Thomas Exner

• Doctor of Philosophy
• Managing Director at Haematex Research Pty Ltd

Direct oral anticoagulants (DOACs) cause unwanted interference in various hemostasis assays, including lupus anticoagulant (LA) testing, where false positive and false negative identification may occur. DOAC Stop (DS) is an activated charcoal (AC) product used to specifically and effectively adsorb DOACs from test plasma. This process normally requ...

We aimed to review the interfering effect of DOACs on tests for haemostatic function and then to discuss overcoming these with activated carbon (AC) products, thereby eliminating DOAC issues from test plasmas. Recent relevant articles were reviewed and are discussed. Laboratory tests for DOACs, lupus anticoagulant, factor assays and APC Resistance...

Introduction: Clotting test results are currently not useful for estimating direct oral anti-coagulant (DOAC) concentrations because baseline results vary. DOAC Stop is a DOAC extracting agent with no effect on clotting factors. We investigated if aPTT (activated partial thromboplastin time) and dRVVT (dilute Russells viper venom time) results mig...

Background The aim of the study was to investigate the specificity of an activated charcoal-based product (DOAC Stop™) initially intended for the specific extraction of direct oral anticoagulants (DOACs) from test plasmas on a range of other anticoagulants. Methods Test plasmas were prepared by adding various anticoagulants to pooled normal plasma...

Aim: To evaluate a simple method using an adsorbent product (DOAC Stop) for extracting direct oral anti-coagulants (DOACs) from plasmas. Method: DOAC Stop was tested on normal and a range of abnormal plasmas initially using activated partial thromboplastin time (APTT) tests and a more DOAC-sensitive Russells viper venom-based clotting test (DOAC...

Background: The dilute Russell's viper venom time (dRVVT) has been suggested for the assessment of the intensity of anticoagulation of all direct oral anticoagulants (DOACs). This study aimed to compare the performance of an optimized liquid-stable dRVVT-based DOAC assay (DRVV-DOAC) on clinical samples before and after mixing these with normal poo...

To investigate the biological effects of foam sclerotherapy in vivo. Ultrasound-guided sclerotherapy was performed using a 3% sodium tetradecyl sulphate or polidocanol. A total of 15 mL of foam was injected. Samples were collected from antecubital veins, target saphenous veins and the adjoining deep veins before, immediately after and 1 hour after...

To investigate the in vitro effects of detergent sclerosants sodium tetradecyl sulphate (STS) and polidocanol (POL) on clot formation and lysis. clot kinetics were assessed in whole blood by thromboelastography (TEG®) and rotational thromboelastometry (ROTEM®). Fibrinogen was measured by the Clauss method in plasma and factor XIII (FXIII) by enzyme...

To investigate the effects of Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on fibrinolytic mechanisms. Measurements were done with serial dilutions of sclerosants in whole blood (WB), platelet rich (PRP) and platelet poor plasma (PPP). Control experiments were done in 5% bovine serum albumin (BSA), spiked with the enzyme/inhibitor. Plasmi...

It has been widely accepted that microparticles expose phosphatidylserine which in turn binds annexin V. It was the objective of this study to compare the antigenic characteristics and phospholipid-dependent procoagulant activity of annexin V positive and -negative subpopulations of platelet-derived microparticles. Annexin V positive and -negative...

One of the mechanisms by which platelet-derived microparticles elicit procoagulant activity is by an increased exposure of phosphatidylserine on their surface. We have previously demonstrated the utility of an activated factor X-based assay for the detection of procoagulant phospholipid activity [Xa clotting time (XACT)]. The objective of this stud...

The objective of this study was to validate a simple factor Xa-based clotting test developed to monitor procoagulant phospholipids (PPLs) and platelet-derived microparticles (PMPs). This assay is easily automated, giving it a major advantage over the more laborious and expensive flow cytometry, electron microscopy and ELISA techniques in general us...

To investigate the in vitro effects of detergent sclerosants on antithrombotic pathways. Proteins C, S and antithrombin (AT) were assayed in normal plasma treated with increasing concentrations of sodium tetradecyl sulphate (STS) and polidocanol (POL). Activated protein C (APC) was investigated by mixing normal plasmas with sclerosants and testing...

