A1c_E Assay and the NGSP secondary reference lab according to CLSI protocol EP09-A3.

Results

The Atellica CH A1c_E Assay is linear from 19.8 to 129.5 mmol/mol HbA1c, repeatability from 0.29 to 0.65% CV, and within-lab precision from 0.62 to 1.09% CV. The method comparison equation Atellica A1c_E Assay = 1.047 – 19.4 HbA1c (r = 1.00) and demonstrated a %TE ≤3.57.

Conclusions

The A1c_E Assay on the Atellica CH Analyser from Siemens Healthineers demonstrates acceptable precision and correlation with the NGSP reference method.

*Under development.

P23: Initial performance data for N latex FLC kappa and lambda reagents on the atellica CH analyser

Ehm M1, Davis J2, Gentzer W1, Mirwaldt C1, Muth D1, Smith V1, Snyder J2, Zander N1.1Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany2Siemens Healthcare Diagnostics Inc., Newark, DE, U.S.

Background

The International Myeloma Working Group consensus guidelines include the use of immunoglobulin free light chain (FLC) determinations for diagnosis and management of clonal plasma cell disorders. We describe reproducibility, imprecision, method comparison, and limit of quantitation (LoQ) data for the application of N Latex FLC kappa and lambda reagents on the Atellica® Chemistry (CH) Analyzer.*

Methods

Latex-enhanced mouse monoclonal antibody reagents (Siemens Healthineers) for FLC kappa (N Latex FLC kappa) and lambda (N Latex FLC lambda) were assayed on the Atellica CH Analyser. A precision study was conducted following CLSI EP05-A3 of four sample pools and two controls for FLC kappa and lambda, with one reagent and one calibrator lot. Each sample was assayed in quadruplicate twice a day for 5 days. A method comparison study was conducted according to CLSI EP09-A3. Results were correlated with data generated on the BN ProSpec® System. The results were analysed using Passing-Bablok linear regression analysis. LoQ was determined for both applications following CLSI EP17-A2.

Results


The within-run CV for the FLC kappa application on the Atellica CH Analyzer ranged from 0.80−2.09% and the total (within-instrument) CV from 1.50−3.78%. For FLC lambda, the within-run CV was 0.81–2.18%, and the total (within-instrument) CV was 1.76–4.75%. Passing-Bablok regression results between the BN ProSpec and Atellica CH Analyser was y = 1.008 x – 0.064 mg/L (r = 0.986) for FLC kappa (n = 175) and y = 1.033 x – 0.695 mg/L (r = 0.973) for FLC lambda (n = 168). LoQ was determined as 1.084 mg/L for FLC kappa, showing a total error of 28.01%. LoQ for FLC lambda was 1.558 mg/L, with a total error of 15.76%.

Conclusions

The application of N Latex FLC reagents on the Atellica CH Analyzer demonstrated acceptable, consistent imprecision. Method comparison showed good agreement with an existing method.

*Under development.

P24: Evaluation of the Atellica CH Assays Sigma Metrics

Pierson-Perry J, Estock K, Pham T.

Siemens Healthcare Diagnostics Inc., Newark, DE, U.S.

Background

The purpose was to evaluate the performance quality of Atellica® CH Assays using Six Sigma philosophy, which uses techniques to identify, quantify, and reduce sources of variability in a product or process. The Sigma metric evaluates the bias between a quality specification and what was observed for that process. This value is expressed in SD. A Sigma metric of 3 is the minimum quality threshold, and a Sigma metric of 6 or greater is superior quality. Allowable total error (ATE) is the quality specification used to evaluate assay performance, it reflects the maximum error in an assay result that will not change the associated medical decision. Typically, within-lab precision data from QC or patient samples—obtained from the assay’s IFU or historical QC measurements—are used in the evaluation of performance. It is common to use a single analyte value, such as a medical decision level, for the calculation. ATE values are difficult to obtain, they vary from source to source and may not be available for all assays.

Methods

Four inputs are required for the calculation of Sigma metric: analyte concentration, ATE value, estimated bias, and estimated precision at the specified concentration. For the Atellica CH Assays, precision and bias input values were obtained from the assay IFU’s, where precision was determined following CLSI EP05-A3, samples assayed in duplicate for 20 days. Bias at the selected concentration was calculated following CLSI EP09-A3. The CLIA table was the primary source for an assay’s ATE.

Results

Seventy-two Atellica CH Assays were analysed, 52 assays

obtained a Sigma level of >6.0, 20 assays obtained a level 3.0 to 6.0, and 0 assays obtained a level of <3.0.

Conclusions

Using Six Sigma philosophy, all Atellica CH Assays analysed obtained the minimum quality threshold of 3.0 or greater and demonstrate clinically acceptable performance.

P25: Bilirubin interference on an enzymatic paracetamol assay and its removal by ultrafiltration

F Alvaro

NSW Health Pathology, Liverpool Hospital, Liverpool NSW 2170, Australia

Introduction

Laboratories have a crucial role in the diagnosis and monitoring of suspected and actual paracetamol overdosage by providing rapid and accurate serum concentrations of the drug. Literature reports of false-positive serum paracetamol concentrations caused by the presence of elevated levels of bilirubin, as well as the listing of bilirubin as an interferent of the Roche cobas c701/702 enzymatic paracetamol assay, have led to uncertainty of the accuracy of results reported by this laboratory. This study looks at the magnitude of the bilirubin interference and the use of ultrafiltration to remove it.

Methods

Aliquots of paracetamol and bilirubin-free serum were spiked with saline (baseline) and increasing amounts of bilirubin and paracetamol stock solutions to give a set of samples with paracetamol concentrations from 0 to 46.5 mg/L and bilirubin concentrations from 0 to 382 umol/L. An aliquot of each sample was centrifuged through a Millipore Centrifree ultrafiltration device to produce a bilirubin-free ultrafiltrate. Both the filtered and unfiltered samples were analysed for total bilirubin and paracetamol on a Roche Cobas c702 analyser. The degree of protein-binding of paracetamol was also determined to correct for paracetamol retained by the filtration device.

Results

Paracetamol-free serum had a measured concentration of 1.7 mg/L at a bilirubin concentration of 145 umol/L. Specimens with theoretical paracetamol concentrations of up to 5 mg/L had >10% positive interference at bilirubin concentrations ³ 95 umol/L; at theoretical paracetamol concentrations between 5 and 15 mg/L the threshold of significant bilirubin interference increased to 382 umol/L. Ultrafiltration removed the bilirubin interference but also produced under-recoveries of paracetamol due to protein binding (measured at 16%), which was successfully corrected for mathematically.

Conclusions

Positive interference by bilirubin on the enzymatic


paracetamol assay, significant in paracetamol concentrations of up to 15 mg/L, can be removed by a quick and simple ultrafiltration step.

P26: Haemoglobin dissolution in collection devices for the OC-SENSOR PLEDIA system

Bahnisch, R; Hosseini, S, O’Loughlin, P

Chemical Pathology Directorate, SA Pathology, Adelaide SA 5031 Australia

Introduction

Faecal occult blood testing is used for the detection of pre-malignancy and malignancy of the large bowel. The Bio-Pharm OC-SENSOR PLEDIA testing system was evaluated to replace a superseded semi quantitative testing system. Evaluation of the new system included assessment of the rate of dissolution of haemoglobin in the PLEDIA proprietary sampling device.

Methods

The PLEDIA system uses latex turbidimetry as its testing principle. Latex particles coated with anti-human haemoglobin agglutinate when presented with faecal sample containing human haemoglobin. The agglutination is proportional to the concentration of the haemoglobin in the sample and measured using turbidimetry. A positive cut off value of 100 ng/mL has been established.

Faecal samples received as fresh faecal collection pots for testing across multiple laboratories for microbiology, virology and/or chemistry testing need to be manually sampled using the PLEDIA sampling bottles for analysis in lab. The rate of dissolution of Hb in the PLEDIA collection device buffer was assessed in 10 patient samples. FOBT analysis was performed hourly from the time of sampling for five hours.

Results

All samples showed increasing levels of Hb detected from time 0. Results showed average increases from initial measurement (time 0) of +18% at 60 mins, +27% at 120 mins, +43% at 180 mins, +97% at 240 mins and +94% at 300 mins. Four of five positive samples were positive at time 0 however one sample was negative at time 0 and 60 mins then positive from 120mins+. In all cases the level of Hb detected plateaued at 240min. Further assessment and analysis will be presented.

Conclusions

Testing for Hb in the PLEDIA sampling device must not be performed before 240min after sample collection.

P27: Audit of clinical testing indications for cerebrospinal fluid neurotransmitter studies

S Bandodkar,1,2 R Dale1,2, S Alvarez,1 L Zeng,1 A Bandodkar2, NJ Perera,1 JZY Chung1,2

1Department of Clinical Biochemistry and Neurochemistry,

The Children’s Hospital at Westmead, Westmead NSW 2145, Australia2Sydney Medical School, The University of Sydney

Introduction

Biogenic amine homeostasis is impaired in inherited disorders of monoamine biosynthesis, metabolism and transport. The synthesis, metabolism, homeostasis and transport of catecholamines, serotonin together with the Pterins and Folate is critical for the accurate diagnosis and treatment of these monoamine neurotransmitter disorders that often present as overlapping clinical phenotypes with other childhood neurological disorders. The Specialist Neurochemistry Laboratory at The Children’s Hospital at Westmead is a CSF Neurotransmitter reference laboratory for most metabolic and neurology clinics in Australia and Pacific rim countries and undertakes the analysis of CSF Homovanillic acid (HVA), 5-Hydroxyindoleacetic acid (5HIAA), 3-O-Methyldopa (3OMD), 5-Hydroxytryptophan (5HTP), Neopterin, Biopterin, Primapterin and 5-Methyltetrahydrofolate (5-MTHF).

Methods

CSF Neurotransmitter panel samples received between July 2015 and July 2017 were audited according to supplied clinical notes. Reference intervals for each of the neurotransmitters is known to vary according to age. For each of the neurotransmitters, obtained results were analysed according to the major categories of supplied clinical testing indications.

Results

There were 953 CSF Neurotransmitter requests with supplied clinical notes. The most common indication for testing was for investigation of possible encephalitis, followed by seizures, movement disorders, autoimmune disease and developmental delay. Where the testing indication was actual or suspected encephalitis, CSF neopterin levels were potentially significantly elevated in comparison to other indications.

Conclusions

CSF neurotransmitter levels stratified according to common clinical testing indications are presented.

P28: Ionised calcium reference intervals in paediatrics by data mining

JZY Chung1,2

1Department of Clinical Biochemistry, The Children’s Hospital at Westmead, Westmead NSW 2145, Australia2Sydney Medical School, The University of Sydney

Introduction

Paediatric reference intervals for ionised calcium are


desirable due to age-related changes in calcium status. We sought to develop suitable reference intervals for ionised calcium in the paediatric population using an indirect data mining approach.

Methods

Ionised calcium results from 2013-2017 were obtained from the laboratory information system. The laboratory provides services to a tertiary paediatric hospital. Ionised calcium measurement was performed by Siemens RapidLab 1265 or RapidPoint 500 instruments. Patients with repeat sampling were excluded. Age partitions were determined by visual inspection. Reference intervals were developed using Bhattacharya analysis. Expected flagging rates were calculated based on the developed reference intervals. Microsoft Excel was used for all data analysis.

Results

A total of 10,699 results for ionised calcium were obtained following exclusion of patients with repeat testing. The following reference intervals were obtained: Age 0 to <1 week (1.02–1.45) mmol/L, 1 week to <6 months (1.24–1.44) mmol/L, 6 months to <2 years (1.17–1.37) mmol/L, 2 years to 18 years (1.13–1.33) mmol/L. Application of the reference intervals to an unfiltered cohort is predicted to yield 28% low results and 3.2% high results.

Conclusions

Paediatric reference intervals for ionised calcium are presented.

P29: Calcium adjusted for albumin formulae in paediatrics

JZY Chung1,2


Introduction

Formulae for calcium adjusted for albumin (cCa) in adults are typically based on total calcium (tCa, mmol/L) and albumin (alb, g/L) with the general form cCa = tCa + A × (B –alb), where A reflects the slope of tCa vs. alb and B reflects the median alb. Application to paediatric populations is complicated by age-related changes in both albumin and the slope of tCa vs alb. We sought to develop and assess formulae for use in a paediatric population.

Methods

The laboratory provides services to a tertiary paediatric hospital. Results for iCa, and paired results for tCa and alb were obtained from the laboratory information system from 2016-2017. Samples were included if collection times were within 10 minutes. Total calcium and albumin (BCG) were measured on the Ortho Vitros 5600. Ionised calcium was measured on the Siemens RapidLab 1265

or RapidPoint 500. Unadjusted tCa was examined with three formulae: Payne (A = 0.02, B = 40), CHW (A = 0.02, B = 35 for age <2 years; A = 0.015, B = 40 for age ≥2 years), Empirical (A and B according to tCa vs albumin). Agreement of each formula with iCa was assessed using the Pearson correlation coefficient, Cohen’s kappa, mean absolute error, and root mean square deviation.

Results

The Payne, CHW and Empirical formulae vs. unadjusted tCa had respective Pearson correlation coefficients of 0.52, 0.61, 0.58 vs. 0.56; weighted Cohen’s kappa of 0.27, 0.37, 0.37 vs. 0.36; mean absolute error of 1.31, 1.04, 1.02 vs. 1.20; and root mean square deviation of 1.88, 1.61, 1.59 vs. 1.72.

Conclusions

The CHW formula better reflects calcium status than unadjusted tCa. Despite its popularity, using the Payne formula gives a worse reflection of calcium status than unadjusted tCa. Candidate formulae for harmonisation should be verified with data where possible.

P30: A simplified method for analysis of sterols and cholestanol in plasma by ultra performance convergence chromatography coupled with mass spectrometer

D M Cox1, L Foyn1, B C McWhinney1, J McGill1,2, SL Stoodley1, J P J Ungerer1

1Department of Chemical Pathology, Pathology Queensland, Royal Brisbane and Women’s Hospital, Queensland 4029, Australia2Chemical Pathology Division, Mater Pathology, Mater Hospital Brisbane, Queensland 4101, Australia

Introduction

Metabolic disorders of the biosynthesis and breakdown of complex molecules often show a slow progression of clinical symptoms and are less likely to cause metabolic crises. These disorders are not usually recognised by basic metabolic analysis and require specific investigations for their diagnosis. The two separate Gas Chromatography Mass Spectrometry (GC-MS) Methods in our laboratory for measuring 7-dehydrocholesterol (7DHC) and cholestanol involve lengthy and complex extractions, and are impacted by the limitations associated with using GC-MS technology. We have developed a simple method to measure sterols and cholestanol in plasma using Ultra Performance Convergence Chromatography coupled with Tandem Mass Spectrometry (UPC2-MS/MS).

Methods

Samples (50 μL) and ethanol containing deuterated internal standards (50 μL) are saponified using potassium hydroxide for 30 minutes at 80oC. Once cooled the samples are centrifuged for five minutes at 3000 rpm, the supernatant is transferred to an autosampler vial and 1


μL is injected onto the UPC2-MS/MS system. The analytes are separated using a BEH 2-ethylpyridine column with a total run time of seven minutes between injections.

Results

The analytes were linear in the range of 0 – 150 μmol/L with the exception of Cholesterol which was 0 – 10000 μmol/L. For each analyte, the coefficient of variation (%CV) was <7.7% (n=20) and <8.0% (n=12) for intra – and inter-run respectively. Results correlated well for 7DHC, Cholestanol and Cholesterol against our current GCMS and Beckman Methods (7DHC R2>0.99; Cholestanol R2>0.97; Cholesterol R2>0.95) and carryover was insignificant (<0.1%). The limit of quantification is suitable for clinical interpretation (Cholesterol <1.12 μmol/L; 7DHC <0.03 μmol/L; all other analytes <0.4 μmol/L).

Conclusions

This new Sterols and Cholestanol method increases laboratory efficiency and improves patient results. With the utilisation of UPC2 technology, the common chromatographic problems for samples possessing a wide range of polarities which require different assays has been overcome.

P31: Audit of procalcitonin retesting intervals in a tertiary paediatric hospital

C Cross,1 V Lodhia,1 NJ Perera,1 JZY Chung1,2


Introduction

Procalcitonin (PCT) is a peptide biomarker produced by many types of cells in the body, often in response to bacterial infections but also in response to tissue injury. The level of PCT in the blood is used as a guide in helping to decide whether antibiotics should be started or stopped for patients with lower respiratory infections and whether antibiotics can be discontinued in patients with sepsis. Elevated PCT features on some hospital sepsis pathway protocols. This increase is particularly marked in bacterial infection within 2-4 hours with maximal levels (up to 100 ng/L) within 12-24 hours. Effective antibiotic therapy is associated with rapid (within 24 hours) reduction in serum/plasma PCT.

The UK Association for Clinical Biochemistry 2013 report on minimum re-testing intervals recommends testing at most once every 24 hours.

Aim

The aim of this study was to determine the current frequency of PCT retesting that occurs within a 24 hour interval in a tertiary paediatric hospital.

Methods

PCT results from 1 January 2017 – 13 June 2018 were obtained and the data analysed for repeat testing. The retesting interval was assessed against the 24 hour recommendation. Data was also analysed according to the testing location.

Results

There were 3792 tests for PCT over the period examined, of which 1351 were repeat tests. There were 459 repeats within 24 hours of the previous result which constituted 34% of all PCT repeat testing.

Paediatric ICU was the largest requester of repeat PCT tests, comprising 1025 (76%) of total repeats. Of these, 381 (37%) were under the recommended 24 hour minimum retesting interval.

Conclusions

Over one third of repeat PCT testing occurred within 24 hours of the previous test, which demonstrates unnecessary retesting of PCT. Optimising retesting frequency can reduce reagent costs and scientist time whilst maintaining clinical value.

P32: Parathyroid hormone measurement: challenging the manufacturer’s stability claims

G Estoesta, D Tran, L Nguyen, MM Salib, C Farrell

Laverty Pathology, North Ryde, NSW 2113, Australia

Introduction

Siemens recently reformulated their Intact PTH assay for Centaur immunoassay systems, moving from polyclonal to monoclonal reagent antibodies. We took this opportunity to test the revised stability claims made by the manufacturer for this assay for samples stored at room temperature (8 hours for serum, 24 hours for EDTA plasma) and frozen (acceptable to freeze serum, but not plasma).

Methods

Serum (SST) and EDTA samples were collected from 10 volunteers. Room temperature stability was tested by comparing baseline results for each sample type to results from multiple time intervals up to 72 hours. Two acceptability criteria were used in assessing differences from baseline values: the desirable specification for bias based on biological variation and at least 90% of results remaining within RCPA QAP analytical performance specifications at each time-point. The acceptability of freezing samples was tested by comparing results before and after a single, overnight, freeze-thaw cycle.

Results

Mean PTH concentrations decreased with time in both serum and plasma samples left at room temperature (p < 0.01). Both acceptability criteria were met up to 8


hours for serum samples, and up to 72 hours for plasma samples. No significant difference was seen in PTH results after a single freeze-thaw cycle for both serum (p = 0.50) and plasma (p = 0.81).

Conclusions

We confirmed the manufacturer’s stability claim for serum samples, but demonstrated that EDTA plasma samples showed significantly greater stability than claimed. Laboratories should critically appraise the stability claims made by assay manufacturers and consider performing their own studies of sample stability.

P33: Development and validation of a hydrophilic interaction chromatography (hilic) UV method for the analysis of vitamin C in serum and plasma

M Fitzpatrick1, VL Nguyen1,2, P Bonnitcha1,2, J Chung1,3, D Sullivan1,2

1New South Health Pathology, Royal Prince Alfred Hospital, Australia2Sydney University, Camperdown, NSW. 2006, Australia3Childrens Hospital Westmead, Australia

Introduction

Ascorbic acid is an essential cofactor in a number of enzymatic reactions and has an important role in wound healing and prevention of scurvy. Accurate measurement of ascorbic acid is complicated by poor analyte stability as well as unsatisfactory retention and separation in reverse-phase HPLC systems. The analysis of ascorbic acid in plasma using Hydrophilic Interaction Chromatography (HILIC) which is more suitable for polar compounds has to our knowledge never before been reported. In this study, we aimed to develop a novel HILIC method for the measurement of Ascorbic acid in plasma.

Methods

Chromatographic conditions and detector settings were optimised after careful selection of analytical column and mobile phases. To extract ascorbic acid, plasma samples were diluted with an internal standard solution containing an antioxidant then passed through centrifugal protein removal columns. The collected filtrate was then diluted with acetonitrile prior to analysis. The method was validated to include recovery, precision, accuracy, linearity, limits of quantification, extract stability and assessment of common interferences. An analysis of RCPA-QAPs and patient samples (n=50) was undertaken to determine the method’s performance.

Results

Ascorbic acid was linear over an analytical range of 5-250 umol/L. The within batch imprecision was less than 3% and the between batch imprecision was less than 7%. Recovery was over 90% for the analyte and its internal standard. Negligible degradation was seen in the extracted

sample over a 48 hour period. This method displayed good agreement with RCPA-QAP medians and against a traditional reverse phase method in patient samples. No interference was encountered with compounds that commonly interfere with ascorbic acid methods.

Conclusions

We have developed a simple, robust method for the measurement of Ascorbic acid in plasma. This novel method has proven to be highly reproducible and accurate with enhanced sample stability.

P34: A protein precipitation LCMS-MS method for 11-NOR-9-CARBOXY-Δ9-tetrahydrocannabinol in urine

S Wilschefski, T Neville, T Smith, G Ward, R Flatman, L Price

Sullivan Nicolaides Pathology, Brisbane, Qld. 4006, Australia

Introduction

Urine drug testing for cannabis primarily targets the main urinary metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol. Most published Methods utilise a time consuming and labour intensive sample preparation, such as solid phase extraction (SPE), liquid-liquid extraction (LLE), or derivatisation, followed by GCMS or LCMS. A high throughput protein precipitation LCMS-MS method for the quantitation of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol has been developed and evaluated.

Methods

The method was evaluated using seventy authentic urine samples. Results were compared to a previously validated LCMS-MS method (an SPE method utilizing positive mode ESI).

Sample preparation involved hydrolysis of glucuronide esters with sodium hydroxide (50°C, 30min), and protein precipitation with methanol. After centrifugation, supernatant was mixed with buffered water 1:1, and injected onto a SciexTM 4500 Q TRAP mass spectrometer operated in negative electrospray mode. A PhenomenexTM Biphenyl column (50 x 3mm, 2.6µm) was used for the 3 minute gradient separation. Mobile phases consisted of formic acid in water (A) and acetonitrile (B).

Results

Protein precipitation method results for patients samples, and a full cycle of an external quality assurance program, compared well with the SPE method (Passing-Bablok y= 0.9887x – 1.055). LOQ is 2.5ng/mL (CV 4.4%, n=12); and LOD is 0.625ng/mL (CV 7.2%, n=12). The signal to noise ratio at the LOD is >150 for both the quantifier and qualifier transition. Matrix effects were minimal. Inter – and intra-run precision was <5% (coefficient of variation). The method was linear to 500ng/mL (R2 1.00).

Conclusions

The method is able to accurately and reliably measure


11-nor-9-carboxy-Δ9-tetrahydrocannabinol in urine, and shows excellent analytical sensitivity. The method is considerably less time consuming than traditional methods, and eliminates the need to use expensive SPE cartridges, and hazardous solvents such as hexane and ethyl acetate which are often used in SPE and LLE methods.

P35: A rapid UPLC-MS/MS analysis of 14 opiates and amphetamine type stimulants in urine

S Wilschefski, S Browning, T Smith, D Allen, O Letsatsi, T Neville, C Reyes, K Mantik, T Forster, N Pham, C Van De Vorstenbosch, M Menzies, G Ward, R Flatman, L Price, D Kanowski


Introduction

In a modern clinical toxicology laboratory, there is an emphasis on providing accurate results in a short turn-around time. Amphetamine type stimulants and opiates are commonly measured in toxicology samples. Many published LCMS and GCMS methods are time consuming, with extensive sample preparation, and extended chromatographic conditions to separate isobaric compounds. A rapid UPLC-MS/MS method for opiates and amphetamine type stimulants in urine has been developed. This method quantifies 14 commonly measured compounds without enzymatic or chemical glucuronide deconjugation.

Methods

The method was evaluated using two hundred authentic urine samples. Results were compared to a previously validated 6 minute LCMS/MS method. Sample preparation involved a simple protein precipitation with methanol and zinc sulfate. Drugs were separated using a reverse phase gradient elution on a PhenomenexTM Biphenyl column (50 x 3mm, 2.6µm). Mobile phases consisted of ammonium formate and formic acid in water (A) and methanol (B). Quantitation was performed on a SciexTM 4500 Q TRAP mass spectrometer operated in positive electrospray mode. The method gradient was designed to separate all isobars and matrix interferences within 2.5 minutes.

Results

Rapid UPLC-MS/MS results correlated well with the slower 6 minute method. Method LOQ ranged from 2.5ng/mL to 50ng/mL. 6-acetylmorphine was linear to 50ng/mL, all other compounds were linear to at least 400ng/mL. Matrix effects and analyte carryover were minimal. Intra-run precision was <5% CV for all analytes. Inter-run precision for pholcodine, pseudoephedrine, and codeine-6-glucuronide was <10% CV; all other compounds were <5% CV.

Conclusions

The method is able to accurately and reliably measure a large panel of common opiates and amphetamine type stimulants in urine, and shows excellent analytical

sensitivity and precision. The short run-time (2.5 minutes) and simple sample preparation allows for a high throughput, rapid analysis. This has significantly improved laboratory workflow and turn-around time.

