Scientific Workshop

Virtual

18th September 2021

 

Thrombosis and Haemostasis Society of Australia and New Zealand.   The Thrombosis and Haemostasis Society of Australia and New Zealand (THANZ), previously named the Australasian Society of Thrombosis and Haemostasis  (ASTH), was  established  in  1994.  The  Society  represents over 300 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 THANZ 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 

Times are show AWST/AEST

9:00/11:00am Welcome: Grace Gilmore (Chair)

9:10/11:10am Emmanuel Favaloro First and second tier testing for VITT: platelet count, D-dimer, fibrinogen and immunological (ELISA) platelet factor 4 assays

9:4011:40am Vivien Chen Third tier testing for VITT: functional procoagulant platelet assay

10:00am/12:00pm Freda Passam

Third tier testing for VITT: functional Multiplate assay 10.20am/12:20pm Tim Brighton

Third tier testing for VITT: functional platelet serotonin release assay

10.45am/12:45pm MORNING TEA 11:00am/1:00pm Dianne Lovelock

A curious case of Heparin Resistance 11:15am/1:15pm Geoffrey Kershaw

Testing in Patients on Emicizumab 11.30am/1:30pm Sandya Arunachalam

RCPQAP - Update on Haemostasis and Point of Care (POC) Programs

11:45am/1:45pm Grace Gilmore

New Rapid test for ADAMTS 13 activity

12:00/2:00pm Closing Remarks

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

2021 THANZ Workshop possible

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Emmanuel Favaloro First and second tier testing for VITT: platelet count, D-dimer, fibrinogen and immunological (ELISA) platelet factor 4 assays COVID-19 (coronavirus disease 2019) represents a pandemic, and several

vaccines have been produced to prevent infection and/or severe sequelae

associated with SARS-CoV-2 (severe acute respiratory syndrome coronavirus

2) infection. There are now many reports of infrequent post vaccine associated

thrombotic events, in particular for adenovirus-based vaccines. These have

variously been termed VIPIT (vaccine induced prothrombotic immune

thrombocytopenia), VITT (vaccine induced (immune) thrombotic

thrombocytopenia), VATT (vaccine associated (immune) thrombotic

thrombocytopenia), and TTS (thrombosis with thrombocytopenia syndrome). In

Australia, the TGA reports the incidence of TTS as one in around every 70,000

AstraZeneca doses administered. In this talk, the initial (first and second ‘tier’)

laboratory test process, as utilised to assess suspected VITT, are reviewed. In

summary, first tier testing typically comprises platelet counts, D-dimer, and

fibrinogen, and second tier testing comprises ELISA based immunological

assessment for platelet factor 4 (PF4) antibodies. In suspected VITT, there is

a generally highly elevated level of D-dimer, a reduction in platelet count, and

PF4 antibodies can be identified by ELISA based assays, but not by other

immunological assays typically positive in HITT. Key to appropriate

identification/exclusion of VITT, and potential differentiation from heparin

induced thrombocytopenia with thrombosis (HITT), is identification of

potentially differential test patterns. Conversely, it is also important to not over-

diagnose VITT, given that not all cases of thrombosis post vaccine will have an

immune basis, and not all ELISA positive patients will be VITT.

   