To investigate the lytic effects of sodium tetradecyl sulphate (STS) and polidocanol (POL) on erythrocytes, platelets, endothelial cells and platelet-derived microparticle (PDMP) formation in vitro and the potential protective effects of serum albumin and agents such as procaine. The effects of sclerosants were studied in blood samples obtained fro...

To investigate the in vitro effects of Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on clotting tests, clotting factors, platelets and microparticles. Platelet rich (PRP) and platelet poor (PPP) plasmas were incubated with varying concentrations of STS and POL. Clotting tests, platelet/plasma turbidity, and microparticle studies were perf...

Diabetes is associated with disturbances in haemostasis that are thought to result in an increased incidence of thrombotic complications and cardiovascular disease. The aim of this pilot study was to monitor activation of haemostasis using specific markers for platelet activation and coagulation. Plasma samples (all blood collected and plasma prepa...

Sickle cell disease (SCD) is considered by many workers to be a hypercoagulable state. The vaso-occlusion in sickle cell disease is complex, involving adhesive processes between sickle red blood cells, leukocytes, and the activated endothelium. Loss of normal membrane phospholipids asymetry with the appearance of phosphatidylserine on the erythrocy...

Increased platelet activation is well documented in patients with acute coronary syndromes and can be detected by various methods, including flow cytometry and enzyme-linked immunosorbent assay. However, such techniques require several steps and cannot provide quick results. Platelet activation ultimately results in procoagulant phospholipid exposu...

Excess platelets shorten most clotting tests for lupus anticoagulant (LA). Often it is not clear if a shortened, normal or slightly prolonged result in a test masks a weak LA in combination with activated platelets, which express procoagulant phospholipid (PPL). Our aim was to investigate a new LA-insensitive factor Xa-activated clotting time (XACT...

An improved activated factor X-based clotting method was used to investigate activity of procoagulant phospholipid (PPL) in blood samples collected into various anticoagulants and in plasmas with a range of abnormalities. The dilute activated factor X-activated clotting time (XACT) was carried out on a mixture of specimen with phospholipid-free por...

The aim of the following presentation is to review certain technical issues complicating clotting tests for lupus anticoagulants (LA). This is a field riddled with incorrect and misleading studies which often make progress difficult and which are difficult to retract. Inconsistent test sensitivity comparisons are sometimes due to incorrect methods...

Circulating anticoagulants are usually defined as pathologic substances interfering with the clotting mechanism. They occur occasionally in coagulation testing laboratories and are often difficult to identify accurately. The following talk aims to clarify laboratory methods available for those inhibitors which are antibodies. There are two main cla...

Recent improvements in testing for lupus anticoagulants (LA) have come about through the availability of more specialised test methods, reagents, references, instrumentation and following the analysis of several multicentre laboratory surveys. Screening tests vary widely in LA sensitivity. Tests are also variably affected by platelets and abnormali...

Until recently the most common laboratory testing for thrombophilia encompassed assays for detecting reduced levels of the antithrombotic proteins antithrombin III, protein C and protein S. These assays were initially based on immunologic, subsequently chromogenic and most recendy on functional methods. The rate of abnormality detection was exceedi...

A method for detecting activated protein C (APC)-resistant factor V, especially factor V Leiden, is described, which uses reagents containing two unfractionated snake venoms. The procedure can be used for testing plasma samples from patients receiving oral anticoagulant therapy, heparin therapy and patients with lupus anticoagulant, and does not re...

Two simple procedures were tested for their potential to identify β2-glycoprotein 1 (β2GPl) or prothrombin cofactor dependence among lupus anticoagulants (LA). The first comprised mixing test plasma 1:4 with β2GP1-deficient plasma instead of with normal plasma. β2GPl deficiency decreased, but did not abolish most LA detectable in KCT, DRVVT and APT...

The method and apparatus are useful for determining a propensity of a blood sample to change from a liquid state to a coagulated state and additionally for measuring the propensity of a coagulated blood sample to lyse. The method includes providing a porous sheet, at least one surface of which contacts an impervious layer; applying the blood sample...

Until recently, laboratory testing foi thrombotic risk has been an expensive and technically-difficult procedure with a very low rate of true abnormal finding. Hypercoagulability may be inherited, such as that due to genetic defects in antithrombin lu, factor V, protein C or S, or acquired, such as in autoimmune disease or after tissue trauma. Test...