P36: TDM of tuberculosis drugs by UPLC tandem mass spectrometry

L Foyn1, ET Connolly1, BC McWhinney1, JPJ Ungerer1, 3, A Burke2, 3 1Chemical Pathology, Pathology Queensland, Herston Hospitals Complex, Herston, Queensland 4029, Australia2Infectious diseases and Thoracic medicine, The Prince Charles’ Hospital, Chermside, Queensland 4032, Australia 3Faculty of Medicine, University of Queensland, Herston, Queensland 4029, Australia

Introduction

Mycobacterial lung infections including tuberculosis and non-tuberculosis mycobacteria (NTM) require treatment with multiple drugs for many months and often years. The development of novel mycobacterial drug assays will allow research into optimising the doses of these toxic and expensive drugs as well as creating future opportunities for therapeutic drug monitoring (TDM) to individualise patient treatment. We have developed two assays for the simultaneous detection of twelve drugs using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The drugs analysed were Azithromycin, Bedaquiline, Cefoxitin, Clarithromycin, Clofazimine, Ethambutol, Imipenem, Isoniazid, Linezolid, Pyrazinamide, Rifampicin and Tigecycline.

Methods

Plasma/serum (50µL) was mixed with methanol, containing deuterated internal standards and centrifuged for 10 minutes. The supernatant was injected (1µL) onto the UPLC-MS/MS system and separated on an amide column (for hydrophilic drugs) or BEH column (for lipophilic drugs). Both assays have a run time of 4 minutes between injections.

Results

Each drug has its own clinically relevant analytical range, up to 500 mg/L. The coefficient of variation (%CV) was 1.1% – 9.3% and 1.5 – 9.4% for intra – and inter-run respectively across 3 concentrations. Recovery for specific analytes was between 97% and 103%. No interferences were found when extracting plasma samples containing various other drugs. All drugs were stable post extraction for 24 hours except Imipenem, which was stable for 2 hours.

Conclusions

The developed methods exhibit excellent analytical range, with good analytical sensitivity and specificity. The data collected during the validation of these two assays demonstrates its appropriateness to be a routine


assay. We believe these TB drug assays will serve to further our knowledge of their pharmacokinetics and pharmacodynamics.

P37: Measurement of salivary biomarkers in patients with congenital adrenal hyperplasia

L Foyn1, BC McWhinney1, T Huynh1,2,3, JPJ Ungerer1,3

1 Chemical pathology, pathology Queensland, Herston Hospitals Complex, Brisbane, Queensland 4029, Australia2 Department of Endocrinology and Diabetes, Lady Cilento Children’s Hospital, South Brisbane, Queensland, Australia3 Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia

Introduction

Measurement of free/bioavailable 17-hydroxyprogesterone (17OHP), androstenedione, cortisol and testosterone in saliva is thought to provide a better means of monitoring patients with Congenital Adrenal Hyperplasia (CAH). Currently monitoring patients with CAH at timed intervals are performed using dried blood spots (DBS) and only to assess 17OHP levels. This sample type, however, is prone to erroneous or unusable samples. We have developed a new salivary steroid method using an integrated on-line Solid Phase Extraction (SPE) system coupled to a UPLC-MS/MS, providing paediatric patients with a less-invasive sample collection.

Methods

Saliva samples were centrifuged and extracted by using 200 µL of sample and adding 100 µL of methanol containing deuterated internal standards. The supernatants were purified by automated online SPE coupled to a UPLC-MS/MS chromatographic system. Analytes of interest were separated using a BEH C18 1.7 µm 2.1x50 mm column with a run time of 3 minutes between injections.

Results

The analytical range for the assay was 0 – 2500 pmol/L for 17OHP, androstenedione and testosterone and 0 – 50 nmol/L for cortisol. The limit of the blank ranged from 2.5 pmol/L for 17OHP to 0.025 nmol/L for cortisol. The intra-run precision of the assay across 3 concentrations was 1.1 – 9.4% and inter-run precision across 2 concentrations was 1.7 – 9.5%. The final extracted samples were stable for 48 hours at 15°C in the autosampler.

Conclusions

This assay is efficient in the measurement of salivary 17OHP, cortisol, androstenedione and testosterone and displays excellent sensitivity over an appropriate analytical range. The development and implementation of this assay will improve CAH patient compliance and monitoring by utilising a simplified sample collection technique.

P38: Investigation of serum, salivary and DBS biomarkers in the monitoring of glucocorticoid therapy in children with congenital adrenal hyperplasia

L Foyn1, BC. McWhinney1, PP. Tanchi2,5, U Visser3, CF. Verge4, T Lafferty3, S Reakes3, C Pretorius1,6, JPJ. Ungerer1,6, T Huynh1,5,6

1Department of Chemical Pathology, Pathology Queensland, Herston, Queensland, Australia2Department of Paediatrics, Lady Cilento Children’s Hospital, South Brisbane, Queensland, Australia3Department of Paediatrics, The Canberra Hospital, Woden, Australian Capital Territory, Australia4Department of Endocrinology and Diabetes, Sydney Children’s Hospital, Randwick, New South Wales, Australia5Department of Endocrinology and Diabetes, Lady Cilento Children’s Hospital, South Brisbane, Queensland, Australia6Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia

Introduction

In congenital adrenal hyperplasia (CAH) treatment monitoring, there is a lack of consensus regarding the most appropriate biomarker(s) and matrices, the value of daily profiling, and whether it offers any additional advantages over routine clinical assessment. Advancements in Mass Spectrometry-based methods allow measurement of multiple steroids within a single sample and in matrices other than serum/plasma – including dried blood spots (DBS) and saliva. Also, recent developments have made the quantitation of free steroid hormones less laborious.

Our aim was to assess to the correlation between 17-hydroxyprogesterone (17OHP), androstenedione, cortisol and testosterone in individual serum, DBS and saliva matrices. Provided suitable data existed, our study would progress to evaluate the correlation of these steroid levels in children with CAH, versus unaffected controls (siblings).

Methods

These steroids were extracted across all matrices using protein precipitation with labelled internal standards in methanol containing zinc sulphate. Supernatant was injected and analysed using Ultra Performance Liquid Chromatography Mass Spectrometry (UPLC-MS/MS). Trial patient samples included a morning serum (day 1, 0800) and daily (Day 1 0800, 1200, 1800, 2200 and day 2 0800) DBS and saliva samples. 17OHP, androstenedione, cortisol and testosterone were quantitated for each of the biological matrices.

Results

Twenty children with CAH and 15 controls were clinically assessed based on set criteria developed by the paediatric endocrinology team. All patients showed excellent correlation between the four steroid hormones in both serum and DBS, and preliminary data for salivary levels are


also supportive.

Conclusions

This research trial has proven that across all matrices, steroid hormone levels can be assessed accurately. The study will continue to assess the value of daily profiling and its potential additional advantages over routine clinical assessment.

P39: Blood lead levels in preeclampsia: a pilot study

R Ghosh, D Golmes, PVSN K Kumar, P Mitra, S Sharma, M Goyal, Praveen Sharma

All India Institute of Medical Sciences, Jodhpur-342005, Rajasthan, India

Introduction

Lead is a toxic heavy metal which accumulates in the body over long term and can result in maternal complications including preeclampsia. There is generation of reactive oxygen species leading to oxidative stress and in turn preeclampsia. Role of lead in pre-eclampsia is not explored in the Indian population. This study aims to find the blood lead levels in pregnant women and its association with preeclampsia.

Methods

The study included 44 healthy pregnant females and 23 pre-eclamptic women. Demographic data and common risk factors for lead toxicity were recorded including age, residence, occupation, husband occupation, passive smoking, use of cosmetics, kajal, surma, receiving supplements/vitamins, history of house remodelling, plumbing, source of potable water, paint in house, use of lead-glazed ceramic and pica. Venous blood was collected and lead level was determined using Atomic Absorption Spectrometry–Graphite Furnace following standard protocols.

Results

The mean blood lead level was 2.38± 2.43 ug/dL in controls and 3.42± 2.18 ug/dL in pre-eclamptic women which was significantly higher (p=0.0132). Significant correlation of BLL was observed with blood pressure in pre-eclamptic women. Preeclamptic patients were observed to be at increased risk of being lead exposed in terms of occupation and living conditions.

Conclusions

As per the findings of this study, higher blood lead level is significantly associated with increased risk of preeclampsia. Patients should be counselled for lifestyle modification to prevent complications.

P40: Evaluation of 25-hydroxyvitamin D assay using Beckman Coulter access II analyser

N Josman, SK Loh, WS Setoh, CKM Ho

KK Women’s & Children’s Hospital, Singapore 229899

Introduction

Vitamin D is a fat soluble vitamin that plays an important role in regulating the absorption of phosphorous and calcium and in bone health. It also has an important role in cell growth and differentiation. Vitamin D deficiency is considered as a global pandemic and it affects roughly 40% of Singapore residents.

In this study, we aimed to study the performance of the 25-hydroxyvitamin D assay on Beckman Coulter Access II analyser.

Methods

We evaluated inter-day imprecision, linearity, limit of quantitation (LoQ) of the assay and its performance in two external quality assessment (EQA) schemes.

Results

Inter-day imprecision, CV (%) over 10 days was 6.8% at 18.7 ug/L (46.7 nmol/L) , 7.9% at 42.7 ug/L (106.6 nmol/L) and 6.3% at 116 ug/L (289.5 nmol/L). The assay demonstrated linearity for measurement between 6.0 to 164.0 ug/L (15.0 to 409.3 nmol/L). Limit of quantitation (LOQ) was found to be 5.7 ug/L (14.2 nmol/L) with CV = 7.8%.

EQA results were comparable with peers participating in the College of American Pathologist and DEQAS proficiency testing.

Conclusions

We have evaluated the performance (imprecision, linearity and LOQ) of the 25-hydroxyvitamin D assay on Beckman Coulter Access II analyser. Assay results were found to be satisfactory compared to peer group in both CAP and DEQAS programmes.

P41: Correlation studies of the Roche AMH plus and Beckman access AMH assays – consistent findings over a four year interval

D Kanowski, B Teis, J Smith, K Young, J Nicolson, S Hunter, R Flatman, G Ward, L Price


Introduction

With the advent of an automated immunoassay for Anti-Mullerian hormone by Roche (the Roche AMH Plus assay) and Beckman (the Beckman Access assay), there have been a number of correlation studies in the literature. A range of correlation values ranging from 0.81 to 1.03 (Roche against Beckman) have been reported. Our laboratory performed a correlation study in 2014 and again in 2018 and obtained consistent correlation values over the two studies.

Methods


In September 2014 we performed an AMH correlation study on 133 samples. The Roche AMH was assayed on the E170 platform and was compared with the Beckman Gen II AMH assay. A correlation of the Beckman Access AMH to the Beckman Gen II AMH was also performed.

In April 2018 the correlation was done again, with the Roche AMH Plus assay on the E602 platform compared with the Beckman AMH Access assay; 110 samples were compared.

Results

In 2014, the following Passing and Bablok correlations were obtained:

• Roche against Beckman Gen II y = 1.23+0.74 x

• Beckman Access against Beckman Gen II y = 1.35+0.92 x

This gave a Roche AMH against Beckman Access correlation of y = – 0.237+0.804 x.

In 2018, the correlation of the Roche AMH on the e602 against Beckman Access AMH yielded y = 0.226+0.793 x

Conclusions

Our correlation studies showed a consistent correlation of 0.80 (Roche against Access). This agrees well with the figure of 0.81 quoted by Roche. It is critical the laboratories maintain consistent results for patient samples over changes in reagent batches. Procedures employed by our laboratory to maintain consistency include:

Maintain internal QC limits to achieve an inter-assay CV of <5%

Checking a range of patient samples with both old and new reagent batches.

Monitoring patient means over time.

P42: Advancing the analysis of plasma free beta-lactam antibiotics utilising the qda mass detector

T Kuhn, B Walker, A Wilce, R Swenson, BC McWhinney, JPJ Ungerer

Chemical Pathology, Pathology Queensland, Herston Hospitals Complex, Brisbane, Qld 4029, Australia

Introduction

Pharmacological efficacy of beta-lactam antibiotics is dependent on the concentration of unbound (free) drug and the duration of exposure to the infection. Maximum efficacy occurs at free concentrations which are ~3-4 times the minimum inhibitory concentration (MIC). Therapeutic drug monitoring of beta-lactams is required to maintain optimal dosage for critically ill patients. Previously, 10 beta-lactams were measured by High Performance Liquid Chromatography coupled to a UV detector (HPLC-UV), and assayed in five groups. We have developed a method which simultaneously detects 14 beta-lactam antibiotics

using Ultra Performance Liquid Chromatography (UPLC) coupled to the Waters QDa mass detector.

Methods

The plasma ultrafiltrate was collected after spinning through a 30K molecular weight cut-off filter. MES Buffer containing labelled internal standards (60µL) was added to the ultrafiltrate (50µL). A 0.1µL aliquot was injected, the analytes separated using reverse-phase chromatography and detected using the QDa mass detector.

Results

The analytes were linear over the range of, 0.1-10mg/L for Ceftriaxone, 0.1-20mg/L for Cefepime, 0.1-25mg/L for Ceftazidime and Ceftolozane, and 0.1-50mg/L for all other analytes. For each analyte, the coefficient of variation (%CV) was <10% (n=10) for intra – and inter-run precision. No interferences or ion suppression issues have been seen across any patient samples. Correlation with the previous HPLC-UV method was excellent (r2>0.96).

Conclusions

We have developed a simple UPLC-QDa assay that measures Cefepime, Ceftazidime, Ceftolozane, Benzylpenicillin, Cephalothin, Ampicillin, Amoxicillin, Meropenem, Flucloxacillin, Cephazolin, Ertapenem, Piperacillin, Ceftriaxone and Tazobactam in biological samples. It has excellent precision, sensitivity and specificity. Implementation of the UPLC-QDa method has improved laboratory efficiency, significantly reduced turn around times and improved patient management.

P43: Eastern health experience of the freelite serum free light chain assays

C-F Lim, C Cocotsi, A O’Neill, A Sutton, A Connell

Box Hill Hospital, Box Hill, Victoria, 3128, Australia

Introduction

Measurement of serum Free Light Chains (sFLC) is intended for the diagnosis and monitoring of treatment of plasma cells disorders such as multiple myeloma, primary amyloidosis and related diseases.

Of the 44 laboratories enrolled in the RCPA QAP for sFLC, only three laboratories were using the c501/c502 analyser platform. In this study, we evaluated the serum Freelite assays on the Roche c502 analyser.

Methods

Freelite Human Kappa Free (Product Code: LK016.CB) and Lambda Free (Product Code: LK018.CB) assay kits were evaluated using the c502 of Cobas 8000 (Roche Diagnostics). Reference method was the Binding Site SPA PLUS assay.

Results

A total of 48 (KFLC) and 41 (LFLC) were analysed using the


reference assay (SPA PLUS) and the test assay (c502). A good agreement between the two assays was obtained (KFLC y=1.0432x+1.0035, R² = 0.998 and LFLC y=1.1193x+2.8437, R² = 0.9926). The interassay precision (%CV; n =42) of the assay was KFLC 4.3% and 3.95% at 17.8 mg/L and 36.2 mg/L, respectively. For LFLC, the interassay precision was 3.1% and 3.4% at 25.5 mg/L and 53.2 mg/L, respectively. As an ongoing result validation, two patients with very high KFLC concentrations were referred to another laboratory using the SPA PLUS assay. Comparable results were obtained between these two assays (c502: 1510 and 2452 mg/L; SPA Plus: 1347 and 2448 mg/L, respectively).

Conclusions

The Freelite assay can be readily adapted to the Roche c502. The assay is precise and easy to use in an automated Biochemistry laboratory. By bringing the assays in-house, we are able to expedite result turn-around for better patient management.

P44: Evaluation of body fluids on the Roche Cobas 8000 platform

Madiha Damlakhi¹, Ailie Connell², C Cocotsi², A. O’Neill², A Sutton², C-F Lim²

¹ RMIT University, Bundoora, Victoria, 3083, Australia

² Box Hill Hospital, Box Hill, Victoria, 3128, Australia

Introduction

Serum, plasma, urine and CSF are the standard body fluids validated for routine use on the Roche Cobas 8000 system. In this study, we evaluated the suitability of ascitic (AS), pleural (PL) and peritoneal dialysis (PD) fluids for the measurement of routine biochemistry analytes.

Methods

Roche c702, c502 and e602 analysers were used for the analysis. A total of 30 Biochemistry analytes (including albumin, total bilirubin (TBIL), conjugated bilirubin (DBIL), tumour markers, sodium and potassium) were measured using AS, PL and PD fluids (n=4). Accuracy was assessed by mixing the fluids with increasing amount of serum (0, 50, 75 and 87.5%). The stability of the analytes in each fluid type (n=1) was measured over a period of 8 days (Day 0, 2, 5 and 8). Replicate measurements (n=5) of each analyte was performed on Day 0 using the same fluid types.

Results

Accuracy study indicated that the calculated concentration of the 30 analytes was within a predefined ±10% limit of the neat fluid concentrations. Relative to Day 0, the percent recovery on Day 8 was ≥92% for AS and PL fluids and ≥91% for PD fluid. Only 3 of the 30 analytes showed less than 90% recovery from day 0. These analytes were TBIL, DBIL and LDH. Repeatability evaluation indicated that the measurement of the analytes was highly reproducible (%CV ≤6.6% for all analytes tested; n≥4).

Conclusions

The close agreement between the calculated and the neat analyte concentrations indicated that AS, PL and PD fluids can be used for routine Biochemistry analysis. The analytes in these fluid types were stable and the measurement was highly reproducible. This evaluation indicated that there was no matrix interference in these fluid types for the measurement of routine Biochemistry analytes.

P45: The determination of serum vitamin K1 concentrations in the new zealand population

CJ McEntyre1, DJ Card2, CW Sies1.1Canterbury Health Laboratories, Christchurch, Canterbury 8140, New Zealand2KEQAS, Nutristasis Unit, Viapath, St. Thomas’ Hospital, London, UK

Introduction

Vitamin K is required for the synthesis of vitamin K dependent proteins (Gla proteins). Gla proteins are essential for blood coagulation, bone mineralisation and regulation of vascular calcification. Vitamin K deficiency increases the risk of chronic diseases such as osteoporosis and vascular calcification and acute, potentially fatal bleeding complications, particularly in patients anticoagulated with vitamin K antagonists e.g. warfarin. Measurement of serum phylloquinone (vitamin K1) concentrations is an established biomarker of vitamin K status. Canterbury Health Laboratories have recently upgraded from HPLC-fluorescence to a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for vitamin K1 determination.

Methods

The LC-MS/MS method utilises calibrators and controls made up in calf serum, spiked with stock solutions determined using UV spectrophotometry. The sample volume has been reduced from 1 mL to 0.35 mL. Results from the LC-MS/MS method were compared with ALTM values from the vitamin K external quality assurance scheme (KEQAS). Patient vitamin K1 results from the previous 5 years were reviewed, with low values (<0.09 µg/L) investigated clinically to identify the likely cause of vitamin K deficiency.

Results

The LC-MS/MS results (n = 10) correlate well with KEQAS ALTM values (Passing-Bablok regression slope = 0.991, r2 = 0.986) and KEQAS assessment criteria indicate satisfactory performance. Vitamin K deficiency was indicated in 1.8% of samples measured. Some of the likely clinical reasons for vitamin K deficiency include: cystic fibrosis (n=3); bariatric surgery (n=3); old age (>70 years) (n=4); Crohn’s disease (n=1), pancreatitis, cholangitis, liver, and kidney disease.

Conclusions


LC-MS/MS provides an improved methodology for vitamin K1 determination. Vitamin K deficiency was associated with conditions that affect vitamin K absorption or inadequate intake. Determination of serum vitamin K1 enables identification and treatment of vitamin K deficiency and screening of high risk groups, e.g. cystic fibrosis is likely to be of benefit.

P46: Pre-analytical acidification for urine calcium and phosphate – is it necessary?

Morrow C, Flatman R, Kanowski D, Ward G, Price L.

Biochemistry Department, Sullivan Nicolaides Pathology, 24 Hurworth St Bowen Hills, Brisbane QLD

Introduction

Pre-analytical acidification of samples for urine calcium and phosphate analysis is widely recommended to prevent falsely decreased values from precipitated salts. The aim of this study was to investigate the commonly accepted benefit of the acidification process.

Methods

Urine calcium and phosphate were measured on 97 patient samples that had been referred to the laboratory for routine testing. Each sample was analysed at four time points: Pre-acidification, 30 min, 60 min, and 120 min post acidification. A final pH of ≤2.0 was achieved by drop-wise addition of 6 mol/L HCl using pH indicator strips (MColorpHast 5.0-10.0 and 0-6.0). The samples were assayed on an Abbott Architect C16000. Pre and post-acidification pH values were recorded for all samples.

Results

Two distinct groups of results were noted. The majority of samples showed a small change (<12%) in results (Calcium: n=91/97, % change – 10.8% to +1.8%, median change – 4.8%. Phosphate: n=95/97, % change – 10.2% to 11.8%, median change – 1.9%).

The remaining samples showed a significant change (>25%) in results (Calcium: n=6/97, % change – 28.8% to 168.4%, median change 55.9%. Phosphate: n=2/97, % change – 28.1% to – 48.6%, median change 38.4%). All of these samples were noted to have pre-acidification pH values of ≥8.0.

The individual change in results from 30 min to 120 min post acidification was noted to be <10% for all patients (Calcium: – 5.2% to 9.0%, Phosphate: – 6.5% to 4.1%).

Discussion

This study demonstrated that acidification is beneficial for a small percentage (<6%) of patients with an initial pH of 8.0 or greater (n=10/97). Studies with a small sample size may easily miss this group of patients that benefit from acidification.

We recommend a contingent approach to acidification. If pH

is <8.0 acidification is not required. If pH is ≥8.0 acidification is required prior to urine calcium and phosphate analysis.

P47: Glucose stability pre and post centrifiguation in various tube types

B Fitzgerald1, S Newton1, MM Salib1 , PE Hickman1,2

1ACT Pathology Canberra ACT 2605 Australia2Australian National University Medical School, Canberra, ACT 2600 Australia

Introduction

At ACT Pathology FE NaFl / K-EDTA tubes are used for glucose tolerance tests (GTTs). Not all centres had centrifuges, resulting large variations in the time to centrifuge post collection. We investigated the effect of sample type and time to centrifugation on the glucose concentration.

Methods

Multiple blood samples were collected into BD PST; BD Barricor lithium heparin, BD rapid clot serum (RST), Greiner FE NaFl / K-EDTA, BD citrate and Greiner FC Mix (citrate/NaFl) tubes centrifuged at varying times post-collection and subsequently assayed at various times for glucose on the Abbott Architect c16000 analyser.

Results

The glycolysis rate is fastest within the first 60 mins post collection across all tube types, with the exception of the Greiner FE NaFl / K-EDTA tubes, which demonstrated no decrease up to 2 hours post collection. The glycolysis rate stabilizes after 60mins in all other tubes. All tubes types demonstrated a stable glucose concentration for up to 6 hours post centrifugation.

Conclusions

When centrifuged promptly, all tube types described are suitable for glucose analysis. The Greiner FC Mix (citrate/NaFl) tube is the only suitable tube type when centrifugation is not possible immediately post collection. ACT Pathology implemented a policy for all GTT NaFl collections to be centrifuged within 10 minutes of collection.

P48: Evaluation of POCT creatinine devices

B Fitzgerald, J Farquhar, S Newton

ACT Pathology Canberra ACT 2605 Australia

Introduction

POCT creatinine devices are often used in radiology departments to assess a patient’s suitability for radiological investigation. We compared two POCT devices against the laboratory enzymatic method.

Methods

Fourty-eight-old patient whole blood specimens were obtained and analysed for creatinine on the Nova Biomedical Creatinine Meter and Abbott i-STAT. The plasma was simultaneously analysed on the Abbott Architect C1600


reference enzymatic assay. The analytical range assessed was 60 – 852 umol/L. Precision was assessed using two patient samples analysed 20 times on each device.

Results

Linear regression equations for the Nova and i-STAT were y = 1.1119x + 15.771, R² = 0.8001 and y = 0.979x + 4.4356 R² = 0.9836, respectively. RCPA analytical performance specifications (APS) (±8 up to 100; ±8 % >100 umol/L) were used to determine acceptability against the laboratory enzymatic method. 15%, 81% and 85% of results were within APS for the Nova, i-STAT and Abbott Jaffe method, respectively. Of the 41 Nova results outside APS, 12 were positively biased and 29 were negatively biased. Nova Precision at 109 and 354 umol/L was 5.5% and 6.5% respectively. When compared against the Architect enzymatic reference value of 74 and 213 umol/L there is a positive bias of greater than 4 and 8 APS, respectively. The i-STAT precision at 78 and 191 umol/L (Architect reference value = 77 and 185 umol/L) was 1.7% and 2.8% respectively.

Conclusions

The Abbott i-STAT show excellent agreement with Architect Enzymatic creatinine method. The Nova Biomedical creatinine meter demonstrates a large degree of positive and negative scatter at concentrations up to approximately 300umol/L. For concentrations greater 300 umol/L there is a strong negative bias. While precision is acceptable for the Nova meter, results cannot be compared against the Abbott Architect enzymatic assay.

