A vitamin K antagonist rapidly reverses a blue toe syndrome in a patient with lupus anticoagulant and antiprothrombin antibodies.

A 30-year old male was admitted to the hospital with extremely painful blueish discoloration of his toes. After clinical and laboratory evaluation the diagnosis of a blue toe syndrome due to primary antiphospholipid syndrome (APS) was made. Complete resolution of the blue toe syndrome occurred within 72 hours following 9 mg phenprocoumon. APS consists of the association of lupus anticoagulant or antiphospholipid antibodies with arterial or venous thrombosis, thrombocytopenia, and spontaneous abortion. The exact pathways leading to thrombosis are still unknown. Our group has previously proposed that membrane-associated immune complexes contribute towards clinical symptoms in the antiphospholipid syndrome. The case presented strengthens that concept.

Monoclonal antibodies against beta-2-glycoprotein I: use as reference material for lupus anticoagulant tests.

The laboratory diagnosis of lupus anticoagulants (LA) remains difficult despite internationally accepted guidelines for its detection. Several interlaboratory surveys have shown poor agreement between laboratories. Further standardization of LA testing will to a large extent depend on better insights on the mechanisms by which LA affect phospholipid-dependent coagulation assays as well as on the availability of well characterized and internationally accepted reference materials and control specimens. We recently raised a series of murine monoclonal antibodies against human beta2GPI (anti-beta2GPI moabs), a phospholipid-binding protein directly involved in the interaction between certain lupus anticoagulants and phospholipids. In this study we report on the use of LA positive anti-beta2GPI moabs as easy to handle reference and control material. The relative LA responsiveness of various phospholipid-dependent clotting assays was determined on plasmas spiked with such moabs and compared well with that determined on LA positive plasma samples. Plasmas spiked with LA positive anti-beta2GPI moabs were also used as control specimens to study the interlaboratory precision of LA testing. With these control specimens, low interassay coefficients of variation were obtained. Our results indicate that LA positive anti-beta2GPI moabs have potential for the production of control specimens that could be made available to routine hemostasis laboratories to assess intra-laboratory precision of LA testing and to manufacturers to control batch-to-batch variability of their reagents.

Beta-2-glycoprotein I dependent lupus anticoagulants form stable bivalent antibody beta-2-glycoprotein I complexes on phospholipid surfaces.

The precise mechanism by which Beta-2-glycoprotein I (beta2-GPI-) dependent lupus anticoagulants lengthen phospholipid-dependent clotting reactions is still poorly understood. In order to study this, murine monoclonal antibodies (moabs) against human beta2GPI were raised. Eight of the 21 anti-beta2GPI moabs, obtained from 2 fusions, fulfilled the criteria for lupus anticoagulant (LA) activity as tested with a variety of sensitive screening assays and confirmatory tests. Seven moabs did not influence any clotting test. The LA positive moabs were found to compete for similar or closely spaced epitopes on immobilized beta2GPI. Two moabs with potent LA activity (moabs 22 F 6 and 22 B 3) and 1 moab without LA activity (moab 16 B 3) were selected to study the interaction between antibody, beta2GPI and phospholipid. Interactions were investigated by real-time biospecific interaction analysis (BIA) based on plasmon surface resonance technology on a BIA-core instrument using a sensor chip coated with phospholipid. When 22 F 6, the moab with the most pronounced LA activity, was allowed to interact with the phospholipid surface at concentrations between 0 and 400 nmol/l, no appreciable binding could be detected. Likewise, no binding could be measured when beta2GPI at concentrations between 0 and 400 nmol/l was passed over the phospholipid coated sensor chip. Combinations of beta2GPI and 22 F 6 resulted in significant binding. Similar results were obtained with 22 B 3, another moab with LA activity. A LA negative Moab, 16 B 3, did not cause binding of antibody-beta2GPI complexes. Fab' fragments, derived from moab 22 F 6, inhibited the binding of beta2GPI-22 F 6 and beta2GPI-22 B 3 in a concentration dependent way, indicating that only bivalent beta2GPI-antibody complexes bind with high affinity to phospholipids. Fab' fragments, derived from moab 22 F 6, also inhibited the LA effect of moabs 22 F 6 and 22 B 3 in diluted plasma. In summary, these experiments indicate that the beta2GPI-dependent LA effect depends on the formation of bivalent beta2GPI-antibody complexes on phospholipid surfaces.

High-dose intravenous immunoglobulin treatment of a pregnant patient with an antiphospholipid syndrome: immunological changes associated with a successful outcome.