Various types of circulating anticoagulant are encountered in coagulation testing laboratories. Those associated with bleeding often cause problems in diagnosis. The most common type of acquired coagulation inhibitor not associated with bleeding is the so-called lupus anticoagulant (LA). Differing from SLE which occurs predominantly in women, prima...

Lupus anticoagulants (LA) have been defined as phospholipid-interfering antibodies. Testing for them has become a frequently requested procedure in coagulation laboratories and new methods have recently become available. Activated partial thromboplastin time (aPTT) reagents with reduced levels or different types of phospholipid provide high sensiti...

To examine the hypothesis that Doppler-defined umbilical placental insufficiency is associated with intravascular coagulation in the fetal circulation. A prospective, descriptive, single centre study. The University of Sydney, Department of Obstetrics at Westmead Hospital. Ninety-one infants were classified on the basis of the systolic:diastolic (S...

Objective To examine the hypothesis that Doppler-defined umbilical placental insufficiency is associated with intravascular coagulation in the fetal circulation. Design A prospective, descriptive, single centre study. Setting The University of Sydney, Department of Obstetrics at Westmead Hospital. Subjects Ninety-one infants were classified on the...

International Reference Preparations (IRP) of Thromboplastins available from the Bureau Communautaire de Référence (BCR) of the European Communities are expensive and inconvenient to import. Therefore, we endeavoured to produce a locally acceptable National Standard Thromboplastin (NST01). Samples of the stabilized, lyophilized human brain thrombop...

Until recently all non-specific circulating anticoagulants (NSCA) were described as “lupus inhibitors”. The adoption of phospholipid or platelet correction tests to distinguish non-haemorrhagic inhibitors (which dissplay positive PL correction) from haemorrhagio inhibitors, which fail to correct with PI and the use of various other tests has led to...

We report here on the usefulness of the 14C-serotonin release assay for the laboratory confirmation of the clinical diagnosis of heparin-induced thrombocytopenia syndrome (HITS). Over the past 3 yrs, some 140 individual serum samples have been tested in our laboratory for heparin-associated anti-platelet activity ('heparin antibodies'). These inclu...

We have developed and evaluated an ELISA-based collagen binding assay (CBA) as an aid in the diagnosis and classification of von Willebrand's disease (vWD). The assay is simple to perform, and appears capable of differentiating Type II vWD from Type I vWD. Using plasma samples from both affected and non-affected patients, or from normal individuals...

A coagulopathy attributable to a deficiency of vitamin K-dependent clotting factors (II, VII, IX, and X) was diagnosed in 3 Devon Rex cats. There was no evidence for exposure to vitamin-antagonist-related rodenticides. The cats did not have evidence of hepatic disease, gastrointestinal disease, or fat malassimilation. Oral treatment with vitamin K1...

Measurement of the plasma concentration of glycocalicin, the extracellular portion of platelet glycoprotein Ib, should prove to be clinically useful in the investigation of causes of thrombocytopaenia and as an indirect method of determining platelet lifespan. We describe an immunoradiometric method for the measurement of glycocalicin in plasma usi...

Six lyophilized plasma samples were sent to 20 "expert" laboratories for assessment of lupus anticoagulant (LA). Four samples contained pooled LA of graded potency mixed with aged normal plasma. One contained LA plus cephalin phospholipid and one contained a nonspecific venom anticoagulant. Sixteen methods were used overall with some participants u...

Six lyophilized plasma samples were sent to 20 “expert” laboratories for assessment of lupus anticoagulant (LA). Four samples contained pooled LA of graded potency mixed with aged normal plasma. One contained LA plus cephalin phospholipid and one contained a nonspecific venom anticoagulant. Sixteen methods were used overall with some participants u...

A simplified dilute Russell's viper venom time (DRVVT) test--in which the venom, trace phospholipid and calcium were combined into a single reagent--was evaluated for the detection of lupus anticoagulants (LA) in 28 plasma samples containing non-specific circulating anticoagulants. In agreement with previous studies, the DRVVT was found to be insen...