P49: Performance evaluation of ALPHA-FETOPROTEIN (AFP), carcinoembryonic antigen (CEA), total prostate specific antigen (TPSA) and free PSA (fPSA) on the Abbott Alinity i analyser

K Nguyen, N Jenkins, J. D’Agostino, HG Schneider

Clinical Biochemistry Unit, Alfred Pathology Service, Melbourne, Australia

Introduction

The Abbott Alinity i Immunoassay analyser will replace the Architect i2000. It uses innovative design to maximize efficiency and specimen throughput and continues with the same chemiluminescent detection technology as its predecessor.

The aim of this study was to evaluate the performance of the Alinity i analyser using 4 commonly performed tumour markers (AFP, CEA, TPSA and fPSA). We assessed (i) assay correlation between the Abbott Alinity i analyser and the Abbott Architect ci16200; (ii) within run and between run precision (iii) assay stability.

Methods

Patient serum or plasma (lithium heparin) samples (n=40) representing the assay linear range, were analysed for

tumour markers on both the Abbott Architect ci16200 and the Alinity i analyser. These samples were analysed over a number of days and across several calibrations. Within-run assay precision was determined using 3 levels of commercial Human Lyophilised Quality Control (QC) materials and 2 patient pools. These samples were analysed in duplicate at 2 different times per day, over 5 days (n=80). Between-run assay precision was determined using the same QC materials and patient pools (n=50) Statistical analysis was performed using Analyse-It.

Results

Linear regression equations for AFP, CEA, TPSA and fPSA were; y =0.9338x+1.716, y= 1.0458x-0.8876, y=0.9681x+0.4741 and y=1.0856x-0.0173, respectively. The correlation coefficient for all four tumour markers was R2=0.99

Within-run precision data for all QC and patient pools showed CV’s <5% for AFP, CEA, TPSA and fPSA.

Between-run precision data for AFP, CEA and TPSA were all <5%. fPSA gave CV= 5.5%.

These findings were in agreement with the Abbott Alinity reagent kit inserts.

Conclusions

Representative clinical immunoassays utilizing chemiluminescent technology tested on the Abbott Alinity i analyser demonstrated acceptable linearity and precision. There was good correlation agreement between the Abbott Alinity i analyser and the Abbott Architect i16200 for AFP, CEA, TPSA and fPSA.

P50: Creatine kinase isoenzymes: when is testing indicated?

K Nguyen, C Farrell, MM Salib

Laverty Pathology, North Ryde, NSW, 2113, Australia

Introduction

The cause for an elevated Creatine Kinase (CK) activity is usually apparent from a patient’s history or physical examination. Creatine Kinase Isoenzyme (CKI) analysis may be requested as part of the investigation or follow-up of an elevated CK activity.

Methods

Data for all CKI requests referred to a private laboratory in Sydney, Australia from Jan 2013 to March 2018 were reviewed. CKI analysis was performed by gel electrophoresis using the Sebia system, with the percentage fractions of CKMM, CKMB, CKBB and MacroCK (if present) reported.

Results

During the 63 month period, there were 707 requests for CKI analysis. 372 (53%) were not performed as CK activity was within the age and gender stratified reference interval. 335 samples were analysed on patients aged 5-90 years


(median age 53 years). Sixty-four percent were male and CK activity ranged from 213 to 121520 U/L (median 488 U/L). 328 (98%) patients had predominantly CKMM (four patients with trace CKBB), one patient had a borderline high CKMB fraction, and six patients had a significant proportion of Type 1 MacroCK. Patients with a Type 1 MacroCK were aged 44-87 years, five of six were female, CK activity ranged from 213-869 U/L and the proportion of MacroCK ranged from 12-92%.

Conclusions

Most requests for CKI analysis were not indicated, likely as they were requested as follow-up after resolution of an acute skeletal muscle injury. Type 1 MacroCK is an uncommon cause for a CK elevation but important to identify to avoid unnecessary further investigations. Testing for MacroCK with CKI analysis should particularly be considered in older women with a persistent mild elevation in CK.

P51: Ganciclovir and valganciclovir in plasma by liquid chromatography mass spectrometry

T. Novos, D. Wong, W. Rawlinson, AR.Horvath

Prince of Wales Hospital, NSW Health Pathology, Randwick 2031, NSW, Australia

Introduction

Cytomegalovirus (CMV) infection is a significant cause of morbidity and mortality in solid organ transplant (SOT) recipients. Within four months after transplantation, at least two-thirds of patients develop infection either through transplant or reactivation of latent virus. The antivirals Ganciclovir and the oral prodrug Valganciclovir are the mainstays for the pharmacological management of CMV infection. Despite pharmacological control options, 0.8% of all SOT recipients and 1.2% of D+/R – (seropositive donor) recipients develop CMV infection during Valganciclovir prophylaxis. Given the low, but significant numbers of transplant recipients failing therapy, antiviral therapeutic drug monitoring is now needed.

We aimed to develop a sensitive liquid-chromatography mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of Ganciclovir and Valganciclovir in plasma, for monitoring treatment in SOT recipients.

Methods

Deuterated internal standards were added to EDTA plasma prior to a protein precipitation. The samples were centrifuged at 13,000g and the supernatant removed and analysed. Valganciclovir and Ganciclovir were then separated by HILIC chromatography, using a Luna silica column. Gradient conditions of 5mM ammonium formate in 90% Methanol/5mM Ammonium formate in 90% Acetonitrile were applied before detection on a SCIEX

API 5500 using electrospray ionization in positive mode. Sample to sample run time was five minutes.

Results

This method is linear up to 20.0 mg/L for Ganciclovir and the lower limit of quantification (LLOQ) is 0.05 mg/L (CV 20%). Between batch precision was: CV 24% (QC1:0.05mg/L), CV 11.5% (QC2: 2mg/L), CV 6.4% (QC3: 20mg/L). Robustness between analysts was 23%, 7.7% and 1.2% at QC 1,2 & 3, respectively.

Conclusions

This method detects and quantifies Ganciclovir and Valganciclovir concentrations in plasma. It can be used to monitor drug inter-individual variability of SOT patients and will provide physicians with more reliable dose-response drug information.

P52: Performance of the Beckman Coulter HBA1C turbidimetric immunoinhibition method with normal haemoglobin and haemoglobin variants

V Papasimeon

Monash Pathology, Dandenong Hospital, David Street Dandenong

Introduction

In our laboratory Hba1C is performed on the ADAMS ARKRAY-6180 using cation exchange HPLC. In the presence of some Hb variants, the samples are sent to a reference laboratory. We investigated a new method (Beckman Coulter AU680) to assess its accuracy, in comparison to the current assay and the reference method (Trinity Biotech Premier Hb9210).

Methods

We analysed 60 EDTA anticoagulated whole blood samples (frozen at – 80 C) from patients with normal haemoglobin (HbA1c 5.2%-18.4%) and 65 samples from patients with Hb variants, Hb S, D, E, A2 (HbA1c 4.3%-19.6%) on 3 analysers. The ADAMS ARKRAY HA-6180, the new Beckman Coulter AU680 method, using turbidimetric immunoinhibition, and the Trinity Biotech premier Hb9210, using boronate affinity HPLC (the reference method).

Results

Passing Bablok regression was used to compare the assays. The ARKRAY and Beckman Coulter Methods correlated well, with intercept A with 95% Cl: – 0.3750 to 0.06034 and slope B with 95% Cl: 0.9655 to 1.0263 on the normal Hb patients and with P=0.68.

Hb variant samples: The correlation between the AU680 and the ADAMS ARKRAY HA-6180, showed good correlation with intercept A (95% Cl) of 0.2000 to 1.3373 and Slope B (95% Cl) 0.8182 to 1.000 and P=0.28.

The correlation between the AU680 and the Trinity Biotech Premier Hb9210 was also good with intercept A (95% Cl)


of – 0.8452 to – 0.1000 and Slope B (95% Cl) – 0.4048 to 0.4048 and P=0.47.

Conclusions

The Beckman Coulter AU680 HbA1C assay correlated well with both the ARKRAY HA-6180 and the Trinity Biotech Premier Hb9210 for both groups of patients, with normal and abnormal haemoglobins. The method could be implemented in our laboratory, pending cost efficiency and investigation of a broader range Hb variants.

P53: Stability of disaccharidases in intestinal biopsy samples

G Pool, M Baxter, D Lal, G Ward, R Flatman, D Kanowski, L Price

Sullivan Nicolaides Pathology, Bowen Hills, 4006, QLD, Australia

Introduction

Measurement of disaccharidase activity in intestinal biopsies is useful in the diagnosis of single or multiple disaccharidase deficiencies. Laboratories expect samples to be transported frozen. On occasion samples are received at fridge (4ºC) or room temperature. According to laboratory protocols, these samples should be rejected. In this study we investigated the stability of disaccharidases at both 4 degrees (1 day stability) and room temperature (1, 3 and 6 day stability).

Methods

Frozen biopsy samples from four sets of 20 patients were analysed for disaccharidases and protein. An extra piece of tissue from each of the 20 patient samples in each set was stored at 4ºC for 1 day and room temperature for 1, 3 and 6 days respectively, then analysed. Protein concentrations (g/L) were determined by a benzethonium chloride turbidimetric method, and lactase, maltase and sucrose activities were determined with established in-house methods, all on a Thermofisher Konelab 30 analyser. Disaccharidase activities are measured in U/L but reported as U/g protein.

Results

Biopsy sample size varies unpredictably, therefore average results across the 20 sample pairs were used to detect changes in protein and enzyme activity. Biopsy samples incubated at 4ºC for 1 day showed an 8% reduction in average protein result, and <4% reduction in average disaccharidase activity (U/g).

Samples pairs at room temperature showed the following changes in average results:

D a y s RT

Protein g/L Lactase

U/L

Lactase

U/g

Sucrase

U/L

1 -14.7% -10.6% +13.2% +9.6%

3 -42.5% -34.3% +20.4% -27.8%

6 -49.6% -44.1% +10.8% -20.5%

D a y s RT

Sucrase

U/g

Maltase

U/L

Maltase

U/g

1 +27.2% +6.1% +22.0%

3 +39.7% -27.8% +38.5%

6 +58.5% -18.9% +62.0%

Conclusions

Intestinal disaccharidase activity appears to be remarkably stable for up to 24hrs at 4 degrees. Samples left at room temperature for any period show significant deterioration and are not suitable for testing.

P54: Urine drugs of abuse screening on the Abbott alinity

JD Pope 1,2, N Jenkins1, MJ Black 1, HG Schneider 1,3

1 Clinical Biochemistry Unit, Alfred Pathology Service, Alfred Health, Melbourne, Australia2 Department of Forensic Medicine, Monash University, Australia3 School of Public Health and Preventative Medicine, Monash University, Australia

Introduction

Drugs of abuse (DOA) screening by immunoassay is typically the first line of investigation in toxicology testing, and can provide important clinical information in the hospital setting. We evaluate the performance of six DOA cloned-enzyme donor immunoassays (CEDIA®) on Abbott’s Alinity ci series analyser.

Methods

Six CEDIA® screening tests (Amphetamines/Ecstasy, barbiturates, benzodiazepines, Tetrahydrocannabinol, cocaine and opiates) were evaluated and compared to assays run on the Abbott Architect ci16200. Positive cut-off levels were based on the AS/NZS 4308:2008 standard. Intra and inter-precision were determined using two commercial human liquid quality controls (QC’s). A further 40 urine specimens were analysed by both the Alinity and Architect immunoassay screening Methods, and these specimens underwent confirmatory testing by a high-resolution mass spectrometry method (liquid chromatography time-of-flight mass spectrometer, LC-TOF-MS).

Results

Intraprecision for all six CEDIA® assays was <4%. Interprecision for the six CEDIA® assays was <10%, except for the negative cannabinoid QC that was 10.4%

Of the 240 immunoassays performed on the 40 urines analysed, there were three discordant results (1.25%).


These were close to the positive cut-off value.

Two urine specimens that tested positive to both amphetamine/ecstasy screens, could not be confirmed by LC-TOF-MS (false positives). There were no confirmed false negative results.

Conclusions

The CEDIA® methodology can be adapted for the Abbott Alinity ci-series analyser, and is suitable for qualitative urinary drugs of abuse screening.

P55: Thyroid function in growing children; the Australian look study

JM Potter1,2, EK Southcott2, F Nouri-Girones2, S Apostoloska2, RD Telford3, PE Hickman1,2.1Australian National University Medical School, Canberra ACT 0200

2ACT Pathology, Canberra Hospital, ACT 2605; University of Canberra, Bruce, 2617Australia

Introduction

Auto-immune thyroid disease is common in the first world. In a recent study of apparently healthy adults 18% of men and 34% of women had raised thyroid antibodies (Hickman et al, 20174). However little is known about the development of, or significance of the presence of antibodies in children. We are undertaking longitudinal studies in children between the ages of 8 and 16 years of thyroid function and antibody concentrations in children who participated in the LOOK (Lifestyle Of Our Kids) study in the ACT. The study enrolled more than 850 8-year old children when commenced in 2005.

Methods

Serum samples that were stored at – 80 deg C were tested for TSH, fT4, anti-TPO and anti-TG on the Abbott Architect ci16200 (Abbott Diagnostics, Sydney, Australia).

Results

Median concentrations of TSH decreased from 1.84 mU/L for 8 year old children to 1.68 for 16 year olds with changes similar for boys and girls with 97.5 percentile varying between 3.8 and 4.2 mU/L over the 8 years. Median fT4 concentrations declined slightly over the same time (14.7 to 13.3 pg/L). Antibodies to both thyroid peroxidase (anti-TPO) and thyroglobulin (anti-TG) were detected in all participants by the age of 10 years.

Conclusions

The data suggest that TFT in growing children are different to those we have previously published for adults (median of 1.3 mU/L and 97.5 percentile of 3.3 mU/L). However, these have not been adjusted for the presence of circulating antibodies.

Hickman et al 2017 Clin Endocrinology (Oxf). ; 86:108-112.

P56: How pre-analytic handling changes a diagnosis and apparent incidence of disease: gestational diabetes mellitus (GDM)

JM Potter,1,2 PE Hickman,1,2 AJ Simpson,1,2 C Oakman,2 C Woods2

1Australian National University Medical School, Canberra ACT 02002ACT Pathology, Canberra Hospital, ACT, 2605, Australia

Introduction

The diagnosis of gestational diabetes mellitus (GDM) is based on the 75 g OGTT and using criteria recommended by ADIPS glucose concentrations on samples collected at t= 0h, +1h and +2h post-glucose ingestion, all have diagnostic significance. As the diagnosis is so important in obstetric management, we instituted strict conditions around collection of all samples during OGTT.

Methods

Routine practice has been to collect venous blood into fluoride tubes and transport samples in a timely manner to the laboratory at the end of the OGTT. In July 2017 stricter handling of glucose samples was instituted, with requirement to centrifuge fluoride tubes within 10 minutes of collection.

Results

In the 29 months prior to change in collections, 7509 OGTT were undertaken with an incidence of GDM of 11.9% overall. After the mandated centrifugation, the average over 9 months increased to 20.9%. This increased incidence is predominantly due to a significant increase in fasting concentrations ≥5.1 mmol/L (4.2% to 10.1%, Κ2 129.4 p<0.001), with a lesser increase at the 1-hr point ≥10.0 (1.5% to 2.7%, Κ2 16.4 p<0.001).

Conclusions

We have inadvertently doubled the diagnosis of GDM in the population of ACT by implementing strict pre-analytic handling. It is well known that glucose stability is an issue, but the consequences for individual patient management are profound. In addition it raises serious questions about variable collection practices in all studies hitherto and development of the published criteria. Although the latter studies recommended handling processes they are very difficult to enforce.

P57: Evaluation of immunoassay drugs of abuse in a laboratory setting

M. Huynh, F. Del Rosso, W. McConnell, S. Sacks, J.Grasko, L. Dusci, G. Bain, C. Quinlan

Clinipath Pathology, Osborne Park, W.A 6017, and Specialty Diagnostix, Beverly Hills, NSW, Australia

Introduction


Immunoassay kits require laboratory evaluation to assess their performance and to determine whether they are suitable as screening tests for the drugs they target.

This study evaluates and compares the Siemens EMIT II assays against the CEDIA amphetamines, benzodiazepines, cocaine metabolites, opiates, 6-Acetylmorphine, and DRI cannabinoids.

Methods

Urine samples were prepared by spiking blank urine with the drugs of interest at the cut off concentration as well as 25% above and below. Both the spiked samples and commercial quality controls were assayed within and between runs to determine precision and accuracy on a Beckman AU 5810. Patient urine specimens that were chosen to target each specific assay were also assayed.

Results

The within run and between run precision for the EMIT II assays for both the spiked and quality control urines was less than 10% for all concentrations tested. The recoveries of drugs from the spiked samples at the cut off concentration ranged from 87 to 109%. The low mean recovery for methyl amphetamine of 87% was due to d,l methyl amphetamine being used instead of d methyl amphetamine in the spiked urine. Although the carboxy tetrahydrocannabinol spike was prepared using alkaline urine, recoveries were low due to adsorption onto non-silanised glass tubes. However recovery from commercial quality control material showed greater than 98%. As expected, there were some discrepancies between results from patient urine samples especially when comparing the amphetamine assays. The EMIT II amphetamine assay showed a low cross reactivity to pseudoephedrine, but lipid-lowering drugs did not cause any false positive results.

Conclusions

The EMIT II assays for the above drugs showed a good comparison to the CEDIA assays as well as the DRI cannabinoid assay. They have excellent precision at all values around the cut-off and are suitable as screening tests for the target drugs.

P58: Evaluation of a new genetically engineered enzyme for the analysis of codeine and morphine in urine

C.Quinlan, M. Huynh, W. McConnell, J. Grasko, S. Sacks and L. Dusci

Clinipath Pathology, Osborne Park. W.A 6017, Australia

Introduction

The analysis of codeine and morphine in urine requires a hydrolysis procedure to break down the glucuronide metabolites. Numerous enzymes containing β-glucuronidase have been used to release the parent drug from its conjugate before analyzing the urine sample

by LCMS. Recently a highly purified genetically enhanced recombinant β-glucuronidase (Kura BG Turbo) has been available. This enzyme has the ability to reduce hydrolysis time and simplify sample preparation.

Methods

We have compared the Kura BG 100 enzyme against the Kura BG Turbo, by spiking blank urine samples with codeine 6 glucuronide, morphine 3 and 6 glucuronide over the range 500ug/l to 10000ug/l. We have also analysed urine samples from patients on codeine and or morphine medication.

Results show that the incubation time can be reduced to 30 minutes when using the Kura BG Turbo with a concentration of 40000 units/ ml of urine. Because the Kura BG Turbo enzyme is highly purified, a column clean up can be eliminated so reducing sample preparation time and cost.

Results

Results have shown recoveries of codeine and morphine from their respective glucuronides to be similar for both enzymes. Patient sample results show a BG 100 to BG Turbo ratio of 1.04 for codeine and 0.94 for morphine.

Conclusions

The Kura BG Turbo is an ideal enzyme capable of the complete hydrolysis of codeine and morphine glucuronides with only 30 minutes incubation.

P59: Acids: it’s a PH thing

MM Sack, BR Cooke

PathWest Laboratory Medicine, Fiona Stanley Hospital, Perth, Western Australia, 6150, Australia

Introduction

Urinary acids are utilised in the diagnosis and monitoring of paediatric neuroblastomas (Hydroxymethoxymandelic Acid (HMMA) and/or Homovanillic Acid (HVA)) and carcinoid syndrome (5-Hydroxyindoleactic Acid (5HIAA)). Our current method utilises High Performance Liquid Chromatography (HPLC) with Electrochemical Detection (ECD), however Liquid Chromatography Mass spectrometry (LC-MS/MS) in negative ion mode provides greater specificity for analyte detection. We have developed a quick, simple Liquid Liquid Extraction (LLE) method for the simultaneous extraction of all three analytes.

Methods

We investigated a variety of extraction Methods including Supported Liquid Extraction (SLE) and dilute and shoot procedures and LLE. Chromatographic investigations were also made into the use of C18 (T3), Pentafluorophenyl (PFP), Biphenyl and phenyl hexyl chemistries with a variety of mobile phases with/without additives.

Results

The most crucial factor for elution recovery was the pH, either of the initial sample (LLE) or the elution solvent (SLE).


HMMA recovery (>70%) was vastly improved when the sample mix was pH 1-2. 5HIAA had optimal recovery using LLE with a sample mixture of pH4, although demonstrated reasonable recoveries at pH 1-2 (>80%) without any detrimental effect on the analyte or internal standard. HVA showed no significant recovery variation with pH changes (>90%). The Phenyl Hexyl column was able to remove interference present in the HVA qualifier ion seen when using the PFP column. As with extraction procedures the mobile phase pH variations were found to have huge effects.

Conclusions

To achieve good recovery for all analytes it was necessary to preacidify the sample to pH 1. In addition optimal chromatographic resolution, better sensitivity, robust ion ratios and good precision was obtained using the phenyl hexyl column and mobile phases at pH2 in both the aqueous and organic solutions. No improvements in recovery or removal of interference were identified when using the more expensive SLE method.

P60: High alkaline phosphatase: dilemma in diagnosis of small duct primary sclerosing cholangitis

Ibrahim M,1 Veerandran P,1 Subasinghe SKCE,2

Teaching Hospital Batticaloa. Batticaloa, Eastern province 30000, Sri Lanka

Introduction

Primary Sclerosing Cholangitis (PSC) affects predominantly men, and its prevalence, although rare, is increasing worldwide especially in association with inflammatory bowel diseases (IBD). IBD is concomitant in 60%-80% of PSC cases and in the absence of IBD it’s diagnosed at a much older age. The absence of IBD may be associated with a better prognosis. The clinical course of PSC is highly variable. 10% of the total PSC population have small duct disease.

Small-duct PSC is a chronic cholesteric liver disease of unknown aetiology characterized by abnormal blood tests and a positive liver biopsy with no typical intra–and/or extrahepatic cholangiographic changes.

Patient

A 27-year-old young Sri Lankan male previously normal found to have abnormal liver function results during his investigations for loss of appetite and loss of weight for 2 month duration.

Results

• AST 75 U/mL;

• ALT 78 U/mL;

• Total bilirubin 7.8 mg/dL Direct 6.1 mg/dl;

• ALP 2162 U/L;

• GGT 513U/L;

• Alb 3.6 Glob 5.2;

• Hepatitis B and C screening–negative;

• AMA, ANA, ASMA, anti-LKM-1 and anti-SLA/LP were negative;

• USS and CT abdomen were unremarkable;

• Liver biopsy revealed mild active chronic hepatitis with cholestasis, presence of bridging fibrosis points towards developing cirrhosis;

• No typical features of PSC;

• MRCP–unremarkable;

• Ileo-colonoscopy with biopsy–no features of IBD;

• Upper GI endoscopy–No features of portal hypertension.

His general conditions and liver profile were improved initially with the treatment of high dose UDCA but within a year patient developed oesophageal variceal bleeding.

Discussion

Generally, the natural history of these patients is favourable. It rarely progresses to large duct disease and progression to cirrhosis is variable along with other potential malignant complications. Patients with established cirrhosis may not show typical histological features of PSC and the treatment of choice would be liver transplantation at the moment for him.

P61: Urinary gamma glutamyl transferase analysis enhances adulteration detection in urine drug testing

D Allen, T Smith, G Ward, R Flatman, L Price, D Kanowski.

Toxicology Section, Biochemistry Dept., Sullivan Nicolaides Pathology, Bowen Hills, QLD, Australia

Introduction

Sample integrity checking is a vital component of urine drug testing. Individuals employ an ever increasing array of adulteration techniques to circumvent drug detection in urine, and laboratories must adapt to improve detection procedures. Urinary enzyme measurement has been previously proposed as an indicator of sample integrity. This study proposes a new marker – gamma glutamyl transferase (GGT), and evaluates its utility as an integrity marker in urine drug testing.

Methods

The effectiveness of GGT as an integrity marker was assessed against a selection of common adulteration scenarios including oxidants, acid, base and synthetic urines substitutes. The performance of GGT against other assays commonly used in adulteration detection was also evaluated. Adulteration detection assays in the study included creatinine, urea, specific gravity, pH, urinary amylase, CEDIA® Sample Check and Axiom-Diagnostics Test True™ Synthetic Urine Assay.


Results

The urine assays for creatinine, urea, pH and specific gravity offered limited utility in the detection of adulterated urine. These urine parameters can also be easily replicated in synthetic urine substitutes. Sample Check and Synthetic Urine assays were successful in detecting a majority of synthetic urines, but failed to detect fruit juice based substitutes. Sample Check was also successful in detecting the presence of most adulterating agents including bleach, glutaraldehyde, acid and base. The Synthetic Urine assay did not detect urines with adulterating agents added. The urinary enzymes amylase and GGT had the highest detection rates for flagging the presence of both adulterating agents and synthetic/fake urines.

Conclusions

Urinary enzyme measurement can provide enhanced adulteration detection. The use of urinary GGT in combination with creatinine and CEDIA® Sample Check provided increased adulteration detection, including the detection of synthetic urine substitutes. This study proposes the use of urinary GGT as a potential marker of urine integrity.

P62: Measurement of neuropeptide hormone, oxytocin utilising UPLC-MS/MS

R Swenson, BC McWhinney, JPJ Ungerer


Introduction

Oxytocin is a mammalian, 9-amino-acid long cyclic peptide hormone produced in the hypothalamus and released by the pituitary gland. The two main actions of oxytocin are to stimulate uterine contraction during child birth and facilitate milk letdown during lactation. Endogenous plasma levels are extremely low (low pg/mL). Current ELISA and RIA methods suffer from cross-reactivity and poor precision at these low levels. In this study, a method for measuring plasma oxytocin was developed using an offline SPE followed by chromatographic separation and detection on a UPLC-MS/MS.

