Scientific Workshop

International Conference Centre Darling Harbour

Level 3 Meeting Room C3.1 Sydney

28th October 2017

Thrombosis and Haemostasis Society of Australia and New Zealand. Previously named the Australasian Society of Thrombosis and Haemostasis (ASTH) is now the Thrombosis and Haemostasis Society of Australia and New Zealand (THANZ) was established in 1994. The Society represents approximately 200 clinicians, scientists and other health professionals committed to promoting and fostering the acquisition, exchange and diffusion of knowledge and ideas relating to normal and abnormal haemostasis. The Society serves as a forum for bringing together a broad array of disciplines, which relate to bleeding, thrombosis and cognate fields. The THANZ Mission Statement Promote excellence in clinical care for people with clotting and bleeding disorders. To lead education and training of scientists and clinicians in the field; To foster innovation through research, discovery and clinical trials; To advocate and develop policies that improve health outcomes. Membership Privileges Newsletters (three per year) Notices, booklets, flyers or brochures of interest to members Copies of Media releases and letters to members from Council Invitations to Society presentations and seminars Attendance to Annual General Meetings Nomination for a Council position THANZ Medal competition eligibility at ASM (45 years of age and under) Voting rights at Council elections, AGMs and subcommittee meetings Access to the ASTH Website and Member’s only area, including Discussion group Discounted registration fees for ASM and Scientific Workshops Further information THANZ Secretariat Email: info@thanz.org.au

8:00 am Registration and Tea/Coffee

9:00 am Welcome: Grace Gilmore (Chair) 9:10 am Gary Moore (St. Thomas’ Hospital – United Kingdom) What have lupus anticoagulant mixing tests ever done for us?

9:40 am Gary Moore (St. Thomas’ Hospital – United Kingdom) Recent advances in lupus anticoagulant detection and the ISTH SSC guideline – time for an update?

10:05 am Sarah Just (NSW – Australia) CP3000 automated coagulation analyser

10:30 am MORNING TEA and TRADE 11:00 am Emmanuel Favaloro (NSW- Australia) VWD testing 11:30 am Geoffrey Kershaw (NSW – Australia) Chromogenic FIX assays 11:50 am Piet Meijer (ECAT – Netherlands) Laboratory guidance for the measurement of Factor VIII inhibitor 12:10 pm

Posters Session

12:25 pm LUNCH and TRADE 1:30 pm Paul Harrison (Birmingham University – United Kingdom) Platelet Function Testing and Antiplatelet Therapy 2:15 pm Lisa Lincz (NSW- Australia) Coumarin and Fish Oil supplement effects on platelet aggregation 2:35 pm Elaine Uhr (NSW- Australia) Application of flow cytometry to diagnose platelet dysfunction 3:00 pm AFTERNOON TEA and TRADE 3:35 pm Helena Liang (NSW – Australia) Platelet Super-Resolution Microscopy 3:55 pm Eileen Merriman (Auckland - New Zealand) Overview of new THANZ website and social media platforms

4:15 pm Closing Remarks 4:30 pm SUNDOWNDER and TRADE (until 5:30 pm)

Sponsored by THANZ

THANZ would like to thank the following sponsors who have made the 2017 THANZ

Workshop possible

Notes

What have lupus anticoagulant mixing tests ever done for us? Gary Moore, St Thomas’ Hospital, UK Lupus anticoagulants (LA) are one of the criteria antibodies for antiphospholipid syndrome and are detected by inference based on antibody behaviour in a medley of coagulation-based assays. Antibody heterogeneity necessitates use of at least two coagulation test types, of different principles, to maximise detection rates. Commonly, they are dRVVT and APTT. The medley for each test type comprises a low phospholipid concentration screening assay to accentuate the effects of LAs, a mixing test to evidence inhibition, and a high phospholipid concentration confirmatory test to swamp LAs and demonstrate phospholipid-dependence. LA are, by definition, invitro inhibitors, so it is entirely logical to follow an elevated screen with a mixing test in the first instance. If an elevated screen is not revealed to be due to an inhibitor, time and resources are wasted performing the confirmatory test. However, whilst standard practice for some time it is now widely acknowledged that mixing tests introduce a dilution factor that can generate false-negative results such that adopting the traditional screen-mix-confirm algorithm can reduce detection rates. This has fuelled acceptance and adoption of the integrated testing model as a faster and more cost-effective LA detection strategy since circumventing the traditional test order permits immediate demonstration of an elevated screen and shorter confirm without relying on (potentially false-normal) mixing tests that would lead to false-negative reporting. However, LA assays are prone to interference by other causes of elevated clotting times, so unless a routine coagulation screen has excluded them, and the confirmatory test is normal, further evidencing that interfering factors specific to that assay system are not present, mixing tests can be crucial to accurate LA detection or exclusion. Specificity can be further increased if a confirm mixing test is also performed as it aids discrimination between potent LA, LA + co-existing abnormality or non-LA abnormality. The important point is that some LA can be detected without a mixing test and the decision on its necessity should be made on a case by case basis. Accepting that mixing tests have a role in at least some patients, efforts are required to maximise diagnostic efficacy in light of test design limitations. Guidelines recommend use of a mixing test-specific cut-off or application of the index of circulating anticoagulant (ICA) calculation to improve detection rates of inhibition and reduce false-negatives. Recent publications suggest that mixing test-specific cut-off is more sensitive than ICA.