Phospholipid procoagulant material mainly derived from platelets interferes or "bypasses" the more specific tests for lupus anticoagulants. Such material in test plasmas can be inactivated with recovery of lupus anticoagulant activity by simple extraction with chloroform. This solvent treatment damages mainly factors VIII, V, VII and IX. Ether and...

Abstract We describe a man and his daughter from a large New Zealand family with the extremely rare variant von Willebrand's (VW) disease type IID. These two patients had a severe bleeding history following minor surgery and displayed easy bruising. However, routine laboratory screening tests and factor VIII studies were essentially normal except f...

The correlation between lupus anticoagulant (LA) potency and anticardiolipin antibody (ACA) ELISA was found to be poor (r = 0.40) in a group of 56 patients accumulated by a haematology department mainly for studies of LA. This correlation was similar whether LAs were assessed by kaolin clotting time or activated partial thromboplastin time incremen...

Recently the significance of a finding of iupus anticoagulant (LA) in a patient has changed. Previously regarded merely as a laboratory curiosity LA are now recognised as potential risk factors for thrombosis and in women appear to be responsible for recurrent fetal loss. Though it was initially suggested that LA and anticardiolipin antibodies (aCL...

A 28-year-old, 16 week primigravida presented with an acute anteroseptal myocardial infarction and a past history of recurrent venous thromboembolism and primary infertility. Although she lacked other clinical features of systemic lupus erythematosus, she had a circulating 'lupus' anticoagulant, anticardiolipin antibodies, a weakly positive anti-nu...

This paper describes the successful fractionation of crude snake venoms by hydrophobic interaction HPLC, with retention of biological activity. A fibrinogenolysin, with its biological activity intact, was isolated from crude E. carinatus venom with use of a hydrophobic interaction column (TSK-Phenyl 5PW). After further purification by ion-exchange...

Human endothelial cells, cultured from umbilical cord veins, have been evaluated for expression of a large number of cell surface antigens with known haemopoietic, particularly myeloid, cell distribution. This was achieved by evaluating endothelial reactivity (using non-fixed cells) with groups of monoclonal antibodies (MAB) belonging to distinct C...

Studies on four copolymers of ethylene and vinyl acetate (Elvax) have shown that with the exception of Elvax 760 (ethylene:vinyl acetate ratio 91:9) these substrates are as efficient as tissue culture grade polystyrene in supporting the attachment and growth of bovine aortal endothelial cells. The importance of extensive ethanol extractions on the...

We report here the use of perfluorosulfonic acid (Nafion) as a substratum for the growth of bovine aortal endothelial cells. This support which can be generated in a number of forms is at least as efficient in maintaining the growth of endothelial and other cell types as tissue culture grade polystyrene (TCP) and represents an advance in this regar...

We describe two novel developments. The first is the preparation of a "cemented" chromatography column comprising siliconized sand-coated by and cemented together with polystyrene thus providing a large hydrophobic surface area suitable for passive antigen absorption. The second is the coating of such a matrix with phospholipid and its use for sepa...

In a routine clinic of patients taking regular oral anticoagulant therapy (warfarin, n= 140) 14% were found to have elevated levels (>4 SD above normal) of anticardiolipin antibody (ACA) assessed by a well standardised enzyme linked immunosorbent assay (ELISA). There was a higher incidence of raised ACA in patients being treated for thrombotic epis...

The presence of the "lupus anticoagulant" in maternal blood is associated with thrombosis in the placental vessels and a high rate of fetal loss. We followed six pregnancies in four mothers positive for lupus anticoagulant autoantibody with Doppler umbilical flow studies. The women were not treated with any drugs, and clinical management was guided...

The protein C activator (PCA) detectable in the venom of Agkistrodon Contortrix Contortrix (ACCV, Southern Copperhead) by specific immunochromometric assay and anticoagulant activity has been isolated and partially characterized. Chromatog raphy of the crude venom on SP-Sephadex followed by Con A Sepharose and finally on hydroxylapatite was necessa...

This case reports the first description of Fitzgerald factor (high molecular weight kininogen) deficiency in Australia. Since this homozygous abnormality was found in an Aborigine it is suggested that the defective gene may be prevalent in some tribes and that abnormal results of clotting tests in Aborigines should be investigated carefully.

