Thomas Exner

Separation of anticardiolipin antibodies from lupus anticoagulant on a phospholipid-coated polystyrene column

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 separating anticardiolipin antibodies from lupus anticoagulant activity in the plasma of a patient with the lupus obstetric syndrome. This is the first description of the separate identities of these two antibodies.

Heparin - induced thrombocytopenia: laboratory investigation and confirmation of diagnosis

Summary 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 included sera from 54 selected (4 positive, 50 negative) controls and a group of 86 patients where the test was requested on clinical grounds. Of 20 patients derived from within our institution, 7 out of 8 patients (88%) with good clinical probability of HITS were confirmed to have heparin platelet antibodies by the serotonin release assay. In contrast, only 2 out of 9 patients (22%) with a low clinical probability of HITS were shown to be positive by this procedure, as were 2 out of 3 patients (66%) deemed to have an ‘intermediate’ clinical probability of HITS. In addition, screening of 50 serum samples forming a ‘negative‐control non‐HITS’ group (either patients on heparin therapy without thrombocytopenia, patients with non‐heparin associated thrombocytopenia [eg. ITP*, other drug related], or normal laboratory volunteers), consistently failed to display heparin associated anti‐platelet activity by the 14C‐serotonin release assay. In addition to the good specificity and sensitivity described above, the 14C‐serotonin release assay was found to be nearly twice as sensitive when compared to the platelet aggregation procedure, and it is therefore a useful diagnostic test for the confirmation of clinically suspected Hits.

Detection of specific proenzyme activators in snake venoms by a new immunoabsorbant-chromogenic substrate method.

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 microtitre tray wells were tested overnight with chromogenic tripeptide substrates known to be relatively specific for the activated forms of the above factors, i.e., plasmin, factor Xa and activated protein C. The immunochromometric assay described detected two new activators of protein C in Agkistrodon piscivorus and Agkistrodon contortrix venoms and a new factor X activator in Agkistrodon rhodostoma venom. Gel filtration of the latter venom indicated that the factor X activator eluted with high molecular weight, was clearly distinct from the peak fibrinogen clotting activity (Ancrod) and appeared to have no procoagulant activity. Although several Bothrops venoms appeared to contain plasminogen activator by this technique, the observed strong chromogenic activity was observed in microtitre wells independently of plasminogen and represented nonspecific amidase activity.

The coagulation system in placental insufficiency: a study in the fetal circulation

Objective To examine the hypothesis that Doppler‐defined umbilical placental insufficiency is associated with intravascular coagulation in the fetal circulation.

Subclassification of non-specific circulating anticoagulants

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 an appreci-aion of heterogeneity among VSCA. We now describe two NSCA which display phospholipid correction but which are associated with minor bleeding problems, clinically unlike typical iupus anticoagulants OLA. Furthermore such haemorrhagio LA can be clearly distinguished from more typical LA by giving negative results in a dilute Russell’s viper venom time test (DRVVT). The first anticoagulant was detected in plasma from an 85 year old man requiring skin grafts after removal of superficial cancers from his legs. It was shown to be an IgM antibody which bound factor VIII weakly in vivo. The second anticoagulant was found in a 55 year old lady with a very long history of minor bleeding episodes associated with “LA”. Neither patient’s plasma displayed antiphospho-lipid antibodies in El.ISA tests. These inhibitors appear to function by interfering with the “tenase” complex rather than with the assembly of the “prothrombinase” comp1ex which is believed to be the target of more-typical “LA”.

Laboratory diagnosis of lupus anticoagulants

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) may be identical phosphotipid-binding immunoglobulins, more recent studies have shown the correlation between aCL and LA to be incomplete. Many patients have raised aCL without LA though the reverse situation is less common. The most sensitive screening tests for LA utilise low concentration of phospholipid for example in the kaolin clotting time, the dilute Russell’s viper venom test or the dilute tissue thromboplastin inhibition test. Prolongation of such tests by LA can usually be corrected by the addition of “excess” procoagulant phospholipid Conversely LA are not corrected by mixing with normal plasma. A LA test appropriate for a particular purpose should be selected. Thus hospital patients often present with other coagulation complications, e.g. anticoagulants, or liver disease, and the LA defect needs to be carefully distinguished from conditions likely to contribute to bleeding. Thus greater specificity is required for this group whereas sensitivity is the most important consideration for detecting LA in otherwise normal females being investigated e.g.for recurrent miscarriages. It is important to carry out LA tests on correctly processed samples particularly avoiding platelet activation which may ‘bypass” the LA defect.

Collaborative calibration of a national standard thromboplastin

International Reference Preparations (IRP) of Thromboplastins available from the Bureau Communautaire de Reference (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 thromboplastin and IRP 67/40 (BCR 099) were sent to eleven major laboratories who were willing to participate in the Australia-wide calibration. Testing was carried out according to the guidelines of the International Committee on Thrombosis and Haemostasis. A panel of four lyophilized, oral-anticoagulant patient plasmas was included for testing as a means of monitoring inter-laboratory variation. Each participating laboratory was instructed to test both the NST01 and the BCR thromboplastins simultaneously on sets of fresh normal plasmas and stable Warfarin patient plasmas (as well as on the lyophilized plasmas provided). The resulting International Sensitivity Index (ISI) of NST01 was calculated as 0.952xa0±xa00.008. This National Standard preparation is now available from the Australasian Reference Thromboplastin Unit (along with the series of Reference patient plasmas) for use by laboratories to improve inter-laboratory comparability of prothrombin time results.