Notes

Recent advances in lupus anticoagulant detection and the ISTH SSC guideline – time for an update? Gary Moore, St Thomas’ Hospital, UK Guideline updates are written in light of research and developments since preceding versions and it can be difficult to know when to initiate a new version. The current ISTH SSC guideline update on lupus anticoagulant (LA) detection was published in late 2009, fourteen years after its predecessor, and considered by many to be overdue. Developments have inevitably occurred in the last eight years and the question is whether there is enough new material, of sufficient substance, to warrant the undertaking. Few would argue with the current recommendations regarding which patients should be tested and how to prepare plasma for testing, and these are unlikely to change. Whilst the recommendation to only employ dRVVT and APTT assays was not without logic, and there is a huge body of evidence supporting the diagnostic efficacy of that pairing, it is fallacious to consider them standardised. Variation persists between reagents and their local applications, warranting further attention. Previous stalwart assays for LA testing, dilute prothrombin time (dPT) & kaolin clotting time were not recommended, for standardisation and technical issues, but are they surmountable? Textarin, Taipan and Ecarin venom-based assays were said to lack standardised commercial assays and require further critical evaluation in the VKA anticoagulant setting. It is important to remember that exclusion from recommendation does not necessarily invalidate an assay, rather there are issues to be resolved before an expert panel can endorse its use. In fact, standardised reagents for dPT, Taipan snake venom time and Ecarin time were commercially available at the time and shown to detect small numbers of LA unreactive in dRVVT or APTT. The statement that integrated tests do not, in principle, require mixing test performance prompted vociferous debate and numerous, valuable publications. It seems that some LA can be detected without mixing tests in some circumstances, but they are invaluable in certain situations, and further guidance is essential. The guideline recommends interpreting mixing tests with a mixing test-specific cut-off or index of circulating anticoagulant calculation and comparative performance has been recently explored. The recommendation to use 99th percentile cut-offs proved controversial and more pragmatic and practical advice is available in BSH and CLSI guidelines. The potential for improved specificity comes at a statistically inevitable cost to sensitivity, which has been demonstrated in some recent publications. The perennial problem of detecting LA in anticoagulated patients has become further complicated by the ever increasing use of direct oral anticoagulants, although it has re-ignited interest in the snake venom prothrombin activator assays. It could be argued that the issue warrants its own guideline to permit sufficient detail, although it continues to evolve, such as recent availability of ‘anticoagulant-insensitive’ dRVVT reagents.

Rapid results and good analytical performance of the CP3000 automated coagulation analyzer Sarah Just1, Diane Zebeljan1, Larry Smith2, Julie R Loats3, Kim Hill3, Gabriella Lakos2 1 Haematology Department, New South Wales Health Pathology (NSWHP) Liverpool, Liverpool Hospital, Sydney, Australia 2 Abbott Laboratories, Santa Clara, CA, USA 3 Abbott Diagnostics, Australia