Methods

Samples (200 µL) were extracted using acetonitrile containing deuterated internal standard. This extract was diluted with 4% phosphoric acid and transferred to a 96 well solid phase extraction plate. Samples were eluted from the SPE plate using 30% acetonitrile containing formic acid then injected onto a UPLC-MS/MS chromatographic system. Quantification was achieved using multiple reaction monitoring on a Waters Xevo TQS mass spectrometer. The time between injections was five minutes.

Results

The analytical range of the assay was 0 – 2500 pg/mL and the limit of the blank was 1.2 pg/mL. The intra-run precision of the assay across 3 concentrations was 2.7 – 8.4% and inter-run precision of the assay across 4 concentrations was 4.5 – 9.7%. The carryover of the assay was <0.1% between injections. The final extracted samples were stable for 48 hours at 15°C in the autosampler.

Conclusions

The combination of SPE sample preparation and the analytical power of UPLC-MS/MS delivered an efficient assay for measuring the small peptide hormone, oxytocin. The method developed exhibits very good analytical sensitivity, in the low pg/mL range, with excellent analytical range and specificity.

P63: TDM of total and free plasma tacrolimus, prednisolone, mycophenolate and MPAG In transplant patients utilising on-line SPE coupled to UPLC-MS/MS

R Swenson, B Espley, BC McWhinney, JPJ Ungerer


Introduction

Quantification of whole blood tacrolimus is the predominant therapeutic tool currently available for management of solid organ transplant recipients. Specific groups of patients, such as the elderly and pregnant women, are difficult to manage due to their altered tacrolimus pharmacokinetics/dynamics. The ability to measure total and free immunosuppressant levels would be beneficial to these groups. The aim of this study was to develop an assay for measuring total and free plasma levels of tacrolimus, mycophenolate, mycophenolate glucuronide (MPAG) and prednisolone.

Methods

EDTA whole blood was incubated and centrifuged at 37°C to separate the plasma. Protein free ultrafiltrate was produced by centrifuging 500µL of plasma at 37°C using a 30000 MW cut-off filter. The samples were then transferred to a 2mL 96 well plate and deuterated internal standards were added. After centrifugation the analytes were measured by automated Solid Phase Extraction (SPE) coupled to an UPLC-MS/MS system. Quantification was achieved by monitoring two transitions for each analyte on a Waters Xevo TQD mass spectrometer. The assay time between injections was five minutes.

Results

Centrifugation of whole blood samples at room temperature resulted in a 38-78% decrease in concentration of total plasma tacrolimus when compared to centrifugation at


37°C. The analytical range of the total assay was 0-3200 ng/L for tacrolimus, 0-500 nmol/L for prednisolone, 0-30 mg/L for mycophenolate and 0-150 mg/L for MPAG. The analytical range of the free assay was 0-200 ng/L for tacrolimus, 0-500 nmol/L for prednisolone, 0-1000 µg/L for mycophenolate and 0-60 mg/L for MPAG.

Conclusions

Due to the temperature-dependent partitioning of tacrolimus between plasma and red blood cells the centrifugation temperature must be regulated during sample preparation. The combination of SPE sample preparation coupled with the analytical power of UPLC-MS/MS delivered a robust and efficient assay which has excellent analytical sensitivity and range.

P64: An evaluation study on the utility of thymol as a urine preservative agent for 24-hour urine collection

Ta n S S , S aw S , Ya n g Z , C h o n g AT, S et h i S K National University Hospital, Singapore, 5 Lower Kent Ridge Road, 119074

Introduction

Twenty-four hour urine collection serves as an important tool for analysis of urolithiasis risk. Within our institution, different types of preservatives such as sodium hydroxide, hydrochloric acid, and boric acid are used depending on the analyte tested, which may render inconvenience to the patient as their urine may be collected thrice for a full panel. The purpose of this study is to evaluate the potential of thymol as a universal preservative for 24-hour urine collection.

Methods

Spot urine samples were randomly selected from eleven healthy freshly voided urine collected in plastic bottles. Each fresh sample of urine was divided into four 4-ml aliquots: one without preservative, one with 0.004 grams of thymol, one with 200µL of sodium hydroxide, and one with 200µL of boric acid. We assessed the concentrations of the paired urine samples of thymol vs current preservative for the following analytes after 24-hours: creatinine, urea, sodium, potassium, chloride, calcium, phosphate, magnesium, uric acid, microalbumin, amylase, pH, citrate, oxalic acid. Citrate and oxalate concentrations were measured using manual spectrophotometry method, pH via glass electrode in a calibrated pH meter, and the remaining analytes measured via spectrophotometry on the Beckman Coulter Olympus AU5800.

Results

The paired student t-test was utilized to compare the concentrations of analytes using thymol versus current preservative in our study. A significant difference was taken

as p < 0.05. Citrate and oxalate yielded a p-value of 0.89 and 0.71, pH p = 0.88, and p-value of the remaining of the analytes ranged from 0.91 – 0.99. The results showed good concordance for all analytes and no significant differences using the preservative of interest.

Conclusions

Based on the results of the study, thymol has minimal effect on the stability of urine and could serve as a universal preservative for 24-hour urinary collection for the analytes of interest.

P65: Interference with several 25-hydroxyvitamin D immunoassays in a patient with IgM PARAPROTEINAEMIA

S Thompson, D Chesher

NSW Health Pathology, Royal North Shore Hospital, Sydney, NSW, 2065, Australia

Introduction

Vitamin D deficiency is often asymptomatic however it can have major clinical consequences if undetected and not treated. The Royal College of Pathologists of Australasia (RCPA) recommends measurement of 25-hydroxyvitamin D (25D) in individuals who are at risk of deficiency. We report the case of an interference in the measurement of 25D which was identified in several commonly used immunoassays and discuss the probable cause.

Methods

We received a vitamin D request taken from an elderly hospital inpatient. Measurement on the Abbott Architect gave an error indicating that the result was above the highest calibrator. The sample was sent for measurement by liquid chromatography-tandem mass spectrometry (LCMS/MS) and returned a result of 67nmol/L. We sent aliquots of the patient’s serum for measurement on three other automated immunoassay platforms. We also carried out dilution studies and polyethylene glycol (PEG) precipitation to further characterise the interfering substance.

Results

Two of the three other automated platforms produced a result which was significantly above the result obtained by LCMS/MS. The specimen diluted in a non-linear pattern, suggesting the presence of an interfering substance in the serum. After treatment with PEG, the Abbott Architect returned a result which was identical to the LCMS/MS result. Review of the patient’s other results revealed the presence of an IgM kappa paraprotein.

Conclusions

The likely cause of the interference is the IgM paraprotein identified in the patient’s serum. Treatment with PEG causes large molecules (such as paraproteins) to be


precipitated, and subsequently we were able to measure an accurate vitamin D concentration. Laboratory staff should be aware of this potential interference and consider 25D measurement by LC/MSMS when immunoassay returns an improbably high result.

P66: A fully automated LCMS method for the measurement of biotin in human serum

K Liu1, W Louey1, H Luu2, H Nguyen1, B Leung1, K Sikaris1, T Yen1, ZX Lu1 and C Trambas1

1 Melbourne Pathology, Collingwood, Victoria, 3066, Australia2 Shimadzu, Rowville, Victoria, 3178, Australia

Introduction

Reports of biotin interference have increased recently, driven by both increased use of high dose biotin therapy and uptake of mg strength biotin supplements in the general community. For laboratories using biotin-affected assays, detection and prevention of interference is a challenge. There are few routinely available methods for biotin assessment. We have developed a fully automated LCMS method for biotin measurement and now describe its evaluation.

Methods

A Shimadzu LCMS-8060 was chosen for its analytical performance and automation technology. Sample preparation consists of acetonitrile precipitation using the clinical laboratory automation module (CLAM-2000) prior to chromatography with gradient elution using sequential mobile phases. Samples are introduced into the LCMS-8060, undergo electron spray ionisation, and biotin is quantitated relative to deuterated internal standard. Our method is calibrated using Sigma secondary reference material traceable to a European Pharmacopoeia Reference Standard. Run time is 4 minutes per sample. We evaluated functional sensitivity by analysing a serial curve of standards from 16ug/L to 1ug/L daily over 14 days. Between run precision was evaluated by comparing our QC at 3, 35 and 75ug/L over the same period.

Results

Our method is linear between 2–40ug/L biotin and higher concentrations can be analysed with dilution. Functional sensitivity (20% CV) is 1.1 ug/L and between run precision CVs are 7.8% at 3ug/L, 5.1% at 35ug/L and 3.5% at 75ug/L. Measured biotin was 41 ug/L in one individual who took 5.2 mg biotin 1½ hours prior to blood collection. In patients with multiple sclerosis on 300 mg daily, peak concentrations in excess of 1000 ug/L were measured.

Conclusions

Our fully automated LCMS method eliminates the need for laborious manual sample preparation and allows efficient,

specific and sensitive quantification of biotin. It thus provides a valuable tool for our laboratory to investigate biotin interference.

P67: Evaluation of EDTA PPT tubes for collection of homocysteine

W Louey, N Asadian, E Capovila, J Nguyen, C Lynch, CM Trambas, K Sikaris, T Yen and ZX Lu

Melbourne Pathology, Collingwood, Victoria, 3066, Australia

Introduction

Pre-analytical issues are paramount for accurate assessment of homocysteine, as its production by red cells continues ex vivo. Collection of an EDTA tube on ice is the current “gold standard” for homocysteine analysis, however this poses pragmatic issues for laboratories, particularly those servicing busy and remote collection rooms. In an extension of a previous study by St Vincent’s Pathology, we evaluated homocysteine collection using the BD K2EDTA Plasma Preparation Tube (PPT) with gel and the gel forms a physical barrier limiting diffusion of red-cell derived homocysteine.

Methods

Homocysteine was measured using the Abbott Architect assay on an i4000SR analyser. Blood was collected from 20 volunteers into BD K2EDTA tubes (controls, collected on ice, centrifuged promptly and analysed immediately), PPT tubes with gel and K2EDTA tubes without gel (both collected at room temperature). PPT tubes were centrifuged within 1 hour of collection and maintained either stationary or on a rocker to simulate transport to a central laboratory. Plasma was sampled from the room temperature tubes at various intervals for 72 hours and data expressed as % increase over controls.

Results

At 24 hours post-collection, average increases were 7.9% (standard deviation 4.6%) for stationary samples and 9.8% (4.8%) for samples maintained on a rocker. At 48 hours the respective increases were 7.1% (5.4) and 10.5% (6.8%). In contrast, plasma from K2EDTA tubes without gel demonstrated average increases of 19.5% (6.7%) and 23.6% (7.6%) at 24 and 48 hours.

Conclusions

The use of a gel barrier reduced homocysteine elevation in K2EDTA plasma samples collected at room temperature. Homocysteine elevation from stationary tubes was no more than 8% above controls, whereas agitation increased homocysteine elevation up to approximately 10% above controls. Collection using PPT tubes with gel offers laboratories a pragmatic alternative to collection on ice for assessment of homocysteine.


P68: Evaluation of a new sensitive oestradiol immunoassay

D Tran1, G Estoesta1, P Stanford2, D Manalansan3, L Nguyen1, MM Salib1, C Farrell1

1Laverty Pathology, North Ryde, NSW 2113, Australia2NSW Health Pathology-East, Randwick, 2031, Australia 3Laverty Pathology, Barrack Heights, NSW 2528, Australia

Introduction

Beckman Coulter recently launched a Sensitive Estradiol assay for their Access platform. We evaluated the manufacturer’s claim that this assay demonstrates improved precision and accuracy compared to standard immunoassays at low oestradiol concentrations.

Methods

Precision was evaluated using three levels of QC material measured in triplicate over seven runs. Low concentration repeatability was assessed by measuring 10 replicates of a patient sample with a low oestradiol concentration. Thirthy patient samples were correlated against the standard Beckman Access Estradiol assay and the Siemens Centaur XP Enhanced Estradiol assay. Samples with oestradiol concentrations below 200 pmol/L were additionally measured by LC−MSMS.

Results

Repeatability precision was <2% and within-laboratory precision <3% at all levels of QC material (mean concentrations 179, 1131 and 2507 pmol/L). At a concentration of 79 pmol/L the repeatability precision was 10.6%. The assay gave comparable results to the standard Beckman and Siemens assays at concentrations above 200 pmol/L: mean difference was −13.5% [95% limits of agreement (LoA) −31.3% to 4.3%] and −6.1% (95% LoA −33.6% to 21.3%), respectively. In contrast, for the 11 samples with concentrations below 200 pmol/L, the mean difference was −55.6% (95% LoA – 123.3% to 12.1%) compared to the standard Beckman assay and −54.7% (95% LoA −118.3% to 8.9%) compared to the Siemens assay. Measurement by LC−MSMS showed that the Beckman Sensitive Estradiol assay was the most accurate: 11/11 results agreed with LC−MSMS within RCPA QAP analytical performance specifications, versus 3/11 for the standard Beckman assay and 1/11 for the Siemens assay.

Conclusions

The Beckman Sensitive Estradiol assay demonstrates superior performance to standard immunoassays for the measurement of low oestradiol concentrations, such as those observed in males and children. Although LC−MSMS remains the preferred methodology for these groups, this new immunoassay may be considered by laboratories without ready access to LC−MSMS technology.

P69: Blood lead levels in occupational exposed workers of

Rajasthan, India

VSN Kiran Kumar Pilla, Raghumoy Ghosh, Prasenjit Mitra, Shailja Sharma, Praveen Sharma.

All India Institute of Medical Sciences, Jodhpur, Rajasthan, 342005, India

Introduction

Lead exposure is a great public health concern due to its effects on morbidity. Lead interferes with a variety of body processes and is toxic to many organs and tissues including the heart, bones, intestines, kidneys, and reproductive and nervous systems. Routes of exposure to lead include contaminated air, water, soil, food, and consumer products. The possibility of lead exposure in humans is therefore of great significance from health point of view. Occupational exposure is a common known cause of lead poisoning in adults, but status of workers exposed to lead in different workplaces is not known. This study focussed on identifying the Blood Lead Levels of occupationally exposed workers of Rajasthan, India.

Methods

One hundred and two participants were selected from various industrial area who were working in metal or paint industries. Blood lead level (BLL) was estimated using graphite furnace – atomic absorption spectrophotometry (GF-AAS).

Results

The mean BLL was 5.26 ± 4.89 μg/dl (range 0.1-30.7 μg/dl) in representative adult population. Out of the total, 10 % were found to be having BLL >10 μg/dl and 39.21% were having BLL > 5 μg/dl which has significantly decreased from leaded gasoline era. Those with increased BLL (>10 μg/dl) were found to have common determinants implicating towards the higher blood lead levels.

Conclusions

Individuals working in paint and battery industries are more prone to lead toxicity, so new policies must be implemented to increase the awareness regarding lead toxicity in these workers.

P70: Is adiponectin levels associated with higher mortality or poorer outcome in multiple myeloma patients?

C Khoo, L Ong, S Saw, S Sethi

National University Health System, Singapore

Introduction

Adiponectin has been known to be associated with insulin resistance, cardiovascular disease, mortality and recently, multiple myeloma (MM) risk. Therefore we conducted a pilot study looking at the relationship between adiponectin levels and disease severity and mortality in MM patients.


Methods

Leftover samples for M band requests between September to October 2016 were tested for adiponectin concentrations using Biovendor ELISA kit.

Disease severity was defined based on presence of serum monoclonal protein, monoclonal protein concentration (M band), disease progression and mortality at 2 years. M band was performed by normal resolution electrophoresis using Sebia Hydragel Protein kit on Sebia Hydrasys II and quantified on Hydrasys 2 Scan.

Results

Fourty-five patients comprising of female/male ratio of 0.73 with mean age of 67 years old were evaluated. There were 25 Chinese, 7 Malays, 2 Indians and 11 from other ethnic groups, with a mean BMI of 24.0.

There was no correlation between BMI and adiponectin concentrations in all patients.

Mean adiponectin concentration was lower in patients with detectable M band (8.8 versus 12.0 ug/mL, p=0.10). There was no mortality during the 2 years follow up. Mean adiponectin concentration was 10.3ug/mL in patients with disease progression compared to 11.4 ug/ml in patients with remission after 2 years.

Conclusions

There is no significant association between BMI and adiponectin levels for MM patients, unlike in other studies.

Patients with detectable M band concentrations had lower adiponectin concentrations, however after 2 years follow up, there was no relationship between mortality or disease progression with initial adiponectin levels.

As the study size is small, future studies can be done in a prospective manner with larger numbers with measurements of adiponectin at different time points to confirm the relationship between adiponectin levels and disease severity in MM patients.

P71: Evaluation of Roche immunoassay for holo-transcobalamin and correlation with Abbott Architect method

E Capovilla, C Lynch, W Louey, C Trambas, K Sikaris, T Yen and ZX Lu

Melbourne Pathology, Collingwood Vic. 3066, Australia

Introduction

Holo-transcobalamin (HoloTC) is the preferred, and a superior marker of cobalamin deficiency. We recently performed a correlation study between the new Roche HoloTC assay on e062 and e801 with the Architect HoloTC assay that has been in use for many years.

Methods

Residual samples from 96 de-identified patients were

analysed for HoloTC on three analysers: Roche e602, Roche e801 and Abbott Architect.

Results

Samples tested were across the range 5 – 125 pmol/L (Architect). Roche e602 and e801 gave the same results. Both analysers achieved excellent within-run CV < 1% with the low (25 pmol/L) and high (52 pmol/L) HoloTC QC (Roche PreciControl ACTB12 Levels 1 and 2). Between run CV (1.7 – 2.5%) was also satisfactory. Passing and Bablock regression analysis comparing the new Roche assays to Architect HoloTC yielded the following equations: (1) Roche_e801 = 0.97(Arch-HoloTC) + 10.6 pmol/L (R2 = 0.986). (2) Roche_e602 = 0.96(Arch-HoloTC) + 11.3 pmol/L (R2 = 0.984). There is an intercept for both Roche assays of approximately 11 pmol/L, and Roche values consistently read higher than the Architect HoloTC. However, Roche provide the reference interval 37.5 – 188 pmol/L that is higher compared to Architect 25.1 – 165 pmol/L. With the supplied cut-offs, 29% of our analysed samples were deficient by the Architect method, and 34% by the Roche methods. All patients classed as deficient by Architect were deficient on the new Roche assays.

Conclusions

Our evaluation of the Roche HoloTC assays demonstrated both correlated well with the Abbott Architect method. However, the assays are not interchangeable with an 11 pmol/L shift in values. Different clinical decision limits to define cobalamin deficiency are required for the Roche and the Abbott HoloTC assays.

P72: Overlooked biotin interference in children

C Tran, A Chiriano, E Haworth, J Lee, S Matthews, T Yen

Laboratory Services, Royal Children’s Hospital, Parkville Melbourne Vic 3052, Australia

Introduction

The earliest reports of “biotin interference” are in neonates with rare biotinidase deficiency, who are prescribed oral biotin. Some infants suspected of having a metabolic disorder may also be started on a “cocktail” of supplements including biotin pending definitive genetic diagnosis. Like all paediatric hospitals we have biotin treated patients and the laboratory performs immunoassays, some with potential for biotin interference. We describe a paediatric patient with congenital kidney disease where falsely high 25-OH vitamin D and falsely low PTH (Roche Cobas) indicated vitamin D toxicity when the patient was vitamin D deficient.

Case details

The 15-month-old child has complex medical problems including congenital kidney disease. Serum creatinine was stable at 125 umol/L (10 – 30 umol/L) and albumin 25 g/L


(29 – 45 g/L). At 12 months of age the patient experienced an acute deterioration possibly due to an underlying metabolic disorder and commenced oral biotin. The patient’s 25-OH vitamin D (Roche) levels were >175 and 143 nmol/L so the team diagnosed vitamin D toxicity. Two months earlier the patient’s results were 67 – 85 nmol/L (> 50 nmol/L). The patient’s PTH was between 13 – 29 pmol/L (Roche; 1.3 – 6.8 pmol/L). Upon an alert to biotin intake, samples were re-analysed and 25-OH vitamin D was verified as 21 nmol/L (Diasorin Liaison XL) and PTH was higher than originally reported (69 pmol/L; Abbott Architect).

Conclusions

In chronic renal failure PTH is elevated and 25-OH vitamin D is usually in the deficiency range. Extremely elevated 25-OH vitamin D is uncommon but occurred twice, and was supported by a PTH not as high as expected for chronic kidney disease. Analytical interferences which impact multiple immunoassays is rare but includes biotin therapy, so these children are vulnerable to erroneous laboratory results. No laboratory is completely exempt from biotin-based interference, so paediatric facilities should consider a system-wide approach to safeguard against this predictable error in a known high risk population.

P73: A student placement case study: endometrial carcinoma

I Thompson,1 M Burley2 1University of Tasmania, Launceston, TAS 7250, Australia2Hobart Pathology, Hobart, TAS 7000, Australia

Introduction

Endometrial carcinoma is the most common invasive cancer of the female genital tract1-6. In 2017, it was estimated to account for 5% of all newly diagnosed cases of female cancer in Australia, making it more common than ovarian cancer1, 7. Endometrial carcinoma can be separated into two types: type I, which are more common, oestrogen-related, generally low grade and have a good prognosis; and type II which are non-oestrogen-related, generally high grade and often recur1, 2, 4, 5, 8. In a significant number of cases, defects in DNA mismatch repair genes are identified, which leads to a more rapid accumulation of mutations that may alter the function of oncogenes and hence drive tumour development1, 4, 5, 8-10. However, there is no current consensus on whether mismatch repair defects have an impact on patient outcome in endometrial carcinoma10.

Case details

The patient presented to her gynaecologist with postmenopausal bleeding. Endometrial curettings revealed that the patient had a type 1 endometrial carcinoma. Subsequently, the patient underwent a hysterectomy with a bilateral salpingo-oophorectomy, which is the primary treatment for this form of the disease4, 5. The endometrial

cavity was found to be significantly distended by the presence of a large, mostly necrotic tumour. Microscopically, the tumour was comprised of closely packed glands with little intervening stroma, indicating a grade 1 endometrial carcinoma, additionally, all surrounding tissues were free of tumour involvement. The tumour had positive expression for oestrogen and progesterone receptors and normal nuclear staining for several mismatch repair proteins. In conclusion the patient was diagnosed with a type 1 grade 1 endometrial carcinoma. The prognosis for the patient should be reasonable, as surgery alone, or in combination with radiation therapy gives an average 90% 5-year survival rate for this form of endometrial cancer1, 4, 5.

P74: The role of dietary nitrates as an ergogenic aid – a literature review

D Hall 1, with thanks to B Perry 2 (Project Supervisor)1Undergraduate, Western Sydney University, Australia2Lecturer, Human Physiology, Medical Science (sosh), Western Sydney University, Australia

Introduction

Variability in the effectiveness of dietary nitrates as an ergogenic aid is used to gain insight into its mechanism of action. Production of nitric oxide (NO) was first described through an enzymatic pathway, whereby nitric oxide synthase facilitated a reaction between L-arginine and oxygen. Yet nitrates ergogenic effects are exerted through the complementary hypoxic pathway, in which oral bacteria reduce nitrate into nitrite, before being further reduced non-enzymatically into NO.

Discussion

Sustained nitrate supplementation has shown to reduce the oxygen demands of submaximal work rates, yet differing mechanistic explanations have been used to account for this. Research focusing on markers of mitochondrial biogenesis and coupling efficiency have produced inconsistent findings. However, the application of 31P-NMR has indicated that there is a reduced rate of ATP turnover during contractions at submaximal work.

Ergogenic effects of dietary nitrates have more recently been demonstrated for higher intensity tasks, such as sprint tests. Additionally, evidence suggests that dietary nitrates exhibit effects most significantly for type II fibers which are less vascular and more conducive to hypoxia. Notably, elite athletes typically experience diminished or negligible ergogenic effects with nitrate supplementation. Given that training has been demonstrated to increase the capillary perfusion of muscles, type II fibers of elite athletes would be less hypoxic and therefore less conducive to NO production.

Conclusions

The review concludes that dietary nitrates are a viable


ergogenic aid for recreational athletes, but are less effective for the elite due to the physiological adaptations to training. Further evidence for the mechanism of nitrates action could be gained from comparative studies of elite and recreational athletes.

P75: The role of the human gut microbiome in polycystic ovarian syndrome associated insulin resistance

E McLemon, O Morton

Western Sydney University, Campbelltown, NSW, Australia

Introduction

Polycystic ovarian syndrome (PCOS) is a chronic condition that affects 12-21% of Australian women and is the leading cause of irregular, painful periods (dysmenorrhea) and infertility in reproductive age women. Women with PCOS are at greater risk of obesity, diabetes and cardiovascular disease. In 2012, PCOS cost up to $400 million dollars in Australia. Diagnosis requires the presence of at least two of three symptoms; menstrual irregularity, hyperandrogenism and “polycystic” ovaries.

Dysbiosis of the gut microbiome has been implicated in chronic inflammation and the insulin resistance associated with (PCOS).

Method

A review of the literature on PCOS and the gut microbiome was performed.

Results

The gut microbiome consists of bacteria, archaea, fungi, protists and viruses that live symbiotically in the gastro-intestinal tract (GIT). Interaction between microbiota and the mucosal GIT lining is essential for immune function and inflammation processes. Dysbiosis occurs when the balance of microbiota is disturbed by diet, stress and antibiotic use which compromises the junctions between the cells that comprise the lining of the gastro intestinal tract. Women with PCOS show greater levels of gut dysbiosis compared to those without.