It is known that patients with thrombosis in autoimmune disease often suffer recurrent thrombotic episodes on withdrawl of oral anticoagulants, frequently have raised ACA and may require long term therapy. In view of the association between lupus inhibitors, phospholipid binding antibodies and thrombotic episodes we investigated a broad group of pa...

The protein C activator in Southern Copperhead (Agkistrodon Contortrix Contortrix) venom was isolated by sequential chromatographies on SP�Sephadex, Con A Sepharose and hydroxylapatite. It was found to be a single chain glycoprotein with an apparent molecular weight of 36,000 and an enzymatic specificity on chromogenic substrates resembling kallike...

Protein C binds readily from human plasma to antibody-coated wells, where it may be quantitated with an iodine 125-labeled antibody to protein C. Treatment with thrombin results in a small reduction in the protein C antigen detectable by this immunoradiometric assay (IRMA). However, activated protein C resulting from thrombin treatment and retained...

In separate experiments, antibodies to plasminogen, factor X and protein C were applied to microtitre trays as commonly used in enzyme-linked immunoassays. After incubation with dilute normal human plasma as a source of the corresponding proenzyme antigen, the wells were exposed to dilutions of various snake venoms. After thorough washing, the micr...

A series of monoclonal antibodies have been produced which recognize different epitopes of the factor VIII molecule. The antibodies were raised in mice against high purity factor VIII (FVIII) and the mouse spleens used in cell fusion experiments. Following cell fusion the hybridoma supernatants were used for screening with a solid phase radioimmuno...

Despite markedly prolonged activated partial thromboplastin times (APTT) patients with Fletcher factor (prekallikrein) deficiency do not clinically bleed. However, there is one reported case of myocardial infarction associated with prekallikrein deficiency. These clinical observations suggest that the contact mechanism has a minor role in normal in...

Potent lupus inhibitors from various patients were mixed with platelet free normal plasma and were compared in activated partial thromboplastin time (APTT), dilute prothrombin time (dil. PT), kaolin clotting time (KCT), contact product clotting time (CPCT), and Russell viper venom clotting time (RVVCT) tests. In the last three tests platelets and p...

Mixtures of various patient lupus inhibitor-containing plasmas and normal plasma were tested concurrently with the dilute tissue thromboplastin inhibition (DTTI) test and the kaolin clotting time (KCT). The KCT was found to be more sensitive to the presence of the lupus inhibitor than the DTTI. The principle of reducing the platelet phospholipid co...

Some properties of a monoclonal antibody generated against the fibrinogen component of a factor VIII preparation were investigated. The antibody bound with equal affinity in solid phase radioimmunoassays to fibrinogens isolated from both normal patients and patients with von Willebrand disease. It reacted in a sensitive immunoassay of plasma fibrin...

Potent lupus inhibitors from various patients were mixed with platelet free normal plasma and were compared in activated partial thromboplastin time (APTT), dilute prothrombin time (dil. PT), kaolin clotting time (KCT), contact product clotting time (CPCT), and Russell viper venom clotting time (RWCT) tests. In the last three tests platelets and pl...

A familial hypodysfibrinogenaemia occurring in four females with occasional haemorrhagic problems in an Adelaide family was investigated. Affected family members had slightly prolonged thrombin time, prothrombin time and Reptilase time tests, and apparently elevated levels of fibrin degradation products (FDPs). Fibrinogen assessed by reactivity wit...

Samples of normal tissues taken from three patients within 24 hours of death were physically disrupted, washed and extracted. Centrifuged supernatants were tested for factor VIII coagulant antigen (VIII CAg) and factor VIII related antigen (VIII RAg) by radioimmunoassays using homologous and heterologous antisera respectively. VIII RAg was readily...

Isoelectric focussing (IEF) in 6M urea/4% polyacrylamide gel of reduced human fibrinogen and the fibrins formed by reptilase and thrombin gave a complex multibanded pattern. Similar results were obtained on IEF in urea/agarose gels containing excess mercaptoethanol. To characterize these multiple bands on the basis of molecular weight secondary ele...

Concentrated, true solutions of ellagic acid (EA) prepared by pH reduction had only a small effect on contact activation initially. However when the EA formed a fine microcrystalline dispersion there was concurrent development of contact activity with turbidity. The dependence of contact activity on EA concentration was found to be a log-log relati...