Objectives Time to first result, user experience and analytical performance of the Sekisui CP3000 were compared against the STAGO STA-R Evolution coagulation analyzer. Methods Time to first result was studied for PT and a panel of three assays in 5 independent runs. User experience was evaluated using weighted scoring survey of analyzer features. Remnant samples were tested on CP3000 for PT and APTT (n=215), TT (n=30), fibrinogen (n=74), D-dimer (DD) (n=40), antithrombin (AT) (n=47) and Protein C (PC) activity (n=33). Anti-Xa (heparin) activity was determined in patients on LMWH (n=34) and UFH (n=10), respectively. Results were compared against STA-R Evolution results using regression, Pearson correlation and qualitative agreement methods. Interference testing was performed using spiked lipaemic, icteric and haemolysed samples for PT/APTT assays. Results Method comparison studies showed correlation coefficients (r) ranged from 0.86 (APTT) to 1.0 (INR) compared to STA-R Evolution. Qualitative agreement were 79.0% and 97% for APTT and TT when STAGO-recommended reference ranges were used, and total agreement for PT results was 90%. Ninety percent of samples in INR range 2.0 to 4.0 had a difference < 0.5 between the two methods in pair-wise comparison (CLSI H57-A criterion) and mean difference in therapeutic range (2.0-3.0 INR) was -0.06. Interference testing showed no effect on PT/APTT results for lipaemia and icterus and minimal effect seen on APTT with both analysers. The average time to result was 133 seconds for PT, and 327 seconds for panel of PT, APTT and fibrinogen. In the user experience survey, CP3000 scored significantly higher for instrument size, QC monitoring, weekly maintenance and troubleshooting, but lower for calibration, QC processing, start-up, and noise. Conclusion Results obtained with CP3000 showed strong concordance with STA-R Evolution results. Time to first result was rapid, and the CP3000 rated favourably for size, QC monitoring, maintenance and troubleshooting.

Notes

von Willebrand factor/disease: From ristocetin to collagen binding and beyond Emmanuel J Favaloro, Institute of Clinical Pathology & Medical Research, Sydney Centres for Thrombosis and Haemostasis, NSW Health Pathology, Westmead Hospital, NSW 2145 The discovery that ristocetin promoted platelet aggregation in a von Willebrand factor (VWF) dependent manner, paved the way for the development of several diagnostic assays based on this finding. Undeniably, the ristocetin induced platelet aggregation (RIPA) and VWF ristocetin cofactor (VWF:RCo) assays are now part of the standard repertoire of laboratory tests for identification or exclusion of von Willebrand disease (VWD), in turn the most common bleeding disorder (arising from defects or deficiency of VWF). Indeed, VWF:RCo is still today considered the surrogate gold standard ‘functional’ or VWF ‘activity’ assay in VWD diagnostics. This presentation will explore VWD diagnostics and recent highlights. The development and increasing maturity of the VWF collagen binding (VWF:CB) assay as a second ‘functional’ or VWF activity assay in VWD diagnostics is one personal highlight. Rather than being a replacement for VWF:RCo, the VWF:CB is actually a supplementary assay that reflects a surrogate of another function of VWF, namely subendothelial matrix adhesion, as part of the process of attaching platelets to damaged tissue, and subsequent thrombus formation. The VWF:RCo, instead reflects a surrogate of a complementary function of VWF, being platelet adhesion, as part of the process of facilitating platelets to attach to each other and to damaged tissue, and thus also aiding subsequent thrombus formation. More recently, this functional surrogate of VWF binding to platelets, aka the classical VWF:RCo assay, has been morphed into a variety of ‘glycoprotein Ib (GPIb) – binding assays’, and has even spawned a new ISTH SSC recommended nomenclature, including terms such as VWF:GPIbR and VWF:GPIbM. These are assays that may or may not use ristocetin, and generally do not even use platelets [1-3].

Notes

Chromogenic FIX assays Geoffrey Kershaw, NSW Measurement of plasma FIX levels is required for the diagnosis and classification of patients with haemophilia B (HB), the post-infusion monitoring of FIX replacement therapy, and for potency labelling of FIX replacement products. Assays of FIX activity have been almost exclusively performed by one-stage APTT-based clotting assay (OSA), which have considerable inter-laboratory variability in results, related in part to the large variety of both APTT reagents and deficient plasma types used. The two currently available commercial chromogenic substrate FIX assays (CSA), from Rossix® and Hyphen Biomed® have been adapted to automated coagulation analysers. The CSAs are two-stage FIX assays: the first being the generation of activated factor X (FXa), the second being colour generation by the action of FXa on a chromogenic substrate. These kits were evaluated for their potential to monitor replacement therapy with recombinant and extended half-life FIX products without requirement for product-specific reagents or calibrators required when using some OSA. The Rossix and Hyphen kits were tested on the Sysmex CS2500 analyser. The Rossix kit and OSAs were tested on the Stago STA-R analyser. Plasma standards were used for all assays. Reference intervals were established from plasma of 128 healthy volunteers and were similar to current OSA levels. Between run reproducibility showed CVs of around 6% or less for all protocols. Recovery of FIX deficient plasma spiked with three recombinant FIX products to five levels from 0.05IU/mL to 1.2IU/mL was consistently within the range 100±25% of expected except for the Hyphen kit which showed slight under-recovery at low spike levels. The CSA protocols were generally more accurate and consistent in obtaining expected recoveries that three OSAs used for comparison. In samples from untreated HB there were some differences between the CSA kits and between CSA and OSA that would lead to re-classification of disease severity according to standard criteria. The CSA were successfully adapted to automated platforms for measurement of FIX in both patient and spiked samples. Full implementation of these assays in the clinical laboratory requires consideration of cost per test, which will be impacted by batch size, reagent stability and kit configuration.