Dysbiosis of Akkermancia muciniphilia, a gut-bacteria, is strongly associated with insulin sensitivity through chronic inflammation. This mucin-degrading bacterium has a role in maintaining the integrity of the intestinal lining. A. muciniphila has a competitive advantage during periods of nutrient deprivation but is disadvantaged with a high fat diet. Low levels lead to ‘leaky gut’ allowing bacterial lipopolysaccharides (LPS) to enter the bloodstream activating the immune system inducing inflammation. Increasing levels of A. muciniphila in the GIT leads to an increase in insulin sensitivity and release of the satiety hormone, GLP-1, which can lead to weight loss.

Conclusions

Current research suggests that further investigation into

the interaction between diet, the microbiome and PCOS disease activity is warranted.

P76: Choosing the right test – a leptospirosis case

CW Leung

Queensland University of Technology, Brisbane, QLD 4000, Australia

Introduction

Leptospirosis is a worldwide zoonosis caused by Leptospira interrogans complex, the pathogenic type of the Leptospira species.

A forty-year-old male was admitted to hospital because of an acute febrile illness. No initial diagnosis was determined apart from an inflammatory condition.

Methods

Full blood count (FBC), electrolytes and liver function tests (eLFTs) were performed on all samples to monitor the patient’s condition. A serological method for detecting IgG antibodies against Leptospiria was performed using a sandwich enzyme linked immunosorbent assay (ELISA). Real time polymerase chain reaction (PCR) detecting Leptospiria by amplifying lipL32 gene was also performed. The patient sample was also sent away to Pathology Queensland Scientific Services (PQSS) for carrying out a Microscopic Agglutination Test (MAT).

Results

The FBC was normal while the eLFTs showed elevated ALP (557U/L), AST (73U/L), ALT (180U/L), GGT (169U/L) and elevated bilirubin. The ELISA and PCR result for the first sample (13/03/2018) were negative and positive respectively. In contrast, the ELISA results were positive in the subsequent samples while the PCR result was negative for the second sample (23/03/2018). PCR was not available for the third sample (collected on 05/04/2018). The test results of the two assays at different time points after onset of the disease were explained by the biphasic nature of the disease.

Conclusions

With the right tests requested and based on the clinical presentation and patient history, leptospirosis was diagnosed with the screening test and then further confirmed with other testing methods.

This case illustrates the strategy of using different testing methods based on the infection timeframe. Different method principles lead to different results. This case also empathised the importance of communication among different departments and between different laboratories.

P77: Use it or lose it – a review of FFP wastage

J. Rowland, K. Rushford, L. Cheng, E. Wood


Monash Medical Centre, Clayton, VIC 3168, Australia

Introduction

Inventory management and minimisation of blood wastage is an important part of good transfusion laboratory practice. Following a policy change implemented at Monash Health to improve fresh frozen plasma (FFP) availability in emergency situations, we analysed product use and wastage.

Methods

Monash Health is an academic tertiary network of hospitals with 1,951 beds over four sites. We audited the impact of having two units of pre-thawed AB FFP available at the two larger sites, which had been implemented aiming to improve the timeliness of FFP provision in the setting of massive transfusion. If not used for emergencies, to avoid wastage the thawed plasma was used on its last day of shelf life in routine cases.

Results

The policy change was made in April 2017. FFP use was compared over two time periods – May 2016 to February 2017, during which a total of 4,584 units of FFP were issued with 211 discarded (4.6%). For May 2016 to February 2017, 3,634 units were issued of which 256 units (7.0%) were discarded.

A subset of five months was reviewed to look at AB FFP use only. 100 units of AB FFP were issued from October 2016 to February 2017, which was 4.9% of total FFP issued during this time. Of this, 2 (2.0%) AB FFP were discarded. In the corresponding period one year later the proportion of AB FFP issued increased to 262 units (17.1% of total plasma issued), and 59 units (22.5%) of AB FFP were discarded.

Conclusions

The policy change to improve availability of FFP for emergency use led to an increase in the proportion of AB FFP used and increased wastage. With these data in mind, and balancing clinical risk against inventory governance, we are now exploring other approaches including having only one pre-thawed unit for immediate release.

P78: Detection of antinuclear antibodies in systemic lupus erythematosus patients using indirect immunofluorescence and line immunoassay Methods

Aye Aye Khin, Win Kalayar Kyaw and Khin Aye Thin

Department of Medical Laboratory Technology, University of Medical Technology, Yangon, Myanmar

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of various autoantibodies. The aim of this study was to detect antinuclear antibodies in clinically suspected SLE patients by indirect immunofluorescence (IIF) and line immunoassay (LIA) methods. ANA patterns were screened by IIF method and specific antinuclear antibodies were detected by LIA

method. A laboratory based cross-sectional descriptive study including 30 serum samples of clinically suspected SLE patients from 500 bedded Yangon Specialty Hospital were tested. Out of total 30 clinically suspected SLE patients, 29 females (96.7%) and only 1 male (3.3%) were found. ANA IIF was positive in 28 (93.33%) cases and LIA was positive in 21 (70%) cases. Percent agreement between the two methods was 76.7%. Four patterns of nuclear fluorescence (homogenous, speckled, homogenous and speckled and nuclear dots patterns) were noted with ANA IIF method. The ANA pattern observed were mostly homogenous (39.28%). Specific antibodies detected in LIA method were dsDNA, histone, nucleosome, Sm, RNP/Sm, SS-A, SS-B, Ribosomal P-protein, Ro-52, Scl-70 and mostly with nucleosome and RNP/Sm (18%) respectively. Intermediate to good agreement (kappa=0.516) was observed between result of ANA (weak positive up to strong positive) by IIF and LIA method. It was statistically significant (p=0.003). However, poor agreement (kappa=0) was observed between result of ANA (trace positive) by IIF and LIA method. LIA negative cases become trace positive in ANA IIF. So that ANA IIF method is more sensitive than LIA test. Sensitivity of IIF was 100% and specificity was 96%. Sensitivity and specificity of LIA test were 75% and 100% respectively. This study found out that LIA method can be used interchangeablely with IIF method in samples from weak positive up to strong positive cases but IIF method should be used in samples negative for LIA to detect trace positivity of ANA.

P79: Performance evaluation of an automated ANA immunofluorescence system and comparison with a current conventional method

A Lee, HS Lim, KR Lee

Seoul Clinical Laboratories (SCL), Yongin, 16954, Republic of Korea

Introduction

Anti-nuclear antibody (ANA) assay is the most popularly used test for screening systemic autoimmune rheumatic diseases. Although the indirect immunofluorescence (IIF) on human epidermoid laryngeal carcinoma (HEp-2) cells is regarded as the gold standard method for ANA detection, there are some drawbacks, such as labor-intensive, requiring skilled operators, and possibility of subjective reading. The authors evaluated the performance of a new fully automated ANA system, and compared it with the currently used method.

Methods

There were 348 serum samples tested with EUROPattern (Euroimmun AG, Lübeck, Germany) automated ANA IIF system, and compared against the conventional IIF (Zeus, New Jersey, USA) with visual reading. Qualitative results (positive or negative), the main IIF pattern recognized (Homogeneous, speckled, nucleolar, centromeric, nuclear


dots, cytoplasmic), and quantitative fluorescence intensity value (equivalent to the end-point titer) were evaluated. Titers with ≥1:80 were counted as positive in both methods. According to the nomenclature of the International Consensus on ANA Patterns, AC1-14 patterns with nuclear staining were considered positive.

Results

The concordance rate between two methods was 75.3% (135/348), with the kappa value of 0.51 (95% CI, 0.42-0.59). While the observed sensitivity of EUROPattern was satisfying (90.2%), the specificity was rather low (60.1%). Regarding pattern recognition, 48.0% (83/173) showed concordant with the conventional IIF. Concerning the estimated titers, visual reading by one expert and EUROPattern reading agreed in 62.6% (218/348) of the cases within the range of ±1 dilution step.

Conclusions

Automated and conventional IIF method for ANA showed moderate overall agreement, revealing much higher positive rate in the automated method than conventional IIF. Although the automated IIF system could reduce laboratory workload for slide preparation and reveal better reproducibility than conventional method, its’ overall performance is still insufficient to replace visual reading. This technique need to be further validated, and its optimal cut-off should be established in clinical studies.

P80: Association of SNAP-25 (rs3746544) polymorphism with lead levels in occupationally lead (Pb) exposed individuals of Rajasthan, India

Prasenjit Mitra, Shailja Sharma, Praveen Sharma


Introduction

Lead toxicity is a major public health issue in developed and developing countries. Both acute and chronic lead exposure has the potential to cause many deleterious systematic effects including hypertension, frank anaemia, cognitive deficits, infertility, immune imbalances, delayed skeletal and deciduous dental development, vitamin D deficiency, and gastrointestinal effects. SNAP25 encodes a protein that is a key part of the soluble N-ethylmaleimide-sensitive factor attachments receptor (SNARE) complex. SNAP25 can deliver neurotransmitter-containing vesicles to the inner plasma membrane. Among several polymorphisms reported in SNAP25 loci, the rs3746544 polymorphism is known to be associated with many diseases. Due to its impact on central nervous system, it was hypothesised that this SNP may have an association with blood lead levels in occupationally exposed individuals. This present study aimed to determine the frequency of SNAP25 (rs3746544)

polymorphism in occupationally lead exposed workers and to check for plausible association of this polymorphism on Blood lead burden.

Methods

Ninety-five occupationally lead exposed workers from different metal and paint based industries were enrolled in this study after obtaining informed consent. SNAP25 polymorphism was genotyped using Taqman SNP Genotyping assay. Blood lead level was estimated using graphite furnace – atomic absorption spectrophotometry (GF-AAS).

Results

The blood lead level of our participants were 5.09 ± 4.92 (range 0.1–30.7 μg/dl). The frequencies of typical homozygotes was 44%, heterozygote was 40%, and homozygotes was 16%. Individuals, who carried at least one G allele, had higher BLL than those with the wild genotype.

Conclusions

SNAP25 polymorphism (rs3746544) may be associated with body lead burden in occupationally lead exposed workers.

P81: Total pathway to method development

G Woollard1,4 B McWhinney2,4 R Greaves3,4 W Punyalack4

1Department of Pathology and Laboratory Medicine, Auckland City Hospital, New Zealand2Analytical Chemistry Unit, Department of Chemical Pathology, RBWH, Herston QLD 4029, Australia3School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC 3083, Australia4Members RCPAQAP Advisory Committees, St Leonards, NSW 2065, Australia

Introduction

The role of medical laboratory scientists in method development extends well beyond the customary bench practices of validation and verification towards involvement with the total process from conception to commission. There is a temporal sequence of activities requiring more than just analytical skills.

Method

Based on the collective experience of the authors, method development logically breaks into three interactive but independent activities pre-development, development and post-development. Each of these phases comprises inputs and outputs variously from pathologists, published literature and administration.

Results

Pre-development phase consists of clinical assessment in terms of purpose and likely demand followed by a business case which covers all clinical and financial elements. A literature review and survey of other clinical labs


accompanies this.

Development of the test itself usually starts with a feasibility study to assess selection and suitability of available equipment. The decision to proceed in-house or use commercial products (if available) is made at this point. Dependent on this, verification or a full validation will be needed. Validation is always required if a new method is designed. Strict criteria following international guidelines must be met for a full validation.

Afterwards costing is done regarding staff time, consumables, instrument and facilities. Quality management is implemented with acceptance/rejection criteria. Associated documentation in terms of sampling and bench method are written. Staff training ensues with competency assignments. Various distributions are written for the lab handbook and test interpretation and to alert potential requestors. Post launch audit commences later to assess test robustness, request rates and utilization.

Conclusions

Total process of method development requires the analyst to engage in a range of activities outside of the bench and exercise a set of skills beyond analytical. The authors support training and continuing professional development in the total method development process.

P82: An optimized method to genotype rs671 SNP through PCR-RFLP for candidate gene association studies

Prasenjit Mitra, Shailja Sharma, Praveen Sharma


Introduction

A single base substitution (GàA) is the basis for rs671 single nucleotide polymorphism (SNP) of ALDH2 gene, in which the 113A allele (formerly known as ALDH2*2) encodes a non-functional or inactive enzyme, whereas the 113G (ALDH2*1) allele codes for a functionally active enzyme. This SNP has been found to be associated with various disorders including alcohol dependence, diabetes, cancer, lead toxicity in various populations. But apart from real time PCR, the reported PCR-RFLP or PCR-CTPP methods are either not easy or cost effective and often provides inconclusive results. The objective of this study was to design a cost-effective and easy method of genotyping rs671.

Methods

Genomic DNA was extracted from whole blood through commercially available kits and quantified. Primers to genotype rs671 were designed using PRIMER-BLAST and a polymerase chain reaction protocol was optimized for proper amplification of the region of ALDH2 gene inclusive of the SNP. Following amplification, search for restriction enzymes able to cut the restriction site was performed and AcuI was selected for restriction digestion. Post PCR-RFLP,

results were observed via agarose gel electrophoresis.

Results

The optimized technique provided good amplification and appreciable results after restriction digestion. Post amplification, a 502 bp band was obtained. After restriction digestion, GG genotype yielded two bands of 297 bp and 205 bp, whereas GA genotype yielded three bands of 502 bp, 297 bp and 205 bp. Bands were appreciably visualized through agarose gel electrophoresis.

Conclusions

We report a modified PCR-RFLP technique for successful rs671 genotyping. This method is relatively easy and cost-effective. Thus, it may be used in laboratories, which do not have facilities for genotyping through Real-time PCR.

P83: A rare missense mutation in lipoprotein lipase gene causing familial chylomicronaemia reported from Sri Lanka BKTP Dayanath,1 UD Senarathne1,2

1Department of Chemical Pathology, Colombo North Teaching Hospital, Sri Lanka; 2Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Sri Lanka

Introduction

Familial chylomicronaemia is a rare genetic disorder of autosomal recessive transmittance. The primary pathology of the disease lies at the level of defective lipoprotein lipase (LPL) enzyme activity with subsequent abnormal chylomicron metabolism. The accumulation of chylomicrons in the blood results in severe hypertriglyceridaemia (usually ≥ 10 mmol/L) with characteristic ‘milky’ appearance in blood. The affected individuals commonly present during the early childhood with recurrent abdominal pain with or without features of acute pancreatitis.

Methods

A one-month-old female who presented with frequent episodes of irritability, poor feeding, and blood mixed stools was found to have ‘milky’ appearance in blood. On examination and further investigation baby was found to have lipaemia retinalis, mild hepatomegaly with ultrasound finding of mild fatty changes, severe hypertriglyceridaemia (534.5 mmol/L) and altered lipid profile (total cholesterol= 62.2 mmol/L, HDL= 0.4 mmol/L, LDL= 22.8 mmol/L). The hypertriglyceridaemia was not responsive to lipid lowering treatment. With the clinical diagnosis of familial chylomicronaemia, samples were sent to overseas laboratory for genetic studies (method: bidirectional sequencing of genomic DNA including all exons and 20 nucleotides flanking each exon of LPL and Apo C2 genes) which reported this patient to be homozygous for LPL c.808>G (Arg243Gly). Both her parents were found to be carriers for the above LPL mutation while older sister was


found not to be carrying the mutation. With diagnostic conformation, the baby was started on dietary fat intake restriction which was able to bring the serum triglyceride levels < 23 mmol/L.

Discussion

The missense mutation of LPL c.808C>G (p.Arg270Gly) is pathogenic for familial chylomicronaemia. This mutation has been reported only two times; first in the above case in 2013 and next in 2015 by ND Sirisena et al, both cases reported from Sri Lanka.

P84: Study of metallothionein polymorphism (MT2A, rs10636) in occupationally lead (PB) exposed industrial workers of Rajasthan, India

Shailja Sharma, Prasenjit Mitra, Praveen Sharma


Introduction

Lead (Pb), a toxic heavy metal, is capable of inducing several adverse health effects following its accumulation in the body. Various proteins are involved in the distribution of metals in human body. Metallothionein is a family of cysteine-rich, low molecular weight (MW ranging from 500 to 14000 Da) proteins, involved in metal transport in the body. It is hypothesized that genetic polymorphisms in metallothionein encoding genes may affect the susceptibility of an individual towards harmful effects of lead (Pb). The present study had an aim to evaluate the genetic effects of the polymorphism of MT2A (single nucleotide polymorphism rs10636; C → G) on blood Pb levels (BLL) of workers from metal and paint industries who are chronically exposed to this metal.

Methods

95 participants were recruited in the study after obtaining informed consent. Venous blood was collected. Blood lead level was estimated by graphite furnace – atomic absorption spectrophotometry (GF-AAS). MT2A (rs10636) genotyping was performed by TaqMan SNP Genotyping assay.

Results

The blood lead level of our participants were 5.09 ± 4.92 (range 0.1–30.7 μg/dl). The frequencies of typical homozygotes (GG) was 45%, heterozygote (CG) was 40%, and atypical homozygotes (CC) was 15%. Individuals, who carried at least one C allele, had higher BLL than those with the GG genotype.

Conclusions

Results obtained from this study support the hypothesis that polymorphisms in genes related to the transport of lead, such as MTs, may modulate the concentrations of the

metal in the body and, consequently, adverse health effects induced by lead exposure.

P85: Body fluid validation using the Abbott Architect analyser

C Vasiliadis1, Q Lam2, M Black3, R Czajko1, M Mohr1, C Chiang1

1Royal Melbourne Hospital, Melbourne, Australia.2Austin Hospital, Melbourne, Australia3Alfred Hospital, Melbourne, Australia

Introduction

Biochemical tests are commonly requested on body fluids, however assays are often not validated for the sample types. These tests become in-house IVD with subsequent need for method validation according to the NPAAC standard. We diluted miscellaneous body fluids into validated matrix type (serum) to validate these sample types across a range of analytes using the Abbott Architect analyser.

Methods

We analysed 12 body fluids (4 x pleural, 4 x drain, 2 x peritoneal dialysis (PD), 2 x ascitic) using serial dilution into serum pool. Analytes were measured on the neat serum pool, neat body fluids, and in 1 in 2, 4 and 8 dilutions. Assays are unaffected by fluid matrix interference if results are linear on dilution, and the difference between the expected and measured results does not exceed the RCPAQAP analytical performance specification set for that analyte. Analytes measured included electrolytes, urea, creatinine, lipids, LDH, bilirubin, and tumour markers.

Results The vast majority of the results were linear and within analytical performance specifications. The analytes which failed the specifications in more than one sample within the same fluid type category included: bilirubin in drain fluids, glucose and lactate in PD fluids. There were also failures which were specific to the individual sample rather than due to fluid type matrix composition interference. For example, one drain fluid was dark green and viscous, resulting in failure across a range of analytes (LDH, lipase, K, urea, Mg, Ca125) in addition to bilirubin. Bilirubin failed in 75% of drain fluid and is likely affected by the fluid matrix.

Conclusions

Different body fluid matrices did not affect performance of the Abbott Architect assays in the clinically relevant areas such as protein and LDH in pleural fluids, albumin in ascitic fluids, and urea in PD fluids.

P86: Stability of lyophilised general serum chemistry qap material

R De Leon1, M Bodenham2, B James1, P Graham1

1The Royal College of Pathologists of Australasia Quality


Assurance Programs (RCPAQAP), St Leonards, NSW, Australia

2Clinipath, Kalgoorlie, WA, Australia

Introduction

The stability of QAP material is a common query received from our participants, particularly when samples may have been exposed to extreme temperatures and delays in transport prior to receipt. This study in collaboration with Clinipath Pathology Kalgoorlie was designed to test the stability of the RCPAQAP material during transport in hot conditions.

Methods

Two sets of RCPAQAP General Serum Chemistry material (Cycle 107) were sent via commercial courier from Sydney, NSW to Kalgoorlie, Western Australia; one shipment at ambient temperature and the other with ice bricks. Data loggers were included with the samples and the temperature data subsequently downloaded. The samples were analysed on a Roche Cobas Integra 400.

Results

The data logger extract showed temperature extremes ranging from 4 to 46 °C and – 6 to 36 °C for the ambient and ice brick shipments respectively. Spiking of temperatures to the upper extremes as the goods moved between courier trucks was also observed. Transport took 3 days.

For the 26 General Serum Chemistry analytes investigated, no results differed by more than the Analytical Performance Specifications (APS). Even the largest differences, seen for some enzymes (up to 2% for CK, ALT, AST, GGT, Amylase; 3% for LD, Lipase and 5% for ALP) were very small and generally within the expected precision of the assay.

Conclusions

While this study has a small sample size, it does demonstrate that lyophilised RCPAQAP material for common chemical pathology tests retains suitable stability during transport in hot climates.

P87: Where to for key indicators in pathology?

SGay1,T Badrick1,K Sikaris2

1Royal College of Pathologists of Australasia Quality Assurance Programs – KIMMS program, Sydney, Australia 2Chair KIMMS advisory committee, c/o Melbourne Pathology, Victoria, Australia

Introduction

A KIMMS workshop was held at St Leonards in March, 2018 with 35 participants representing most pathology sectors in Australia. Issues discussed included the need for laboratories to change their concept of measuring quality from compliance towards a risk based system. Discussion also included the question of what further audit surveys could be performed, including whether the current KIMMS report could be adjusted to help laboratories achieve risk

reduction goals.

Methods

The format for the workshop was 4 formal presentations followed by 4 x 30 min round-table discussion groups and finishing with a summary session. Each delegate attended all four discussions which allowed all delegates to contribute to the conclusions from the workshop.

Results

Participants agreed that laboratory “stewardship” of pre and post analytical errors was required, particularly where risk needs to be managed together with clinicians. A high risk pre-analytical area requiring survey was the quality of pathology request forms and in particular the provision of necessary clinical information with the request. Clinical notes with the request form assist test selection and interpretation and therefore the need is dependent on the complexity of testing and interpretation. A high risk post-analytical area requiring further survey was the consistency of high risk result notification. It was acknowledged that selected tests that are high risk may be targeted. Participants in the workshop volunteered to help put together surveys, and/or conduct follow up studies.

Conclusions

The workshop showed that RCPAQAP through KIMMS should conduct further targeted surveys to help laboratories ascertain their exposure to specific risks. The data KIMMS produces can be used to establish benchmarks, and to engage with clinicians to work with pathology to bring about changes that benefit patient outcomes.

P88: Introduction of a body fluids external quality assurance program

B James1,2, GRD Jones2, J Calleja2, F San Gil2, KW Choy2, P Graham1,2

1The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP), St Leonards 2066 NSW, Australia2Members of the AACB RCPAQAP Body Fluids Working Party, St Leonards 2066 NSW, Australia

Introduction

The Royal College of Pathologists Australasia Quality Assurance Programs (RCPAQAP) introduced a Body Fluids External Quality Assurance (EQA) program in March 2018 with input from the Australasian Association of Clinical Biochemists (AACB) / RCPAQAP Body Fluids Working Party (BFWP). This was largely in response to requests from RCPAQAP participants to assist with the validation of their body fluid testing, given most in-vitro diagnostic device (IVD) providers have no validation claims for these matrices.


Methods

Samples designed to imitate a range of body fluids were obtained from Aalto Scientific, USA. The material is a liquid preparation with diluted human plasma as a base. Where necessary, the material has been supplemented appropriately. The first two samples issued were consistent with a normal pleural fluid and a parapneumonic effusion. Results from 70 participants who reported values for various chemistry and tumour marker analytes were assessed.

Results

The first survey report in the Body Fluids program was issued in May 2018 after consultation with the BFWP. Based on BFWP opinion the APS applied to the results were generally set at twice the width of the corresponding serum APS a reduce need for analytical accuracy.

Method differences were observed for creatinine (Jaffe vs. enzymatic), Lactate (dry chemistry analysers), and CA 19-9. A wider-than-expected range of pH values were reported for the parapneumonic effusion sample. Significant method differences were not apparent for the other , these may be revealed as a wider variation of samples are tested. Tightening of some APS’s may also highlight method differences.

Conclusions

While recognising the Body Fluids program has some limitations, the initial results indicate that it should be a useful tool to assist laboratories in validating their Methods for analysing various body fluids. It is encouraging that most laboratories and platforms show concordance in measured analyte concentrations in the first samples.

P89: Implementation and evaluation of QC patient risk management software in a regional laboratory in Tasmania

D McPhie,1Dr L Prentice,2 Dr P Vervaart2

1Diagnostic Services Pty Ltd, North West Pathology, Burnie, Tas. 7320, Australia; 2Diagnostic Services Pty Ltd, Hobart Pathology, Hobart, Tas. 7001, Australia

Introduction

In recent years, a variety of risk management strategies involving QC rule and frequency selection have been described in the literature to minimise patient harm. One such strategy has been developed that claims to objectively mitigate patient harm by calculating a Risk Management Index (RMI) to assist laboratories in quantitating such patient harm. As part of an early adopters program we were asked to implement and evaluate Bio-Rad’s Mission: Control 2.0™ software (MCS).

Methods

Parameters were entered into the MCS for each candidate

analyte. These included; Reference Mean, Allowable QC Rules, Allowable QC Frequency, Number of QC Per Day, Number of Patients per Day, Current QC Rules, Total Error Allowable, Mean Time Between Failures, Severity of Harm and Probability of Harm. Unity™ Central Data was then imported and the resultant calculations for each analyte were displayed in either, the Approved QC Plan or Recommended Updates screens. QC Rule and Frequency combinations were then chosen that minimised the patient risk by selecting, where possible, a RMI below the budgeted RMI. A practical QC schedule (PQS) was then developed based around each QC material and implemented. Weekly patient comparisons were evaluated between the MC instrument line and the reference instrument line to monitor analytical stability during the evaluation period.