Vivien Chen Third tier testing for VITT: functional procoagulant platelet assay

Freda Passam

Third tier testing for VITT: functional Multiplate assay Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) has emerged as a serious prothrombotic complication of the ChAdOx1 nCoV-19 (AstraZeneca) vaccine. Patients with VITT develop anti-PF4 antibodies that can activate platelets via the Fcgamma RIIa receptor causing thrombosis and thrombocytopenia. The mechanism of VITT shares similarities with heparin induced thrombocytopenia (HIT). HIT functional assays have been employed to aid in the diagnosis of VITT. Methods: A VITT functional assay by Multiplate was performed on samples received from 45 Australian patients referred for functional VITT testing with probable VITT: confirmed thrombosis within 4-42 days ChAdOx1-nCov-19 vaccination, D-Dimer > 5x upper limit of normal and platelets < 150 x 10^9/L [1]. The same samples had been tested on PF4/heparin ELISA (Asserachrom HPIA IgG Assay, Stago Diagnostics). Whole blood in hirudin was received from HIT antibody-responsive donor (as determined by reactivity to the anti-CD9 antibody, ALB6). The Multiplate method was based on Morel-Kopp, et al, JTH, 2016 [2]. Serum samples were heat inactivated (56oC for 45 min). Donor blood was incubated with normal saline, or heparin in normal saline, within the multiplate cell for 2 min at 37 degrees. The final concentration of heparin was 0, 0.5 and 100 U/ml. Patient serum was added to the cell and platelet aggregation was recorded over 6 min. Platelet aggregation was determined as the area under the curve (AUC) on the Multiplate readout. Serum from patients with confirmed HIT were used as positive control [2,3]. The normal range of AUC was determined by a group of 30 healthy and patient samples (without HIT or VITT). The average + 3SD AUC in the no-heparin condition was used as cut-off for Multiplate positivity in the samples of probable VITT. A positive result for VITT was reported for AUC >= 19.8 in the no-heparin condition. Results Multiplate testing had a fast turn-around time with Multiplate run time of 6 minutes for 1 patient sample at 3 doses of heparin (0.0.5, 100 U/ml). The test requires 450 ul of patient serum. Out of the 45 samples tested, 20 samples were reported positive by Multiplate (positive rate 44.4%) whereas 29 samples were positive by PF4 ELISA (positive rate 64.4%). The average inhibition of platelet aggregation with heparin 100 U/ml was 58.1% (range 21.4-84.6%). The accordance of Multiplate with PF4 ELISA was 68.9%. Conclusion: The Multiplate Analyzer provides a relatively fast functional assay which aids in the diagnosis and management of patients with VITT.

 

 

Tim Brighton Third tier testing for VITT: functional platelet serotonin release assay The Serotonin Release Assay (SRA) is considered a gold-standard assay for the diagnosis of Heparin-Induced Thrombocytopenia & Thrombosis (HITT). The method today is almost identical to original publication by Sheridan et al in 1986. C-14 labelled serotonin is incubated with known "reactive donor platelets and then subjected to patient's heat-inactivated serum. Activation of platelets via Fcy receptors by the anti-PF4 antibodies in the applied serum only in the presence of low dose (0.1 IU/mL) heparin leads to serotonin release. With recognition of VITT and potential similarities in pathogenesis to HITT we have made modification in our SRA test in 2021. In this talk I will discuss modifications made to the assays and our experience over the last few months is evaluating patients with VITT. Correlations with anti-PF4 antibody and other functional assays will be explored. Our observations suggest multiple pathogenic mechanism occur in apparent VITT.

Dianne Lovelock A curious case of Heparin Resistance Unfractionated heparin (UFH) is a sulphated glycosaminoglycan used intravenously as an immediate acting anticoagulant in acute thrombosis. The anticoagulant effect of UFH is routinely monitored using the activated partial thromboplastin time (APTT). Heparin resistance is defined as the requirement of high doses of UFH (>35000 IU/day) to achieve therapeutic APTT values. In the acutely ill patient, numerous plasma substances may interfere with both the action of heparin and the accurate measurement of the APTT, causing both true and apparent heparin resistance. We present the case of a 65-year-old man who was admitted to the emergency department with abdominal pain. He was diagnosed with acute pancreatitis complicated by splanchnic vein thrombosis. He had a long-standing history of chronic renal impairment which contraindicated direct oral anticoagulant (DOAC) use, and UFH therapy was initiated. However, despite increasing doses of UFH over several days his APTT remained subtherapeutic. Our investigations highlighted some of the issues in heparin monitoring. In particular, our testing showed extremely high factor VIIIc levels in this patient, ranging from 2.61 to 5.51 IU/mL. Factor VIII is often elevated in the acute phase reaction, thus complicating heparin monitoring in acutely ill patients. Our testing also demonstrated the effect of a possible variant antithrombin on heparin monitoring. Safe and effective management of UFH anticoagulation is most often dependent on the APTT being a reliable measure of anticoagulation. Heparin resistance, both true and apparent, can complicate monitoring of heparin, particularly in acutely ill patients. While the use of DOACs are increasing, the use of UFH is unavoidable in a subset of patients where these agents are contraindicated. Therefore, the limitations of the APTT in monitoring UFH tool must be well understood by the laboratory to ensure optimum care for patients.