Notes

Laboratory guidance for the measurement of Factor VIII Inhibitors

Piet Meijer,

ECAT Foundation, Voorschoten, The Netherlands

In up to 45% of severe haemophilia A patients the development of Factor VIII (FVIII)

inhibitors may occur as a result of treatment with Factor VIII products. Reliable and

reproducibly inhibitor testing is important as detection and monitoring of inhibitors is crucial

in therapy management.

The most widely used method for the detection of FVIII inhibitors is the Bethesda assay [1].

A modification of this method with improved specificity and sensitivity, the Nijmegen assay,

was described in 1995 [2]. This method is recommended by the International Society on

Thrombosis and Haemostasis as the reference method for Factor VIII inhibitor testing.

The assay methodology for FVIII inhibitors is rather complex and a number of variables may

influence the test result. The complexity of the assay system may result in undesirably high

within- and between-laboratory variation in test results.

Considerable between-laboratory variation for FVIII inhibitor testing (30 – 60%) has been

shown by several providers of external quality assessment programs. These observations

demonstrate the lack of standardization for Factor VIII inhibitor testing. It has been shown

that significant improvement in the comparability of test results between laboratories is

possible [3].

The results of this workshop demonstrate that standardization is feasible.

In the presentation the laboratory test for Factor VIII inhibitor will be discussed step-by-step

and important aspects for standardization will be highlighted.

The International Council for Standardization in Haematology (ICSH) has decided to draft a

laboratory guideline for testing of Factor VIII Inhibitor. This guideline is currently under

development. With the introduction of this guideline in laboratory practice it is assumed that

further improvement in the comparability of test results between laboratories can be

obtained.

Notes

Should personalized antiplatelet therapy become part of routine clinical practice? Paul Harrison

Birmingham University, UK

Antiplatelet therapy with aspirin (ASA) and a P2Y12 receptor antagonist is the mainstay of therapeutic

management in patients with cardiovascular disease. Several studies have shown that not all patients

benefit from the treatment to the same degree and that either high or low on-treatment platelet

reactivity is associated with increased risk of thrombotic or bleeding events respectively. Reduced

efficacy of ASA is frequently observed in Type 2 diabetes (T2DM), compared with the general

population. Indeed, the prevalence of “true” ASA poor responders is thought to be rare in the general

population but is certainly more frequent in T2DM. Failure to respond to ASA therapy is normally a

result of a combination of factors including genetic polymorphisms, drug interactions and poor

compliance. Despite this, current guidelines do not recommend laboratory testing for monitoring and

personalizing ASA therapy. In contrast, T2DM is a pro-thrombotic state characterized by increased

platelet reactivity and turnover, increased hepatic synthesis of fibrinogen and plasminogen activator

inhibitor (PAI-1) and quantitative changes in the glycation and oxidation of clotting factors.

Accordingly, it is possible that higher or more frequent doses of ASA might be required to potentially

overcome metabolic barriers in T2DM, particularly in conditions where platelet turnover is increased.

In contrast, considerable pharmacodynamic responses are observed with the P2Y12 receptor

antagonist clopidogrel with many patients giving a poor response. Unlike ASA, clopidogrel is a prodrug

that is converted to an active metabolite by several cytochrome P450 enzymes (CYP) and

polymorphisms of the CYP2C19 gene are associated with reduced efficacy. The newer P2Y12

antagonists prasugrel and ticagrelor are more effective in inhibiting platelet function but are also

associated with increased risk in bleeding. Many studies have demonstrated that the presence of

either high or low on-treatment platelet reactivity with these drugs as measured by ADP platelet

aggregation is associated with a higher incidence of thrombosis or bleeding respectively. This

supports the concept of a therapeutic window and the personalization or optimization of antiplatelet

therapy for individual patients. Despite this, the majority of tailored pharmacotherapy trials have

failed to demonstrate any clinical benefit in personalized therapy based upon platelet function

monitoring. The clinical benefits to this approach therefore remain uncertain. Study design, choice of

primary endpoints, choice of patient population and timing of platelet testing could explain these

results. For example, many trials were conducted in low-risk stable populations and exclusively

utilised the VerifyNow assay as the platelet function test of choice. Although the results have been

disappointing, this does not mean that we have totally disproven the potential utility of platelet

function or genetic testing in this setting. Furthermore the current platelet function tests are

inadequate for optimally identifying all patients at high risk of bleeding and thrombosis.