Results

A PQS was developed using the MCS and implemented for a period of two weeks. During this time, patient comparisons were within RCPA Allowable Limits of Performance and a decision was made to continue the evaluation for a total of three months.

Conclusions

The MCS provided a semi-objective way to assess patient risk using easily obtainable laboratory parameters. The resultant RMI and other calculated parameters allowed selection of a PQS in our laboratory that resulted in no patient harm events during the evaluation period as recorded in our non-conformance quality system.

P90: A five-year review: external quality assurance of hereditary haemochromatosis testing

N. Pillay1, S. Chai1, M. Horan1, T. Badrick1, B. Bennetts2

1RCPAQAP Molecular Genetics, St Leonards, NSW 2065, Australia2Children’s Hospital at Westmead, Westmead, NSW 2145, Australia

Introduction

The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) plays an integral part in offering external quality assurance (EQA) on a range of inherited genetic disorders. Hereditary haemochromatosis (HH) EQA has been offered by the RCPAQAP since 2003. HH is an autosomal recessive disorder that is associated with variants in the HFE gene and iron overload. Three HFE gene variants (c.845G>A, c.187C>G and c.193A>T) have been characterised and associated with HH.

Methods

An EQA was developed to assess inter-laboratory performance on HFE genotyping. A total of 44 DNA samples were distributed to 150 laboratories during the years 2014-2018. Laboratories were requested to report on the gene


variant detected and the zygosity status in each sample tested.

Results

The EQA data from the last five years are presented here and represent the levels of genotype reporting for the three HFE gene variants; c.845G>A, c.187C>G and c.193A>T. A comprehensive qualitative report was generated and includes individual laboratory performance for HFE genotyping. From 2016, participating laboratories were requested to include all specific testing Methods performed for HH genotyping analysis. These data were included in each participant survey report.

Conclusions

Participation in an EQA is essential to ensure that testing and reporting standards are maintained. This review has identified that over the last five years, participating laboratories have consistently produced good quality data for HH testing. Based on these data, the risk to patients being mis-diagnosed for HH is likely to be reduced.

P91: The importance of low level quality control for high sensitivity troponin assays

PE Hickman1,2, G Koerbin3, C Oakman1, T Badrick4, JM Potter1,2

1ACT Pathology, The Canberra Hospital, Canberra, ACT, 2614, Australia2Australian National University Medical School, Canberra, ACT, 0200, Australia3Australian National University College of Medicine and Health, Canberra, ACT, 0200, Australia4RCPA Quality Assurance Programs, St Leonards, Sydney, NSW, 2065, Australia

Introduction

With the advent of the new high-sensitivity troponin assays, it is becoming critical to measure troponin accurately to low concentrations. To ensure assay performance is acceptable, appropriate Quality Control (QC) must be run.

Methods

In addition to the routine use of commercial QC materials, we prepared pools of human QC material, with low troponin concentrations close to the limit of quantitation (LoQ), <10 ng/L, measuring the concentrations regularly on our laboratory analysers. We use the Abbott Architect high sensitivity Troponin I assay (hs-cTnI), The quoted Limit of Detection (LoD) for this assay is 1.9 ng/L with a 20% CV at 1.8 ng/L. Our pooled human QC had a mean value over two batches of 4.6 ng/L (CV 11%) and 3.6 ng/L (CV 14%) respectively.

Results

Over three years we found no drift or shift in our hs-cTnI

assay. Data was analysed using unpaired student t-tests and ANOVA as well as Passing-Bablok linear regression analysis comparing analysers at different locations and using different lot numbers. We found that only the very low concentration human QC material gave warning of precision problems with the hs-cTnI assay. At the time of the documented poor assay precision, the higher concentration QC material indicated satisfactory performance. The low human QC level, however, produced a CV of >30%.

Conclusions

Choice of QC material with an appropriate concentration is important for any assay. For hs-cTnI assays, it is of particular importance to use control material with a concentration near to the limit of quantitation.

P92: Investigating the effects of lyophilsation on general serum chemistry EQA material for routine measurands

W Punyalack, P Graham, T Badrick

The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP), St Leonards, NSW, Australia

Introduction

Verifying commutability in external quality assurance (EQA) material remains paramount for the proper assessment and evaluation of clinical chemistry methodologies.The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) in association with the Australasian Association of Clinical Biochemists (AACB) have previously demonstrated commutability in a select range of endocrine measurands when base pool serum has been lyophilised and fortified.

The source material for the RCPAQAP General Serum Chemistry and Therapeutic Drugs (GC) EQA program is subject to various forms of manipulation (dialysis, filtration, fortification and lyophilisation) to produce the desired product. Each stage in this production cycle potentially changes in the base material such as protein denaturation .

The RCPAQAP intend on conducting a broad study to characterise the GC EQA material, exploring the effect each stage of production has on the degree of commutability. This initial project aims to determine the impact lyophilisation has on a selection of routine biochemistry measurands: calcium, creatinine, glucose, potassium, urea and enzymes.

Methods

Pre-lyophilised aliquots of the 2018 GC material were obtained from the manufacturer and stored at – 30. Samples were distributed to 27 volunteering laboratories, representing a variety of instruments and methods, who were requested to analyse the samples for routine biochemistry measurands. The returned results were


compared to the corresponding lyophilised sample in cycle 107 of the 2018 GC program.

Results

The mean percentage difference between the pre-lyophilised and post-lyophilised recoveries ranged from 0.6-4.4 for calcium, 3.5-9.1 for creatinine, 2.0-3.5 for glucose, 1.5-4.8 for potassium, 1.7-3.7 for urea and 0.7 – 57for enzymes.

Conclusions

The results suggest that lyophilisation of EQA material has limited impact on the recovery of calcium, creatinine, glucose, potassium and urea as results were comparable to post-lyophilisation recoveries. Enzymes displayed greater variability.

P93: Icterus assessment in an external quality assurance program

Samantha Shepherd1, Peter Graham1

1Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP), Sydney, NSW, Australia

Introduction

The icteric index provided by many automated chemistry analysers is used as an estimate of bilirubin concentration to provide an alert to potential bilirubin interference in a range of chemical pathology assays.

There are known differences between icterus assessment across automated analytical platforms. Reporting units contribute to this variability, where icterus may be expressed semi-quantitatively (e.g. +, ++, +++), mass units (g/L), molar units (µmol/L) or a unitless absolute value (e.g. 1, 2, 3 …20).

The Royal College of Pathologists Australasia Quality Assurance Programs (RCPAQAP) introduced icterus into the General Serum Chemistry program in 2018. We sought to provide participating laboratories with a means to assess the accuracy and harmonisation of icterus assessment compared to their peer group. Further, the results from this study could potentially be used to design a Serum Indices QAP.

Methods

In 2018 participants who enrolled in the RCPAQAP General Serum Chemistry program were given the option to report their icterus results when analysing their survey material. Over 170 participants reported their icterus results quantitatively and over 90 reported qualitatively. The samples covered an expected range of total bilirubin between 17-101 µmol/L.

Results

There were no significant method differences found for labs reporting icterus qualitatively across all levels

While the quantitative results correlated with the

measured total bilirubin concentration, icterus differences between platforms tended to match differences in bilirubin measurements; e.g. Abbott Architect participants showed a similar low bias for icterus and total bilirubin compared to the all lab median/target.

Similarly, platform variation was found for the labs reporting “unitless” values, particularly at higher levels.

Conclusions

This study indicates generally acceptable performance of most methods and confirms the value of an Indices QAP to assist with monitoring and harmonisation of icterus assessment across different platforms.

P94: Evaluation of quality assurance by way of Six Sigma strategy in a high volume clinical chemistry laboratory

Sivasooriar Sivaneson1, Gobhy Ramaloo1, Thuhairah Abdul Rahman2, Martin Giddy1, Seelan George1

1Quantum Diagnostics Sdn. Bhd., Wisma Kemajuan, Jalan 19/1B 46300 Petaling Jaya, Selangor.2Centre for Pathology Diagnostics and Research Laboratories (CPDRL), Faculty of Medicine, Universiti Teknologi MARA, Jalan Hospital 47000 Sg. Buloh, Selangor

Introduction

Six Sigma strategy measures the degree to which any process deviates from its goal and is used to ensure the quality of laboratory testing. This study aims to determine the Six Sigma performance in Quantum Diagnostics laboratory.

Methods

Internal quality control (IQC) data was analyzed retrospectively over 6 months (June-October 2017) for 26 biochemistry tests analyzed on COBAS 8000 (Roche Diagnostics, Germany). Sigma was calculated using the equation TEA-bias/CV where TEA is obtained from the Westgard’s website (2014).

Results

‘World-class’ sigma values (>6) were obtained for 15 (57.7%) analytes for Level 1 QC and 17 (65.4%) analytes for Level 2 QC, signifying less stringent QC rules application whereas satisfactory sigma of >3 were observed in 9 (34.6%) analytes for Level 1 QC and 7 (26.9%) analytes for Level 2 QC signifying more QC rules implementation. Poor sigma of < 3 were seen in only 2 (7.7%) analytes in Level 1 QC (sodium and magnesium) and Level 2 QC (sodium and chloride) which highlights the need for evaluation and improvement of these methods. However, it should be noted that the biological variability targets for these analytes are very stringent and despite having poor sigma levels, their CV is <2%.

Discussion

Six Sigma strategy is a useful quality assurance tool that


significantly.

O1: Appropriateness of pathology requests according clinical notes supplied with ferritin requests using the metric of mean abnormality rate

Sikaris KA, Trambas C, Yen T, Lu ZX,

Melbourne Pathology, Melbourne, Victoria, 3084, Australia.

Introduction

Increasing pathology utilisation has put a focus on the clinical appropriateness of pathology requests. Unnecessary testing can be harmful, especially as 95% reference limits are usually designed to cause false positive results in 5% of healthy individuals, and these abnormalities can cause unnecessary clinical follow up the higher the mean abnormality rate (MAR), the more likely the testing has caused true positive results of potential clinical value. It is often assumed that requests with no clinical notes have little justification compared to requests with clinical notes. We sought to investigate the MAR according to the clinical notes provided for the common scenario of ferritin testing.

Methods

We reviewed the clinical notes provided for 127,522 serum ferritin requests (mainly as ‘iron studies’) in late 2015. Two thirds of these were performed in women, and for both men and women, the percentage of requests without clinical notes was about 30%.

Results

When clinical notes were provided, the MAR for low ferritin (<30 ug/L) in women was 23.5% and in men 7.0%. When clinical notes were not provided, the MAR for low ferritin was 22.8% in women and 6.6% in men.

When clinical notes were provided, the MAR for high ferritin in women was 4.1% (>300 ug/L) and in men 8.1% (>500 ug/L). When clinical notes were not provided, the MAR for low ferritin was 4.7% in women and 7.4% in men.

The clinical notes with high MAR (>30%) for low ferritin were microcytosis, anaemia, vegetarian and pregnancy and those with high MAR (>20%) for high ferritin were liver disease, haemochromatosis and haematological malignancies.

Conclusions

The results suggest there is little difference in the MAR whether clinical notes are provided or not. The findings of this study suggests the absence of clinical notes does not represent an absence of reason for testing.

O2: Appropriateness of pathology requests according clinical notes supplied with TSH requests using the metric of mean abnormality rate

Sikaris KA, Trambas C, Yen T, Lu ZX,

Melbourne Pathology, Melbourne, Victoria, 3084, Australia

incorporates IQC and EQA data to derive an allowable deviation from established targets. Furthermore, application of the Six Sigma allows the laboratory to streamline the Westgard multirule which improves the efficiency and cost effectiveness of IQC programs.

P95: Improving turnaround time of urgent tests by improving integration processes between laboratory sections in a high throughput laboratory, Quantum Diagnostics

Gobhy Ramaloo1, Sivasooriar Sivaneson1, Thuhairah Abdul Rahman2, Martin Giddy1, Seelan George1

1Quantum Diagnostics Sdn. Bhd., Wisma Kemajuan, Jalan 19/1B 46300 Petaling Jaya, Selangor2Centre for Pathology Diagnostics and Research Laboratories (CPDRL), Faculty of Medicine, Universiti Teknologi MARA, Jalan Hospital 47000 Sg. Buloh, Selangor

Introduction

One measure of efficiency in a diagnostic laboratory is promptness in producing results. Turnaround time (TAT) has become a conspicuous indicator reflecting a lab’s efficiency. Delays in TAT in a high throughput laboratory such as Quantum occurs during the handing over of samples from serology to biochemistry section. This study aims to determine the difference in TAT prior to and after the implementation of improved work processes in sample handling between these two sections.

Methods

Urgent tests TAT, which includes H. pylori and EBV tests alongside routine biochemistry tests, were collected from July 2017 till January 2018. Prior to August 2017, priority was given to the biochemistry tests on Cobas 8000 (Roche Diagnostics, Germany) before handing over samples to serology to run H. pylori (Immulite 2000, Siemens) and EBV (Snibe, BMS) tests. Delayed TAT was identified to originate from holdup at the biochemistry section. Work processes were then reorganized to prioritize serology tests first, where samples were loaded on respective analyzers and transferred to the biochemistry analyzer immediately upon aspiration of sample. In addition, activation of similar biochemistry tests on two analyzers were done to ensure no analytical disruption occurs that would affect TAT.

Results

Percentage TAT achieved was 40.4% in July of 2017. Subsequent TAT achieved in the following months of September, October, November, December 2017 and January 2018 were 78.4%, 77.5%, 86.2%, 85.2% and 88.2% respectively.

Discussion

Lab indicators are important to identify poor work processes and formulating plans to overcome them improves TAT


Introduction

Increasing pathology utilisation has put a focus on the clinical appropriateness of pathology requests. Unnecessary testing can be harmful, especially as 95% reference limits are usually designed to cause false positive results in 5% of healthy individuals, and these abnormalities can cause unnecessary clinical follow up The higher the mean abnormality rate (MAR), the more likely the testing has caused true positive results of potential clinical value. It is often assumed that requests with no clinical notes have little justification compared to requests with clinical notes. We sought to investigate the MAR according to the clinical notes provided for the common scenario of TSH testing.

Methods

We reviewed the clinical notes provided for 512,976 serum TSH requests (mainly as ‘thyroid Function Tests’) in 2015/2016. Two thirds of these were performed in women and the percentage of requests without clinical notes was 28% in women and 33% in men.

Results

When clinical notes were provided, the MAR for low TSH (<0.5 IU/L) in women was 10.5% and in men 6.5%. When clinical notes were not provided, the MAR for low TSH was 7.5% in women and 5.5% in men.

When clinical notes were provided, the MAR for high TSH (>5.0 IU/L) in women was 7.0% and in men 5.6%. When clinical notes were not provided, the MAR for low TSH was 6.0% in women and 4.2% in men.

The clinical notes with highest MAR for TSH were hyperthyroid, goitre, thyroiditis and weight loss. The clinical notes with the lowest MAR for TSH were anxiety, fatigue, hair loss and weight gain.

Conclusions

The results suggest there is slightly lower MAR when clinical notes are not provided. The MAR also reflects the pretest likelihood of thyroid dysfunction in the selected population.

O3: Assessment of methylmalonic acid compared to holotranscobalamin in serum samples

T Smith, K Mantik, C Reyes, D Allen, G Ward, R Flatman, D Kanowski, L Price.

Biochemistry Dept, Sullivan Nicolaides Pathology, Bowen Hills, QLD

Introduction

Measurement of methyl malonic acid (MMA) has been proposed as an accurate indicator of vitamin B12 deficiency. The aim of this study was to develop and evaluate an LC-MS-MS Method for measuring serum MMA and compare results with holotranscobalamin levels.

Methods

Preanalytical preparation involved sample precipitation, transfer, drydown and reconstitution before running a 2.6 minute water (0.2% formic acid): acetonitrile gradient elution using a Thermo Fisher Aquasil C18 column with a Sciex 6500 QTRAP LC-MS-MS.

External QAP samples were used for method evaluation of MMA as well as 896 patient samples submitted for holotranscobalamin testing with a subset of 630 samples also analysed for total vitamin B12 and homocysteine. Holotranscobalamin, total vitamin B12 and homocysteine were measured using Abbott Architect immunoassays.

Results

The serum MMA LC-MS/MS Method had an LOQ of 0.05µmol/L (CV=20%). Linear regression analysis (Passing-Bablok) of external RCPA QAP median results (n=44) and LC-MS/MS MMA gave a slope of 0.96 with an r2 of 0.982.

Of samples with a holotranscobalamin <26pmol/L, 46% had an elevated MMA (>0.5µmol/L).

On average 12% of holotranscobalamin results >26pmol/L had an elevated MMA regardless of the actual holotranscobalamin level.

Conclusions

MMA is a small, highly polar molecule within a complex matrix which increases the challenge for analysis by LC-MS/MS. Our method is suitable for routine laboratory testing.

MMA has an inverse relationship with holotranscobalamin and has utility in the diagnosis of metabolic vitamin B12 deficiency. As found in other similar studies, holotranscobalamin and MMA levels do not perfectly correlate. This may be due to a series of factors not limited to: incomplete treatment history, individual genetic variation, residual enzymatic activity and renal function.

Correction factors (eg. eGFR, combination/ratio of homocysteine, total B12 and holotranscobalamin) and cut-offs other than >0.5 µmol/L should be considered for assessment of vitamin B12 status.

O4: Measurement of MGMT methylation in glioblastoma

DG Burke1, D. Lynch2, A. Cooper2, J. W. Po2, T. Roberts2, P. de Souza2 and T. Becker2.1National Measurement Institute Australia, 36 Bradfield Rd, Lindfield NSW 2070, Australia2Centre for Circulating Tumour Cell Diagnostics and Research, Ingham Institute for Applied Medical Research, 1 Campbell St, Liverpool NSW 2170, Australia

Introduction

MGMT promoter methylation is a predictor of temazolamide response in glioma patients. The current diagnostic test is based on extraction of DNA from tumour tissue, but tissue is not always available and does not provide the opportunity


to monitor treatment effectiveness through serial biopsies. Thus a method for measurement of methylated MGMT in gliomas from blood biopsies is needed.

Methods

We have developed methylation assays without bisulphite conversion for two genetic regions, one in the first exon and another in an upstream region that correlates with overall survival. The assays measure concentration of methylated and unmethylated target and with appropriate validation may produce measurements that are traceable to the mole in the International System of Units thus making results globally comparable.

Results

The assay targeting the upstream region had a limit of detection (LOD) of methylated MGMT around 2 copies/µL in a background of 2000 copies/µL of unmethylated DNA and thus has the potential to detect methylated MGMT in blood of patients with glioblastoma. The assay that targeted the exon one region had a higher LOD but was adequate for effective comparison between target regions and with the current diagnostic test.

The new assays were applied to twenty tumour tissue samples from the CONCERT Biobank, four of which had been tested with the standard clinical methylated MGMT test. The low LOD of the new test enabled reliable detection of methylation in small samples and in those with methylation ratios of 2% and lower. Results from the upstream region were consistent with the standard test, but interestingly, methylation of exon one was detected in all samples.

Conclusions

Since circulating tumour DNA, extractable from blood, reflects the tomour of origin methylation pattern, this test for methylated MGMT will be applied to analysis of blood after analytical validation using reference materials.

O5: Body fluids testing: verification of the performance of serum-validated assays in various fluid matrices on a Roche COBAS c701 analyser

Calleja J,1 Sargeant E,1 Louey W,1 Yen T,1 Trambas C,1 Sikaris K,1 Lu ZX1,2

1Melbourne Pathology, Melbourne, Victoria, 3084, Australia.2Department of Medicine, Monash University, Melbourne, Victoria, 3084, Australia

Introduction

Analyses of body fluids may provide useful information to aid clinical diagnosis. Most commercial assays are not validated by the manufacturers for many body fluids other thanserum and urine. We aimed to evaluate the performance of the serum-validated assays in various body fluid matrices on a Roche COBAS c701.

Methods

Fluid specimens with volume >5 mL were de-identified and stored at – 20°C for up to three months, then thawed and centrifuged on the day of analysis. Characteristics of the fluids were recorded. A serum pool (400 mL) was created and filtered to remove debris and fibrins. Recovery studies were performed by spiking the pooled serum sample with increasing proportion of a fluid sample (0%, 20%, 40%, 60%, 80%, 100%). These were analysed in duplicate for common tests. A linear regression equation for each analyte was obtained. The analyte concentrations calculated from the regression equations were compared to the measured concentrations for the neat fluid samples, RCPAQAP allowable limits were used as acceptance criteria.

Precision studies were performed on selected fluid samples by measuring 15 replicates within run for each anlayte. Biological variability precision goals were used as acceptance criteria.

Results

Twenty-six fluids (Ascetic:8; drain:7; pleural:7; synovial:2; cyst:2) were studied. Regression lines for all the analytes in the fluids tested in the recovery study were linear (most achieved r2 >0.95). Measured concentrations in neat fluid samples showed results within ±1ALP of the calculated results in all the fluid types for the analytes studied (Na, K, Cl, Creatinine, Urea, urate, Glucose, Protein Albumin, LDH, CK, Amylase, Lipase, Bilirubin, Ca, PO4, Mg, Cholesterol, Triglyceride). Precision for most of the analytes met the desirable/optimal biological goals.

Conclusions

Our recovery and precision studies demonstrated that many Roche c701 assays for common analytes are suitable for routine analysis of various non-serum body fluids.

O6: Discovering the faecal microbiome of pre-term neonates in the NICU at Townsville hospital and health service

D Rudd1 H. Jaa Kwee,1 J Westaway1, R Huerlimann1, T Kosch1, R Norton4, D Watson2, Y Kandasamy1,2,3

1James Cook University, Townsville, QLD, Australia2Townsville Hospital and Health Service, QLD, Australia3The University of Newcastle, NSW, Australia4Pathology Queensland, Townsville Hospital, QLD, Australia

Background

From the perspective of a Neonatal Intensive Care Unit (NICU) it is important to understand neonatal microbiome and its assembly immediately following birth in order to tailor treatment regimens. Understanding and preserving this delicate microbial ecosystem through evidence-based interventions could be an important link to improving


health outcomes for these babies.

As part of a larger study, developing an understanding of the neonatal microbiome caused by pre-term birth and probiotic interventions, this pilot study was designed to develop protocols for the collection, transport and analysis of faecal samples.

Methods

All pre-term babies (<32 weeks and >32 weeks) were recruited from the THHS NICU (Oct – Dec 2017). Faecal samples were collected during the first 3 days and just prior to discharge. The microbiome was identified using both culture dependent and independent methods. DNA extraction was optimised (Bioline Isolate Faecal DNA kit) and amplification/16s library preparation was performed using two PCR cycles using 785F/800R primer combination targeting V3 and V4 regions (Illumina MiSeq System) to identify individual microbiome make up for each neonate at each time point.

Results

The sequencing of the16s DNA coding for the ribosomal 16s RNA (16S meta-barcoding) provided reproducible data. Where DNA extraction was unsuccessful, all samples showed no growth on traditional culture analysis. Intra individual variation in microbiota was seen between babies and the microbial diversity increased upon discharge. We have established an effective protocol for the collection, transport and analysis of faeces from pre-term neonates within the NICU at THHS.

Conclusions

This study confirms and validates current collection and analysis protocols. Information gained from this study will contribute to the current knowledge and clinical practices undertaken to preserve the fragile microbial ecosystem of babies admitted to NICU and therefore improve the health and quality of life for these babies.

O7: Clinical application of nuclear magnetic resonance (NMR) spectroscopy in biochemical genetics

Chun-yiu Law1, Ching-wan Lam2

1Department of Pathology, Queen Mary Hospital, Hong Kong2Department of Pathology, University of Hong Kong

Introduction

There are more than >1,000 different types of inborn errors of metabolism (IEM) had been described and various laboratory methods had been developed to detect the diagnostic metabolite(s) to assist the biochemical diagnosis. In this work, we described our local experience in the application of Nuclear Magnetic Resonance (NMR)-based urinalysis in the diagnosis of various biochemical genetic disorders.

Methods

Positive IEM amples were recruited in this laboratory after metabolic and/or genetics analysis. NMR-based urinalysis was performed according to our in-house protocol published previously. In brief, the sample preparation was simple (i.e. 2 steps) and only 600 µ L urine sample was needed. The analytical time was around 15 minutes which a standard proton NMR spectrum would cover almost >200 different compounds with quantitative information.

Results

We had created our own NMR library and database using >100 control samples from non – IEM subjects to assist clinical diagnosis. Positive identifications of diagnostic metabolites were shown in various IEM conditions, including beta-ketothiolase deficiency, beta – ureidopropionase deficiency, propionic acidemia, holocarboxylase synthetase deficiency, fructose 1,6 bisphosphatase deficiency, hyperornithinemia-hyperammonemia–homocitrullinuria syndrome, citrin deficiency, methylmalonic aciduria, 3-hydroxyisobutyric aciduria, succinate semialdehyde dehydrogenase deficiency, etc.

Conclusions

Our works suggested the clinical use of NMR in biochemical genetics. Urine analysis is non – invasive and only a very small amount of sample is needed. The overall analytical time is fast (almost comparable to blood gas analysis) and this therefore makes NMR an essential and useful clinical instrument to support acute care in paediatric patient with metabolic decompensation.