Geoffrey Kershaw Testing in Patients on Emicizumab Emicizumab is a recombinant, humanised bispecific antibody which binds to and bridges FIXa and FX, thus acting as a FVIII mimetic. It is given as a subcutaneously and has a half-life of around 30 days. While it is given without the need for routine laboratory monitoring, assay of plasma emicizumab levels be needed in suspected non-adherence, unusual bleeding or suspected anti-drug antibodies. The mean therapeutic level in the pivotal HAVEN 1 trial was 50µg/mL, with a range of 35-70µg/mL. Being an antibody, emicizumab is essentially in an active state in the circulation, unlike FVIII which requires conversion to the active state by thrombin. As a consequence, even low plasma concentrations of emicizumab produce a dramatic shortening of the patient’s baseline APTT, which has flow-on effects to other intrinsic pathway tests such as Bethesda assays for FVIII inhibitors. The haemostasis laboratory therefore needs a strategy to test plasma containing emicizumab in different scenarios. The plasma emicizumab assay itself has proven to be quite robust and reliable by one stage clotting assay on multiple analyser platforms and with many different APTT reagent-FVIII deficient plasma combinations. This has been validated by in-house studies and confirmed by an Australian field study of emicizumab measurement, and more recently by external quality assurance programs. The latter found interlaboratory coefficients of variation were about half that seen in similar surveys of FVIII measurement. While a human-component chromogenic FVIII assay can also be used to quantitate emicizumab, it has little practical advantage over using a clotting assay. To measure plasma FVIII levels or FVIII inhibitor titres in presence of emicizumab the required method is a chromogenic FVIII assay with bovine components, as there is no cross-reactivity with emicizumab. Rare cases of anti-drug antibodies have also been reported, but an assay for anti-drug antibodies is not currently commercially available.

Sandya Arunachalam RCPQAP - Update on Haemostasis and Point of Care (POC) Programs There are currently 19 haemostasis and POC programs that the RCPAQAP offers. Over the past year, there have been significant developments made to the online portal for data entry and the reports. An overview of results and performance of D-Dimer, POC-INR and ROTEM / TEG will be presented. With POC tests, the industry is lagging in giving importance to EQA. This presentation will highlight the role of EQA in such settings. Changes that can be anticipated for 2022 will also be revealed.

Grace Gilmore New Rapid test for ADAMTS 13 activity Thrombotic thrombocytopenia purpura (TTP) is a rare life-threatening disease and timely diagnosis is critical. An ADAMTS13 activity level <0.1IU/mL is used to differentiate TTP from other TMA’s. Current test iterations based on FRET and ELISA assays take several hours at specialised central laboratories. A point-of-care screening test would expedite diagnosis. Suspect TTP patient plasmas (n=122) from 6 diagnostic laboratories in Australia and New Zealand were collected for the study. Local and central measurement of ADAMTS13 activity levels, were conducted using. TC Technozym ELISA assay and TC TECHNOSCREEN screening assay. Additionally, the central study site also included a Hemosil AcuStar ADAMTS13 assay. The qualitative screening test indicates if ADAMTS13 is at one of four level indicator points: 0, 0.1, 0.4 and 0.8IU/mL. And both TECHNOZYM and Hemosil gave a normalised gradient value between 0.0 – 1.0 IU/mL. ADAMTS13 activity levels from local and central study sites were comparable, r= 0.78. As were the levels of Hemosil with ELISA, r= 0.86. TECHNOSCREEN results were interpreted in binary, either above or below 0.1, the TTP clinical threshold. All but 1 of the screening results of 0 gave quantitative ELISA results <0.1IU/mL. There was 1 result of 0.4 that was <0.1IU/mL and no results at 0.8 resulted in activity levels less than <0.1IU/mL. The screening test provides a time critical qualitative ADAMTS13 evaluation. The simple assay operation makes it suitable for all laboratories, especially small and regional laboratories where quantitative levels are not performed.