Improvements in platelet function testing and the understanding of the molecular events involved in

thrombus formation may result in the development of new tests with enhanced clinical value.

Ongoing and better designed trials are also in progress. Therefore individualization of antiplatelet

therapy may still be the future.when assessing the exosite II aptamer using the TEG with normal

blood.

Notes

Curcumin and fish oil effects on platelet aggregation and coagulation Lisa Lincz, NSW Dietary supplements are becoming increasingly popular, with >60% of Australians choosing to consume daily tablets that promise to improve their health and wellbeing. While some complementary medicines may be harmless, others can interfere with normal biological functions. Two of the more common supplements are fish oil and curcumin, both touted for their anti-oxidant and anti-inflammatory properties, with conflicting reports on their potential anti-thrombotic effects. Fish oils are a rich source long chain omega-3 polyunsaturated fatty acids including eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), and our work has shown that their anti-platelet effects are both differential and gender specific. Curcumin is the major active polyphenolic compound (comprising 2-5%) responsible for the yellow colour of Turmeric, a commonly used spice made from the rhizome of Curcuma longa. Most currently available preparations of curcumin contain approximately 77% curcumin, 18% demethoxycurcumin (DMC), and 5% bisdemethoxycurcumin (BDMC). These are broken down in the liver to form the main metabolite, tetrahydrocurcumin (THC). The bioavailability of curcumin is extremely low, but several invitro studies have shown it to inhibit platelet aggregation and a single study has demonstrated remarkable effects on coagulation, with both curcumin and BDMC able to prolong PT and aPTT by 2-3 fold. However, the effects of other turmeric derivatives, DMC and THC, on the coagulation system or platelet aggregation, have not been investigated. Any potential anticoagulant and/or anti-platelet effects of fish oil or curcumin could have profound effects for patients already taking anti-thrombotic medications for these purposes. Alternatively, these features could be exploited for therapeutic use. Thus we have investigated the individual effects of EPA and DHA supplementation in healthy individuals as well as curcumin, DMC, BDMC, and THC invitro on platelet aggregation and coagulation.

Notes

Application of flow cytometry to diagnose platelet dysfunction Elaine Uhr Institute of Haematology, Royal Prince Alfred Hospital, Sydney, Australia Platelet flow cytometry is a more specific technique than light transmission aggregation where the results for initial screening for a platelet abnormality are “suggestive of” a platelet disorder and prior to molecular characterisation of a specific platelet receptor genetic mutation. It utilizes single cell analysis to identify and quantitate inherited disorders as absence or reduction (heterozygotes) of platelet surface membrane receptors and, post platelet activation, secretion of platelet granule proteins onto the surface membrane. Efficient platelet washing pre assay enabled the development of flow assays for alloantibodies matched to WHO Standard and also for platelet specific auto antibodies. Aspects to be discussed include the requirement for strict adherence to assay technical conditions to produce valid results and examples of assay applications that demonstrate clinically useful information.

Notes

Platelet super-resolution microscopy: Helena Liang, NSW

The 200 nm optical diffraction limit of visible light prevents our use of conventional light microscopy to study a number of highly relevant biological structures in platelets such as the platelet mitochondria, its super-fine filopodia and other structures currently only visible through the use of high-resolution transmission and scanning electron microscopy (EM). Such EM methods are able to provide detailed morphological information, but challenging for identification of specific proteins within the revealed structures. More recent super-solution microscopy techniques such as direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM) are powerful optical methods able to resolve fluorescence below the 200 nm diffraction limit. We have been able to use an inexpensive home-built dSTORM system to visualize actin and tubulin within filopodia and changes in the platelet membrane after activation to approximately 20 nm resolution. SIM has resolution only to 50nm, but has the capacity for live imaging. These modalities will be important tools for direct imaging of changes in platelet membrane structure and sub-micron organelles after agonist exposure in order to enhance our understanding of these processes in platelet biology during thrombosis and haemostasis.

Notes

THANZ in 2017: website and social media Eileen Merriman, NZ THANZ Councillor This session will give an overview of the updated THANZ website, including the interactive Clique community, and social media links.

Notes