O8: Early morning salivary cortisol assesses HPA axis suppresssion in children with atopic dermatitis on topical glucocorticosteroid

M Harrop1, M Kim2, A Yim2, JC Su2, R Czajko1, M Mohr1,2, C Chiang1 1Royal Melbourne Hospital, Melbourne, VIC, Australia2Royal Children Hospital, Melbourne, VIC, Australia

Introduction

Topical corticosteroids (TCS) are first-line treatments for atopic dermatitis (AD). High volume usage can result in Cushing’s syndrome and hypothalamic-pituitary-adrenal (HPA) axis suppression. Conversely, parental phobia with TCS‘s side effects lead to non-compliance and poorly controlled disease. Early morning salivary cortisol (EMsCort) correlates with circulating free cortisol and is a potential non-invasive marker of HPA status. The aim of this study is to establish the relationship between EMsCort and TCS use in children with AD, and to assess the clinical utility of EMsCort as a triage test for HPA axis suppression or TCS non-compliance.


Methods

EMsCort were collected prospectively from AD subjects and healthy siblings. Exclusion criteria were: age < 9 months, oral glucocorticoid treatment, contaminated samples or collection after 10:30am. Clinical data extracted included age, gender, weight, height, eczema severity (EASI score), use of topical corticosteroid (TCS), and use of occlusive wet dressings.

Results

Fifty-six saliva samples were available for analysis (AD subject n = 34, healthy sibling control n = 22). EMsCort did not differ between sibling controls and AD subjects on low volume TCS (9.6 vs 13.1 nmol/L, p = 0.07). EMsCort in high volume TCS users was 56.7% lower compared to low volume TCS users (p < 0.001). EMsCort correlated inversely with TCS use, pearson coefficient = – 0.76 (CI: – 1.24 to – 0.27), p = 0.003. Five AD subjects had very low EMsCort < 3.0 nmol/L, 3 had concurrent routine blood test for serum cortisol measurement, all of which returned low values (15 to 200 nmol/L).

Conclusions

EMsCort is a non-invasive, convenient marker to monitor HPA axis in children on TCS. A value greater than 7 nmol/L excluded HPA axis suppression, whereas value less than 3 nmol/L flagged potential complication of TCS therapy and prompted earlier clinical evaluation for steroid sparing therapies.

O9: Excess mortality in the presence of hyperhomocystenemia and vitamin B6 deficiency – the role of oxidative stress, systemic inflammation and telomere shortening

M Herrmann 1,2, I Pusceddu2,3, M Kleber4, W März,5,6, W Herrmann3

1 Medical University of Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Austria; 2 Laboratory of Clinical Pathology, Hospital of Bolzano, Italy; 3 Departement of Clinical Chemistry, University of Saarland, Homburg, Germany; 4 Medical Clinic V, University of Heidelberg, Mannheim, Germany; 5 Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Germany;6 Synlab Academy, Synlab Holding Deutschland GmbH, Mannheim, Germany

Introduction

Accumulation of DNA damage is a key component of aging. Telomeres and B-vitamins are essential for the maintenance of genomic integrity. Short telomeres and B-vitamin deficiencies have been proposed as risk factors for age-related diseases and mortality that interact through

oxidative stress and inflammation with each other. However, available data is inconsistent.

Objectives

We aimed to investigate the predictive role of relative telomere length (RTL) in blood leucocytes, plasma HCY and B vitamins for mortality in cardiovascular patients. Potential relationships between RTL, HCY, B vitamins and indices of inflammation were also explored.

Methods

RTL, vitamin B6, B9, B12, HCY, interleukine-6 (IL-6) and high-sensitive C-reactive protein (hs-CRP) were measured in baseline samples from 3316 participants of the Ludwigshafen Risk and Cardiovascular Health Study (LURIC). Median follow-up was 9.9 years. B vitamin status including vitamin B6, B9, B12, HCY, and markers of inflammation including interleukine-6 (IL-6) and high-sensitive C-reactive protein (hs-CRP) were quantified.

Results

All-cause mortality increased gradually with higher baseline concentrations of HCY and lower concentrations of vitamin B6. Patients in the 4th quartile of RTL, HCY and vitamin B6 had hazard ratios (HR) for all-cause mortality of 0.579 (95%CI:0.478-0.700), 2.842 (95%CI:2.354-3.431) and 0.376 (95%CI:0.312-0.454), respectively, when compared to those in the 1st quartile. Adjustments for confounders did not substantially change these results. Age-corrected RTL was significantly correlated with HCY (r=-0.060; p<0.001) and vitamin B6 (r=0.04; p=0.031). Subjects with the longest telomeres (4th quartile) had highers concentration of vitamin B6, but lower concentrations of HCY, IL-6 and hs-CRP. Multiple regression analyses identified HCY and IL-6 as independent negative predictors of age-corrected RTL.

Conclusions

Short telomeres, high HCY and low vitamin B6 are risk factors for all–cause mortality. Our results suggest that hyperhomocysteinemia and vitamin B6 deficiency increase mortality through accelerated telomere shortening caused by oxidative stress and systemic inflammation.

O10: False positive HBV core and E antibody results due to biotin interference

CM Trambas, K Sikaris, T Yen, ZX Lu, L Waring

Melbourne Pathology, Collingwood, Victoria

Introduction

The Introduction of high dose biotin therapy for the treatment of progressive multiple sclerosis (MS) has exposed these patients to the risk of biotin interference. Whilst attention has largely focussed on thyroid function test (TFT) derangement, biotin interference affects extensive assays across various platforms. The possibility of erroneous serology results is of particular concern: biotin


interference causes falsely low results for sandwich assays such as HBV surface antigen (HBsAg) and falsely high results for competitive assays such as core and e antibody. We now report two MS patients where high dose biotin therapy affected HBV serology results.

Methods

Our patients were brought to attention through electronic surveillance strategies implemented to prevent biotin interference in our laboratory. HBV serology was requested for each patient and was initially carried out using our routine, biotin-affected Roche method. To investigate biotin interference, biotin was depleted from the samples by adsorption against streptavidin-coated magnetic beads. Parallel testing was also conducted using the biotin-unaffected Architect platform.

Results

Each patient demonstrated positive core and e antibody results when initially tested, however biotin interference was suspected due to a characteristic biotin-affected TFT pattern, prompting further investigation of the serology results. Re-analysis following depletion of excess biotin demonstrated negative core and e antibody results, as did analysis using the Architect method, confirming biotin interference.

Conclusions

MS patients on high dose biotin are at risk of false positive core and e antibody results. In the context of negative HBsAg, this may be misinterpreted as latent HBV infection. In addition to causing psychological stress from (false) diagnosis, this may misguide clinical management as latent HBV is a contraindication to immunomodulator therapy. Ongoing risk mitigation is required to prevent harm from biotin interference in MS patients and biotin should be withheld for at least 72 hours prior to blood tests.

O11: Fasting and non-fasting lipid profile among health care workers at teaching hospital batticaloa Sri Lanka

M Ibrahim 1, P Veerandran 1, M Umakanth 2

1,2Teaching hospital Batticaloa, Batticaloa, Eastern province, 30000, Sri Lanka

Introduction

Measuring lipid profile is routine test to evaluate the cardiovascular risk, given the high correlation of hypercholesterolemia and cardiovascular risk. Based on the density the cholesterol is classified into low-density lipoproteins (LDL), high-density lipoproteins (HDL) and very low-density lipoproteins (VLDL). The aim of the study is to see any difference between the fasting and non-fasting lipid profile measurements.

Methods

This is a cross-sectional study conducted among health care

workers of both genders, at Teaching Hospital Batticaloa, Sri Lanka. The written consent for fasting and non-fasting blood sample for lipid profile test were taken. We included all health workers, those who had age of 25-year-old or higher. After an overnight (12-14 hours) fast, blood samples were taken for lipid profile. Following weeks, we collected non-fasting blood samples 2-4 hours after breakfast.

Results

Only 84 health workers participated in both fasting and non-fasting lipid profile. The mean fasting total cholesterol (TC), HDL, LDL, non-HDL cholesterol measurements are higher than non-fasting measurements, the fasting measurements are 194.99mg/dl for TC, 46.05mg/dl for HDL, 123.43mg/dl for LDL, and 148.94mg/dl for non-HDL. The mean non-fasting measurements are 189.34mg/dl for TC, 44.11mg/dl for HDL, 111.13mg/dl for LDL, and 145.23mg/dl for non-HDL cholesterol. However, mean non-fasting TG is higher than fasting measurements, 176.53mg/dl and 132.40mg/dl respectively. The TC, HDL-cholesterol, non-HDL, LDL, and TG showed the significant (P<0.05) difference between before and after meals with the mean difference of – 5.65mg/dl, – 1.94mg/dl, – 3.71mg/dl, – 12.3mg/dl and +34.13mg/dl respectively.

Conclusions

In this study, significant different were seen between fasting and non-fasting blood sample. More significant different were observed in Triglyceride. When we consider range of risk categories of lipid profile, this minimal change of fraction not alter the management plan. However, non-fasting lipids are sufficient for general screening of cardiovascular risk.

In future, more data is also needed assessing individuals from different ethnic and social background.

O12: Harmonisation of IGF1 reference intervals across a single laboratory network

P Ward1, D Chesher1, L Ding1, C Farrell2, J Chung3,4, R Horvath5, P Stanford5

1 NSW Health Pathology-North, St Leonards, 2065, Australia 2 Laverty Pathology, North Ryde, 2113, Australia 3 NSW Health Pathology – SW, Camperdown, 2050, Australia4 The Childrens’ Hospital, Westmead,2145 , Australia 5 NSW Health Pathology-East, Randwick, 2031, Australia

Introduction

Insulin Like Growth Factor 1 (IGF1) is measured at five laboratories within NSW Health Pathology (NSWHP). Age and sex related reference intervals, proposed as part of the AACB-RCPA Harmonization of Reference Intervals project were evaluated for use by the pathology network.

Methods

Each of the five NSWHP laboratories supplied data from routinely tested samples, including IGF-1 results (measured


by Liaison XL in 4 and Liaison in one laboratory), age and sex. The IGF-1 results were then compared to the Bidlingmaier study ranges, those provided by Diasorin and the proposed harmonised RIs, derived from transformation of the Bidlingmaier study ranges using Method comparison data.

Robust statistical methodology was also used to derive the upper limit of the pooled data. Proportion of outliers from the NSWHP laboratory data were compared to a similar analysis provided by Laverty Pathology from their predominantly community based practice.

A sample swap was conducted through the NSWHP laboratories. Each laboratory circulated five specimens to each of the other laboratories resulting in 25 samples measured in 5 different laboratories.

IGF-1 results matched with a Growth Hormone result were obtained from two of the laboratories.

Results

Significant scatter of results was observed in the hospitals’ data above all of the reference intervals, across all ages and for both sexes but appeared more pronounced in males. The scatter appeared less pronounced in the community based data supplied by Laverty Pathology.

The Liaison had a significant negative bias as compared to the Liaison XL.

Using the proposed reference intervals, approximately 10% of patients with a suppressed Growth Hormone have elevated levels of IGF1.

Conclusions

IGF1 data sourced from a hospital population does not allow for an easy verification of proposed reference intervals. The Liaison reads statistically lower than the Liaison XL analysers possibly limiting the application of common reference intervals.

O13: Laboratory diagnosis of acquired factor inhibitors

AU Herath Mudiyanselage

Monash Medical Centre, Clayton, Victoria, 3168, Australia

Introduction

Coagulation factor inhibitors are immunoglobulins arising either in congenitally factor deficient patients post treatment, or as an autoimmune response in previously normal subjects. They can be associated with catastrophic bleeding and require prompt detection, accurate measurement and monitoring by the coagulation lab. The most common factor inhibitors are those directed against factor VIII (FVIII), with inhibitors to other coagulation factors encountered less frequently. We report two cases of Acquired Haemophilia A displaying type I and type II kinetics respectively and the case of an acquired Factor V (FV) inhibitor.

Methods

Laboratory assays included coagulation screening tests (prothrombin time and activated partial thromboplastin time), mixing studies, factor assays and inhibitor titres performed by modified Bethesda assay. .

Results

Case 1 demonstrated a normal PT, prolonged APTT of 96 seconds (22-32) and non-correction on immediate mixing with normal plasma (53 seconds), extending on incubation at 37oC (66 seconds). FVIII level was <0.01 IU/mL (0.5-1.5) with a FVIII inhibitor titre of 104 Bethesda Units (BU) (0-0.5).

Case 2 presented with a normal INR, prolonged APTT (56 seconds), partial correction on immediate mixing studies (35 seconds), but non-correction post incubation (41 seconds). FVIII level was 0.07 IU/mL with a FVIII inhibitor titre of 5.5BU. The Bethesda assay showed non-parallelism between plasma dilutions, demonstrating complex type II kinetics.

The final case presented with a prolonged INR (1.7) and APTT (41 seconds). A reduced FV level of 0.161 U/mL (0.6-1.5) and a FV inhibitor of 4.6BU was determined.

All cases demonstrated altered waveform patterns with routine tests analysed on the optical ACL TOP instrument. Both FVIII inhibitor patients presented with bleeding symptoms, whereas the FV inhibitor patient experienced thrombotic complications.

Conclusions

Acquired coagulation factor inhibitors are relatively rare but potentially life-threatening, making prompt detection and accurate quantitation paramount to patient care.

O14: Laboratory quality control in infectious disease serology testing – are we truly out of control or jumping at shadows?

G Vincini, W Dimech

NRL, Melbourne, Australia

Introduction

External quality control (EQC) samples are used to monitor the performance of test systems. The “traditional approach” to EQC uses the mean ± standard deviation (SD) of the first 15-20 EQC test results to establish acceptance ranges for a particular EQC lot; all further EQC results are plotted on Levey-Jennings charts and have Westgard rules applied. Variations can be found in German RiliBÄK, USA CLSI and UK Standard methods. These standards require scientists to set ranges, with little room for alternative interpretation. Auditors expect compliance; however there are reasons why the traditional approach cannot be applied to infectious disease serological testing. QConnect Limits, an alternative approach to establish quality control limits


for infectious diseases testing, has been use for over five years.

Method

A total of 73 QC datasets, obtained from five different infectious disease serology analytes from 17 laboratories were parsed through traditional methods for establishing statistical control limits and compared to QConnect Limits. The number of QC datasets with more than 20% of data failing for each method was compared.

Results

The number of datasets having more than 20% of QC results failing Westgard rules when the first 20 and then first 100 results were used to calculate the mean ± SD ranged from 1 (1.4%) for R4S to 38 (52.1%) for 10X rule, and from 0 (0%) for R4S to 20 of 36 sets (55.5%) for 10X, respectively. In contrast, the number of datasets with greater than 20% failing RiliBÄK control limits was 18 (24.7%). No datasets had more than 20% of results outside the QConnect Limits.

Conclusions

The failure rate of EQCs using QConnect Limits was more applicable for monitoring infectious disease serology testing compared with other traditional methods, as these alternatives reported an unacceptably high percentage of failures across the 73 datasets.

O15: Lipidomic signatures in cancerous pleural effusions and its clinical translation for cancer diagnosis and prognosis

CY Law1, CW Lam2CY

1Department of Pathology, Queen Mary Hospital2Department of Pathology, The University of Hong Kong

Introduction

Previously, we identified a lipidomic signature in cancerous pleural effusions (PE) through a metabolome wide association study (MWAS). Free fatty acids (FFAs) 16:0, 18:1 and 18:2 were significantly increased in cancerous PE compared with non-cancerous PE. Herein, we hypothesize such lipidomic changes could be revealed by measuring total FFAs in PE. Its diagnostic and prognostic efficacies were evaluated in this work.

Methods

69 and 156 PE samples were recruited in the Proof-of-Concept (PoC) and a validation studies respectively. Samples were grouped into cancer and non-cancer after reviewing clinical, laboratory and radiological data. Total FFAs were measured by enzymatic-colorimetric assay, followed by statistical analysis on its diagnostic and prognostic significance using MedCalc (Version 18.5).

Results

In PoC, 44 and 25 subjects were classified as cancer and non-cancer accordingly. The mean FFAs in cancer

is 2.00-fold higher than non-cancer with p-value <0.01 using independent t – test. Since the PoC supported the diagnostic use of FFAs in cancer, a larger validation study was followed which recruited 84 cancerous and 72 non-cancerous samples. The mean FFAs in cancer is 1.92x higher than non-cancer with p-value 0.01. Using receiving operating characteristic curve (ROC) analysis, the area-under-ROC was 0.66 (p-value <0.001). High and low FFAs-cancer groups were defined using the optimal cut-off determined by ROC analysis. Kaplan-Meier survival analysis showed a significant increase in event (i.e. deceased) in the high-FFAs vs low-FFAs cancer group with a median survival of 93 days vs 242 days and a Hazard ratio of 3.59 (95% confidence interval: 2.14 – 6.04, p-value <0.0001).

Conclusions

Our validation study provides further evidence substantiating the role of FFAs in cancer. Understanding of such changes would require further experimental works in cancer biology. Nevertheless, we demonstrated the application of a simple and affordable Method to study FFAs in cancerous PE with impact on diagnosis and prognosis.

O16: Parathyroid hormone: the case for age-related reference intervals

C Farrell, L Nguyen, MM Salib

Laverty Pathology, North Ryde, NSW 2113, Australia

Introduction

Parathyroid hormone (PTH) has been found to increase independently with age. We used data mining techniques to evaluate whether the PTH changes that occur with age meet criteria for stratifying the reference interval.

Methods

Data from a four-year period were extracted from a private pathology laboratory database. Patient results were included in the study if they were 18 years of age or older and had simultaneous measurement of PTH, albumin-adjusted serum calcium, eGFR and 25-hydroxyvitamin D (25-OHD). Subjects with an abnormal albumin-adjusted calcium, eGFR <60 mL/min/1.73m2 or a 25-OHD <75 nmol/L were excluded. If a subject had more than one instance of testing in the study period, only data from the first episode of testing were included. Patients were partitioned by decade of age. The acceptability of using a single reference interval for all age-groups was assessed against the criteria of Harris and Boyd, as well as Lahti et al.

Results

After applying the exclusion criteria, results were available from 10,788 subjects. The mean PTH concentration of each age-group was compared with all others. Each age-group showed a statistically significant difference with at least one other. The proportion of each age-group falling outside the upper and lower reference limits of the manufacturer’s


range were compared to the acceptable limits of Lahti (0.8-4.1%). These limits were exceeded at either the upper or lower reference limit for all age-groups. Similarly, the portion of results falling outside of a reference interval derived by Bhattacharya analysis exceeded the limits of Lahti for all but two age-groups.

Conclusions

Age-related changes in PTH concentration across adulthood are of sufficient magnitude that a single reference interval did not perform acceptably in categorising patient results as normal versus abnormal. Laboratories should consider partitioning reference intervals by age to facilitate accurate interpretation of patient results.

O17: Reference change values for critical results – is my patient getting worse?

GRD Jones1, JZY Chung2

St Vincent’s Hospital, Darlinghurst, 20101 and Children’s Hospital at Westmead, 21452, NSW, Australia

Introduction

The standard application of the use of reference change values (RCV), also known as a critical difference (CD) is to assess whether a patient’s numerical pathology result is different between two measurements. The assumption being tested is whether the second result indicates a change in the patient not accounted for by random variation. In management of unwell patients however a different question may be considered, rather than “are they different from previously?”, it may be “are they getting better/worse?” We have created a model to estimate the probability of a change in the specific direction of concern.

Methods

A spreadsheet model was created for two-tailed probability (ie has there been a significant change in either direction) and for one-tailed probability (ie is the patient likely to have improved/deteriorated). A serum sodium of 120 mmol/L, where the concern was that the sodium concentration may have fallen, was modelled.

Results

From a starting sodium of 120 mmol/L, a result of 119 mmol/L is likely to indicate a true change with a probability of 54%, but also indicates that there is a 77% chance that the sodium in the patient has fallen. At 118 mmol/L there is an 86% chance of a true difference, but there is a 93% probability of a worsening serum sodium. Indeed a repeat sample with a sodium of 120 mmol/L indicates a zero probability of a true change, but a 50% chance that the sodium is now lower, and a sodium of 121 still has a 23% chance of a fall in sodium.

Conclusions

Depending on the clinical question, the probability

calculations for the likelihood of change in a patient may need to be considered differently. RCV calculations may need to incorporate the clinical issue under consideration.

O18: Reference limits for methylmalonic acid for detecting vitamin B12 deficiency

F Bahadory, P Stanford, AR Horvath

NSW Health Pathology, Prince of Wales Hospital, Randwick, NSW 2031, Australia

Introduction

Elevated methylmalonic acid (MMA) concentration is a sensitive and relatively specific marker of metabolic vitamin B12 deficiency in patients with normal renal function. The decision limits for MMA in the assessment of Vitamin B12 deficiency are variable across published studies (0.21 – 0.47 µmol/L) (Aparicio-Ugarriza et al 2015). Our study aimed to define the reference limits for MMA measured by liquid chromatography mass spectrometry (LC-MS/MS) in a healthy population.

Methods

Holo-transcobalamin (HTC, Abbott Architect) and MMA (LCMS/MS) were measured in 143 healthy individuals (36% male, 64% female, age range: 19–68 years). Distribution of results was reviewed and outliers excluded by the Method of Dixon-Reed. Samples with high creatinine (male >110 µmol/L, female >90 µmol/L, a known reason for elevated MMA) were excluded. Only samples with HTC >50 pmol/L (indicative of B12 sufficiency) (n=126) were included. MMA reference intervals were calculated using Analyse-it® (Estimator: Harrell–Davis Quantile, Bootstrap method).

Results

MMA results had a non-normal, right-skewed distribution. Of the 143 samples tested, 13 with HTC <50 pmol/L, and 1 with creatinine >110 µmol/L were excluded. Three extreme outliers were excluded. The upper reference limit (97.5th percentile) calculated from the remaining samples was 0.36 µmol/L. In this population, the lower reference limit for HTC was 52.6 pmol/L, close to the cut-off point for B12 replete status (>50 pmol/L) currently used in our laboratory.

Conclusions

Based on data from 126 healthy B12 replete individuals, the upper reference limit for MMA was established as 0.36 µmol/L. This is in agreement with some published literature (Vogiatzoglou et al., 2009; Aparicio–Ugarriza et al., 2015). Further studies are required to establish reference limits in other age groups and in patients with renal dysfunction.

O19: Total iron binding capacity calculation – to be revisited

G Rathnayake, J Ashford

Royal Darwin Hospital, NT 0811, Australia


Introduction

Territory Pathology is on Ortho Clinical Diagnostic for iron studies except for ferritin. By observing results on 2017 liquid serum chemistry program on QAP it was noted total iron binding capacity (TIBC) was significantly high compared to everybody. TIBC (µmol/L) is calculated from transferrin (g/L) with a multiplication factor of 25.1 which is in use historically based on molecular weight and iron binding capacity of transferrin.

Methods

Serum iron, transferrin, TIBC and % transferrin saturation on 2017 QAP Liquid serum chemistry was reviewed on “data analysis” option of RCPAQAP website.

Results

Eighty serum iron results were closely located. Transferrin results of 77 laboratories showed only slight dispersion. Even though results of serum iron and transferrin for Territory Pathology were on high side, those are within overall median. TIBC, (24 laboratories) showed significantly high results for Territory Pathology. Percentage transferrin saturation was reported by 53 laboratories. Territory Pathology has not reported percentage transferrin saturation. Offline calculation of saturation demonstrated lower results compared to overall medians. RCPAQAP TIBC is calculated based on 6 different calibrator categories; serum based – Analyser specific 0 labs , Aqueous-Analyser specific – 2 labs, factor 25-2 labs, factor 22-1 lab, 23 2, other factor 16 labs.

Conclusions

Ortho clinical diagnostic transferrin method is traceable to higher order reference material (ERM-DA470) which is listed in JCTLM. TIBC calculation from transferrin is based on different factors. Even though reporting TIBC is discouraged it affects results of transferrin saturation which is reportable. According to RCPA “iron studies standardised reporting protocol” raised percentage transferrin saturation may be the earliest indicator of iron overload. However high TIBC would have negative impact on transferrin saturation, which could underdiagnose iron overload. Therefore the factor to calculate TIBC should be closely reviewed and may be it is the time to harmonise it.

O20: Vitamin D status of new-born infants; LC-MS/MS Analysis of archived new-born screening dried blood spots

R Zakaria1, 2, RF Greaves1, 2, JJ Koplin2, J Pitt3,5, N Tzanakos5, P Roche1, KJ Allen2, 3, 4

1 School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC2 Murdoch Children’s Research Institute, Parkville, VIC, Australia3 Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia

4Department of Allergy and Clinical Immunology, Royal Children’s Hospital, Parkville, VIC, Australia5 Victorian Clinical Genetics Services, Parkville, VIC, Australia

Introduction

The population median vitamin D levels are known to vary based on variables of ethnicity and latitude; with higher level for Queensland compared to the Victorian population. Studies linking vitD insufficiency with numerous pathologies in both children and adults, including allergic disease, are well documented. In Victoria, the prevalence of both vitamin D insufficiency and challenge-proven food allergy in infants is high and the two have been linked. Potentially, the vitD status at birth has implications on the prevalence of food allergy and other pathologies. We have therefore aimed to determine the frequency of vitamin D insufficiency in Melbourne, Victoria newborns.

Methods

Three thousand dried blood spot (DBS) samples were retrieved from archived newborn screening (NBS) cards from the Victorian Clinical Genetics Services (VCGS) at the Murdoch Children's Research Institute (MCRI) (HealthNuts ethics ref, 1006215). One 3 mm DBS disk was eluted, then subjected to supported liquid extraction and derivatisation prior to LC-MS/MS analysis. The assay has the lower limit of quantitation of 0.2 nmol/L and the upper limit of linearity of 353 nmol/L, with a between run imprecision, assessed from the in-house DBS control samples of 9.8% at 45 nmol/L. Measured of 25hydroxy-vitaminD3 is traceable to NIST972. Results are interpreted as insufficient when the corrected vitamin D is between 50 and 25 nmol/L and deficient when less than 25 nmol/L.

Results

Of the 3000 DBS samples retrieved, 514 have been analysed so far for vitamin D (25hydroxy-vitamin D3 plus epi – 25hydroxy-vitamin D3; no 25hydroxy-vitamin D2 was identified). The preliminary data demonstrate a median vitamin D level of 26 nmol/L with results ranging from 4 to 142 nmol/L. 44% of newborns were considered to be vitamin D deficient.

Conclusions

This is the first, broad-based study of vitamin D insufficiency in newborns in Victoria. This information can potentially be used in a longitudinal study of the association between early life vitD status and the risk of paediatric food allergy.

NHMRC grant ID: 1041420

O21: Vitamin D test requests: are the MBS criteria effective?

C McGow,1 PD O’Loughlin,2,3 HA Morris1,2

1 School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, 5000, Australia2SA Pathology, Frome Road, Adelaide, 5000, Australia


3School of Health Sciences, University of Adelaide, Adelaide 5000, Australia

Introduction

An audit of vitamin D test requests examined compliance to Medical Benefits Schedule (MBS) criteria introduced 1 November 2014 as well as the appropriateness of these criteria for identifying increased risk of vitamin D deficiency.

Methods

A sample of pathology test requests for vitamin D assessment was extracted for the months September 2014, March 2015, and January 2018. Request forms were examined for clinical notes justifying a vitamin D assessment and patient’s previous biochemistry and vitamin D test results were reviewed. Special notice was taken of patients who were pregnant or were previously vitamin D deficient.

Results

Two hundred and eight test requests were examined for each month and 16.8% met the criteria in 2014 compared with 25% in 2015 and 26.4% in 2018, an increase of 8% and 9% respectively. The audit identified that for each month 17 to 38% of patients whose requests met the criteria were deficient (serum 25 hydroxyvitamin D < 60nmol/L) compared with 16 to 32% of patients who did not meet the criteria (P>0.05 chi-square). Ninety-nine of the 624 test requests were for assessment during pregnancy while 165 patients were previously vitamin D deficient.

Conclusions

The introduction of the new MBS criteria was associated with a 9% increase in the proportion of test requests meeting these criteria. However, the criteria do not appear to be appropriate for identifying risk of vitamin D deficiency. Neither pregnancy nor vitamin D deficiency is included in the criteria although a significant proportion of requests were for these conditions.

O22: When prophylaxis fails

C Jiang,1 K Rushford2

1Monash Health Pathology Australia, Moorabbin Hospital, Australia2Monash Medical Centre, Clayton, Vic. 3168, Australia

Introduction

The use of routine antenatal and postnatal Rh D immunoglobulin has reduced the incidence of haemolytic disease of the fetus and newborn. We describe a case where this approach was unsuccessful.

Case report

A 28-year-old G1P0 female with no history of blood transfusions or major surgery presented with PV bleeding at 6/40. Laboratory testing was performed and she was noted to be O Rh(D) negative with no antibodies detected by indirect antiglobulin test (IAT). 250 IU Rh(D) immunoglobulin

(RhDIg) was administered. At 17/40 she suffered small retroplacental haemorrhage and a further 625 IU RhDIg was administered. Kleihauer testing was not performed to quantitate the fetomaternal haemorrhage, as ANZSBT guidelines state that the standard dose is sufficient. Routine antenatal prophylaxis was given at 28/40 and a sample sent for IAT antibody screening. Anti-D was detected with a titre of 32. Isoimmunisation was presumed and follow up monitoring and regular titres were recommended. At 32/40 anti-C and anti-E were now present, and the anti-D titre was 512. The titres of all three antibodies are continuing to rise, but monitoring by Doppler ultrasound of the fetal middle cerebral artery peak systolic volume is reassuring to date.

The cause of failure to prevent isoimmunisation in this patient is not known. However, causes could include clinically silent other bleeding episodes, etc. Her BMI is noted to be normal.

Conclusions

Clinical studies showed that the administration of Rh (D) immunoglobulin to an Rh (D) negative mother reduces incidences of Rh isoimmunisation significantly. In this patient, despite appropriate antenatal care and RhDIg prophylaxis, significant isoimmunisation has occurred.

O23: Lactate dehydrogenase (LDH) activity in tumor extracellular microenvironment strongly associated with overall survival

CW Lam1, CY Law2

1Department of Pathology, The University of Hong Kong2Department of Pathology, Queen Mary Hospital, Hong Kong

Introduction

There is no effective treatment for malignant pleural effusion (MPE), a common presentation of lung cancers with a median survival of 3 – 4 months. Here, we hypothesize cancer-related inflammation would affect the overall survival and the degree of inflammation can be reflected by a single biomarker, lactate dehydrogenase (LDH).

Methods

Twnety-four samples with MPE and 14 samples with non-MPE were recruited. The diagnosis of adenocarcinoma was based on cytology, histopathology, clinical and radiological assessment. All cancer cases recruited here were collected before any anti-cancer treatments.

Results

The mean LDH ratio (PE-to-blood ratio) for MPE and non-MPE were 1.37 (95% CI: 1.06 –1.69) and 1.92 (95% CI: 1.18 – 2.66) respectively. There is no statistical difference between the two group using t-test with p-value of 0.16. Interestingly, Kaplan-Meier survival analysis using LDH ratio showed a significantly longer survival in patients with higher LDH ratio (median: 7.6 months, 95% confidence interval


(CI): 5.5 – 13.3) than patients with lower LDH ratio (median: 3.3 months, 95% CI: 2.0 – 3.9) with p-value of 0.0427 using a cut-off of 1.41. The hazard ratio was determined to be 2.63. To investigate whether the changes of LDH ratio in longer and shorter survival groups were related to pleural permeability, we had compared the PE total protein ratio (PE-to-blood ratio) between the two sub-groups. The PE total protein ratios in the two sub-groups were both equal to 0.69, indicting the changes of LDH ratio is independent to pleural permeability.

Conclusions

LDH ratio is a well-established inflammatory biomarker in PE. This ratio between PE and blood circulation is a differential normalization which quantitates the extent of inflammation inside the pleural cavity. A suppressed LDH ratio in cancer patients with short survival strongly suggested an active immunosuppression process and the underlying pathomechanism of such cancer-related inflammation would require further research works.


Australian Journal of Medical Science August 2016 Vol. 37 No. 3 97E-mail: [email protected]


SOUTH PACIFIC CONGRESS

17–19 September 2019Gold Coast Convention and Exhibition Centre

SPONSORSHIP AND EXHIBITION PROSPECTUS

NEW

WAV

E SC

IENC

E


2019 RCPAQAP/AIMSMALARIA/MORPHOLOGY WORKSHOP

Registration forms for the 2019 RCPAQAP/AIMS Malaria /Morphology Workshops will be available on the RCPAQAP

Haematology website on Monday the 29th April 2019.

The RCPAQAP/AIMS Morphology Workshop is run over 2.5 days,with sessions on setting up a microscope, Malarial Parasite

identification, Staining, RBC morphology, Bone Marrows, Leukaemia,Myelodysplastic syndromes, Thalassaemias and Paediatric

morphology.

VENUE: Charles Perkins Centre, The University of Sydney, John Hopkins Dr, Camperdown NSW 2006

Wednesday (10:00 - 17:00): Malarial Parasite WorkshopThursday and Friday (8:30 - 17:00): Morphology Workshop

Speakers include prominent pathologists and scientists who volunteer their time and donate slides and notes for each workshop.

Previous experience in Haematology Morphology is desirable.Registrants should expect an intensive program which provides the

opportunity for hands on review of cases and assistance fromexperts, where required. On departure, participants will take away

session notes and a full set of digital images, scanned by virtualmicroscopy.

2019 scheduled dates for the Workshop is:31st July, 1st, 2nd August 2019

T 1300 78 29 20 F 02 9356 2003 E [email protected]


BOOKS FOR REVIEW

Following is a list of books available for review by resource consultants and members of the Institute with particular expertise in the field. The reviewer is invited to retain the complimentary copy of the book once the review is received.

As per our agreement with the book publishing companies, complimentary books are submitted to the Institute provided that all reviews are published in the Australian Journal of Medical Science. These reviews must be of a high quality as buying decisions and the reputation of the book and author are important considerations.

Books not requested will be allocated at discretion of the Editors for the Australian Journal of Medical Science. Reviews should be 300 to 700 words depending on the volume of the book. Time limit for return of review is six weeks.

Please send your request to: Australian Institute of Medical Scientists PO Box 1911 Milton Qld 4064 Tel: (07) 3876 2988 Fax: (07) 3876 2999 Email: [email protected]

1. Bifidobacteria: Genomics & Molecular Aspects edited by B. Mayo, & D. Van Sinderen. Caister Academic Press. xii + 260 pages.

2. Medicine and Sport Science Volume 55: Cytokines, Growth Mediators & Physical Activity in Children during Puberty edited by J. Jurimae, A.P. Hills & T. Jurimae. Karger. viii+178 pages.

3. Digestive Diseases The Keys to IBD 2010: Treatment, Diagnosis & Pathophysiology. Edited by G. Rogler & W. Sandborn. Karger. 188 pages.

4. Else Kröner-Fresenuis Symposia Volume 1: Molecular Mechanisms of Adult Stem Cell Aging edited by K.L. Rudolph. Karger. xii+108 pages.

5. Endocrine Development Volume 24: Hormone Resistance and Hypersensitivity edited by M. Maghnie, S. Loche, M. Cappa, L. Ghizzoni & R. Lorini. Karger. viii + 160 pages.

6. Frontiers of Hormone Research Volume 41: Endocrine Tumor Syndromes and Their Genetics edited by C.A Stratakis. Karger. xii + 187 pages.

7. Frontiers of Hormone Research Volume 39: Kallmann Syndrome & Hypogonadotropic Hypogonadism edited by R. Quinton. Karger. x+174 pages.

8. Frontiers of Neurology & Neuroscience Volume 27: Neurological Disorders in Famous Artists: Part 3 edited by J Bogousslavsky, MG Hennerici, H Bäzner, C Bassetti. Karger. 240 pages.

9. Generic: The Unbranding of Modern Medicine by Jeremy A. Greene. John Hopkins University Press. 368 pages.

10. Human Pathogenic Fungi: Molecular Biology and Pathogenic Mechanisms edited by Derek J. Sullivan & Gary P. Moran, Caister Academic Press. x + 342 pages.

11. Internal Medicine: A Doctor's Stories by Terrence Holt. Black Inc. 273 pages.

12. Intolerant Bodies: A Short History of Autoimmunity by Warwick Anderson and & Ian R. Mackay. John Hopkins University Press. 250 pages.

13. More Than Hot: A Short History of Fever by Christopher Hamlin. John Hopkins University Press. 400 pages.

14. Pediatric and Adolescent Medicine Volume 19: Metabolic Syndrome and Obsesity in Childhood and Adolescence edited by W. Kiess, M. Wabitsch, C. Maffeis, A.M. Sharma. Karger. x + 202 pages.

15. Phage Therapy - Current Research and Applications edited by Jan Borysowski, Ryszard Miedzybrodzki & Andrzej Gorski. Caister Academic Press. 368 pages.

16. Shigella: Molecular and Cellular Biology edited by William D. Picking & Wendy L. Picking. Caister Academic Press. 280 pages


Australian Institute of Medical Scientists

Immunohaematology Quality Assurance Program

Meet your accreditation requirements with the AIMS Immunohaematology Quality Assurance Program (QAP)

• Designed to suit both small and large laboratories

• Includes blood grouping, antibody screening/identification and compatibility testing

• Runs bi-monthly starting in July at the beginning of each financial year

• The reports provide participants' own results along with graphical representation of results of their peers (de-identified) allowing for easy comparison and analysis

For enrolment enquiries please contact the AIMS National Office:E-mail: [email protected]

Phone: (07) 3876 2988Postal Address: PO Box 1911, MILTON QLD 4064, AUSTALIA

Or contact Steve Mackay:E-mail: [email protected]


AUSTRALIAN JOURNAL OF MEDICAL SCIENCE

Instructions to authorsThe following instructions are based on the “Uniform Requirements for Manuscripts Submitted to Biomedical Journals”, also known as the Declaration of Vancouver, and on the Australian Government Style manual: for authors, editors and printers, 6th edition, 2002. URLs were correct on September 29th, 2008.

Manuscripts that do not fully comply with the following ‘Instructions to Authors’ may be returned for revision before they are considered for publication.

The Australian Journal of Medical Science (AJMS) will consider for publication any paper relevant to the field of Medical Science. Disciplines include blood banking, clinical biochemistry, haematology, histopathology, immunology, microbiology and molecular biology. Areas of general interest to medical laboratory scientists, including toxicology, epidemiology, public and community health, and professional and management issues will also be considered.

Papers published in the AJMS are in the form of:

• Review Articles• Original Articles• Brief Communications• Technical Notes• Case Studies• Letters to the Editor• Book Reviews

Articles submitted for publication are understood to be offered only to the AJMS and those accepted become the property of the AJMS.

All individuals listed as authors must have made a substantial contribution to the conception and design of the study, the acquisition of data or the analysis and interpretation of data; the drafting of the article or revising it critically for important intellectual content; and final approval of the version to be published. The corresponding author must take responsibility for obtaining permission from all the authors for the submission of any version of the manuscript and for any changes in authorship.

When the manuscript is submitted the authors must disclose any potential conflict of interest and/or commercial support.

Requirements & preparation of manuscriptsGeneralArticles should be submitted in electronic format to [email protected]. If an article is too large to be submitted by email, it should be submitted on a CD or USB stick.

Number pages consecutively commencing with the title page.

Arrange the article in the following sequence:

• Title page• Abstract and key words• Main Text • Acknowledgements• References• Tables - each table, complete with title and footnotes,

on a separate page• Legends for illustrations.

Authors should ensure that their manuscript communicates their ideas and concepts simply and clearly so that the article is easily read and understood. Authors are strongly recommended to refer to the recommendations on reporting standards as outlined in the statements and checklists of the CONSORT group (see: http://www.consort-statement.org/) and similar groups such as STARD (see: http://www.stard-statement.org/). The principles outlined in these standards may be used as general guidelines and not just as applied to clinical trials and diagnostic studies.

Title pageThe title of the article should not exceed three lines (40 characters per line), including punctuation and spacing. All authors must be identified on the title page (e.g., William Smith, Susan Yeo, …”). Where applicable, the title page should also include the name of the institution with which each author is affiliated and to which the work should be attributed. In the case of multiple authors, the name, postal address, email address, telephone and facsimile number of the author responsible for correspondence relating to the manuscript should be indicated.

Abstract & keywordsThe abstract should be approximately 150 words and should make sense when read alone or in conjunction with the article. The abstract should be a concise overview that describes the important details of the article including the purpose of the study/investigation, basic procedures (study subjects/experimental animals/observational and analytic methods) and the results and principal conclusions. New and important aspects of the work and its implications may also be included. References should not be included.

Three to ten keywords may be listed. Authors are advised to comply with the terms from the Medical Subject Headings (MeSH) list from Index Medicus (see http://www.nlm.nih.gov/mesh/). Keywords should be given below the Abstract.

TextThe style of writing should conform to acceptable English usage. Do not use slang, medical jargon or unnecessary abbreviations. Accepted spelling is the first choice given in the latest edition of the Macquarie Dictionary.


Wherever possible, observational or experimental articles should be divided into sections headed:

• Introduction• Materials and methods• Results• Discussion• References

For other types of articles such as commentaries, reports and reviews, use an appropriate format or consult the Editors for guidance. Do not include a separate section for conclusions, these should be given in the discussion.

IntroductionClearly state the purpose of the article leading the reader from the known to the unknown. Summarise the rationale for the study and state the question to be answered as appropriate. Give only strictly pertinent references, and do not review the subject extensively.

Materials & methodsPresent the materials and methods in a logical sequence. Describe the selection of the observational or experimental subjects (patients or experimental animals, including controls) clearly. Notification of ethics approval must be given where relevant. Identify the methods, apparatus and procedures in sufficient detail to allow other workers to reproduce the results. Give references to established methods, including statistical methods. Adequately describe new or substantially modified methods. Identify precisely all drugs and chemicals used, including generic name(s), dosage(s), and route(s) of administration. Do not identify patients or hospitals without consent.

ResultsPresent the results in the same sequence as given in the Materials and methods; use tables and illustrations where these will help the reader understand the work being presented. Do not repeat in the text all the data in the tables or illustrations.

DiscussionIndicate the new and important aspects of the study and emphasise the conclusions that follow. Do not repeat in detail data given in the Results section and do not add new data. Include in the Discussion the implications of the findings and their limitations and compare the observations to other relevant studies. Recommendations may be included if appropriate. Link the conclusions with the goals of the study and answer the experimental question stated in the Introduction. However, avoid unqualified statements and conclusions not completely supported by your data. Avoid claiming priority and alluding to work that has not been completed. State new hypotheses when warranted, but clearly label them as such.

AcknowledgementsAcknowledge individuals who have made substantial contributions to the study including technical work and financial support. Authors are responsible for obtaining consent from all the individuals acknowledged by name as inclusion may be interpreted as an endorsement of the article’s contents.

ReferencesThe AJMS uses a modified Harvard System (author-date system).

Throughout the body of the manuscript cite the author/s name and the publication year in parentheses as in the following examples:

(i) Research in this area (Jones 1999) …

(ii) It has been successfully demonstrated that (Smith and Brown 1981; Auteur 1995; Scienziato et al 2007).

(iii) Following further investigation, Wetenschapper (2002 highlighted the difficulties inherent in…

Where there are three or more authors, acknowledge only the first author, e.g., (Smith et al 2007). For two authors the following style should be used: (Smith and Brown 2007).

The reference list should be in the format described below. Journal titles should be abbreviated in Index Medicus format (see: ftp://nlmpubs.nlm.nih.gov/online/journals/ljiweb.pdf ) using standard abbreviations from the ISSN List of Title Word Abbreviations (see: http://www.issn.org/en/node/344) All authors should be given in the reference list.

Do not use abstracts as references. “Unpublished observations” and “personal communications” may not be used as references, although references to written, not verbal, communications may be cited (in parentheses) in the text. Include in the references manuscripts accepted but not yet published, designate the journal followed by “in press” (in parentheses). Information from manuscripts submitted but not yet accepted should be cited in the text as “unpublished observations” (in parentheses).

Examples of the correct form for references are given below:

Journal Reference:

Stein MK, Downing RW, Rickels K 1978. Self-estimates in anxious and depressed outpatients treated with pharmacotherapy. Psychol Rep 43: 487-492.

Personal Author(s) of a book:

Osler AG 1976. Complement: mechanisms and functions. Englewood Cliffs: Prentice-Hall.

Editor, Compiler, Chairman as Author:

Rhodes AJ, Van Rooyen CE, comps. 1968. Textbook of virology: for students and practitioners of medicine and the other health sciences. 5th ed. Baltimore: Williams and Wilkins.

Chapter in Book:

Weinstein L, Swartz MM 1974. Pathogenic properties of invading microorganisms. In: Sodeman WA Jr, Sodeman WA, eds. Pathologic physiology: mechanisms of disease. Philadelphia: WB Saunders; 457-472.

Online documents:

National Center for Biotechnology Information. OMIM: o n l i n e M e n d e l i a n i n h e r i ta n c e i n m a n . htt p : / / www.ncbi.nlm.nih.gov/omim. Accessed February 25, 2007.


TablesNumber tables consecutively with Arabic numerals and supply a brief title for each. Give each column a short or abbreviated heading. Place explanatory matter in footnotes, not in headings. Explain in footnotes all non-standard abbreviations used in each table.

For footnotes, use the following symbols in this sequence:

* † ‡ § ¶ ** ††

In preparing tables, consideration should be given to the page width of the Australian Journal of Medical Science. All tables should be prepared for publication vertically. In the text, cite each table in consecutive order, and mark in the margin of the text its approximate location.

If data from another published or unpublished source is used, written permission must be obtained and a copy must accompany the manuscript.

IllustrationsColour illustrations may be submitted on a CD. Images should be scanned at a minimum of 300 dpi.

When plotting points, the following symbols are preferred:

= o

In most instances, figures will be reduced to one column in width. All letters and numbers should be drawn to be at least 1.5 mm high after reduction, symbols at least 1.0 mm. Titles for illustrations belong in the legends for illustrations and not on the illustrations themselves.

Photomicrographs must have internal scale markers and the magnification must be stated. Symbols, arrows, or letters used in the photomicrographs should contrast with the background.

Cite each figure in the text in consecutive order, e.g., “Figure 1 illustrates …” or “… as shown (Figure 2)”. If a figure has been published, acknowledge the original source and submit with the manuscript written permission from the copyright holder to reproduce the material. Permission is required, regardless of authorship or publisher, except for documents in the public domain.

Legends for illustrationsWhen symbols, arrows, numbers, or letters are used to identify parts of illustrations, identify and explain each one in the legends. The figure legend must contain a boldface (a) name (“Figure” + arabic figure number) and (b) substantive title.

AbbreviationsUse only standard abbreviations (see list of commonly used abbreviations).

Avoid abbreviations in the title. The full term for which an abbreviation stands must precede its first use in the text unless it is a standard abbreviation for a unit of measurement.

Report measurements in the units in which the measurements were made. In most countries the International System of Units (SI) is standard.

Commonly used abbreviationsAbbreviation or Symbol Standard Units of Measurement

g gramg gravityHz hertzh hourIU international unitK kelvinkg kilogramL liter, litrem meter, metremin minM molarmL millilitremol moleN newtonnm nanometrep probability rpm revolutions per mins secondwk weekyr year

Additional informationThe following are useful sources of information. The first two publications are used by the AJMS as standard references.

Style Manual Committee. Council of Biology Editors. Scientific style and format: the CBE manual for authors, editors, and publishers. 6th ed. Cambridge University Press, 1994.

Style manual for authors, editors and printers. 6th ed. John Wiley & Sons Australia Ltd, 2002.

O’Connor M, Woodford FP. Writing scientific papers in English: an ELSE-Ciba Foundation guide for authors. Amsterdam, Oxford, New York: Elsevier-Excerpta Medica, 1975.

Day RA. How to write and publish a scientific paper. Philadelphia, Institute for Scientific Information Press, 1979.

Zeiger M. Essentials of writing biomedical research papers. 2nd ed. New York, McGraw-Hill, 2000.

Matthews JR, Matthews RW. Successful scientific writing: a step-by-step guide for the biological and medical sciences. 3rd ed. Cambridge, Cambridge University Press, 2007 [Also available in eBook format.]


Topic: GASTROINTESTINAL MANIFESTATIONS in PRIMARY IMMUNE DISORDERS Guest Speaker: Dr Theresa Cole

The AIMS Fellowship is an attractive and highly competitive option to academic post graduate degrees

TRANSFUSION SCIENCE

The Fellowship is recognised by the Department of Health and Ageing as meeting the requirements for the supervision of GX and GY laboratories

CLINICAL BIOCHEMISRTY

CYTOLOGY

HAEMATOLOGY

ANATOMICAL PATHOLOGY

IMMUNOLOGY

MICROBIOLOGY

GENERAL (including Core Laboratory)

Qualification for the Fellowship is by EXAMINATION in one of eight disciplines

Candidates for the Fellowship must have been AIMS members for a minimum of two years and must meet certain other criteria

The Fellowship Program is modular – candidates must complete: Two compulsory modules Two elective modules A viva voce examination A scientific dissertation Candidates have up to five years to complete the Fellowship Program

To enrol in the Fellowship Program or for further information please contact the AIMS National Programs Manager:

Phone: +61 7 3876 2988.

Email: [email protected]


Australian Journal of Medical Science August 2016 Vol. 37 No. 3 97E-mail: [email protected]


Con

cept

ion

agen

ceL2

R.c

om -

©20

15 D

iagn

ostic

a S

tago

- A

ll rig

hts

rese

rved

- N

on-c

ontra

ctua

l pho

tos

- 03/

2016

MaxInnovation

MaxProductivity

MaxVersatility

Max Reliability

Max



Con

cept

ion

agen

ceL2

R.c

om -

©20

15 D

iagn

ostic

a S

tago

- A

ll rig

hts

rese

rved

- N

on-c

ontra

ctua

l pho

tos

- 03/

2016

MaxInnovation

MaxProductivity

MaxVersatility

Max Reliability

Max


A

ugus

t &

Nov

ember

20

18Vo

l. 3

9 N

o. 3

& N

o. 4

AU

GU

ST

& N

OV

EM

BE

R 2

01

8 V

OL. 3

9 N

o. 3

& N

o. 4

Pa

ge

s 5

5 - 1

42

AU

ST

RA

LIA

N JO

UR

NA

L O

F M

ED

ICA

L S

CIE

NC

E

REGULAR FEATURESAIMS AACB NATIONAL SCIENTIFIC MEETING 2018

Scientific PresentationsPoster and Oral Presentations

8

REGULAR FEATURES AIMS AACB NATIONAL SCIENTIFIC ...

Documents

Transcript of REGULAR FEATURES AIMS AACB NATIONAL SCIENTIFIC ...