A patient with a history of habitual abortion, deep venous thrombosis, thrombocytopenia, high titer IgG anticardiolipin antibodies and a clearly positive lupus anticoagulant, was treated during her seventh pregnancy with high-dose intravenous immunoglobulins (IVIg) from the third month onwards. Every month, a daily infusion of 400 mg immunoglobulins per kg body weight was given during five consecutive days. The patient's pregnancy ended preterm with a live birth, delivered by caesarian section because of a placental abruption. The 1070 g (P20-P25) weighing girl was in good health, apart from a bradycardia, due to dysfunction of the atrioventricular conduction. Each treatment with IVIg resulted in a slight reduction of both anticardiolipin antibodies and lupus anticoagulant levels and in an increase in platelet count. During the six-month observation period, a gradual decline in antiphospholipid antibodies and an increase in platelet count was found. The potential role of anti-idiotypic antibodies, present in the IVIg used for treatment, was studied. In vitro, IVIg were able to reduce the binding of the patient's anticardiolipin antibodies to cardiolipin coated microtiter plates. The presence of anti-idiotypic antibodies in IVIg was further documented by affinity chromatography and by realtime biospecific interaction analysis (BIA) on a BIA-core instrument. Affinity purified anticardiolipin antibodies were retarded on a column of insolubilized IVIg and a weak interaction was found between IVIg and affinity purified antiphospholipid antibodies, coupled to the BIA-core biosensor. In addition, the same technology revealed increased levels of anti-antiphospholipid antibodies in the patient's plasma following IVIg therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimization of the dilute prothrombin time for the detection of the lupus anticoagulant by use of a recombinant tissue thromboplastin.

The dilute prothrombin time (dPT) is a widely accepted sensitive screening test for the lupus anticoagulant (LA). In general, Simplastin (Organon Teknika), a rabbit brain thromboplastin, diluted 1/500, is used in this test. Recently, human tissue thromboplastin obtained by recombinant DNA technology has become available and we have evaluated the usefulness of one such preparation, Innovin (Dade), for the detection of the LA. dPTs, using several dilutions of Innovin were determined on plasmas from 18 normal individuals and 15 patients with a well-documented LA. The dPT ratios, calculated as the individual result divided by the mean normal, were statistically compared. Innovin in dilutions from 1/100 onwards was found to be significantly more responsive to the LA than Simplastin (P < 0.05). Intra-assay coefficients of variation (CVs) ranged from 1.05% to 14.8% with Innovin, versus 2.7%-24.3% with Simplastin (P < 0.05); inter-assay CVs ranged from 5.6%-11.8% with Innovin, versus 4.2%-33.8% with Simplastin (P < 0.001). Analysis of 316 consecutive plasma samples from non-anticoagulated patients, on which a LA determination was requested, showed a 100% sensitivity and a 96% specificity of the dPT performed with Innovin, as compared to 81% and 93% respectively for the dPT using Simplastin.

A new lupus anticoagulant neutralization test based on platelet-derived vesicles.

We here present an easily standardizable and reproducible procedure which clearly separates lupus anticoagulants (LA) from coagulation factor inhibitors. This new LA neutralization test makes use of platelet-derived microvesicles which were prepared as follows: gel-filtered platelets (4 x 10(5)/microliters) were incubated with 60 microM of the calcium ionophore A23187 for 20 min at 37 degrees C. The vesicles were separated from the platelet aggregates by centrifugation at 1000 g for 10 min. The vesicle containing supernatant was then spun down at 15,000 g for 15 min, lyophilized and stored at -20 degrees C until used. The vesicles were resuspended in plasma from normal individuals, from patients with LA activity, from patients with factor VIII inhibitors, from patients with congenital factor deficiencies and from patients receiving oral anticoagulants or intravenous heparin. A kaolin clotting time was performed in the absence (KCT) or presence of these vesicles (KCTves) and the ratios of these times to their respective mean normal times were calculated. Segregation of LA patients from all remaining patients except heparinized ones could be made with a high degree of accuracy. A thrombin time was needed to separate LA from heparinized patients. The method was highly reproducible and only minor (negligible) differences in potencies were observed between different vesicle preparations. Both the intra-batch and the inter-batch coefficients of variations on the KCTves were lower than 6%.

Recombinant human erythropoietin increases blood pressure, platelet aggregability and platelet free calcium mobilisation in uraemic children: a possible link?

Recombinant human erythropoietin was administered to 10 uraemic children on chronic haemodialysis, all of whom responded by correcting their haemoglobin. In addition, they showed an increase in blood pressure; platelet aggregations, subnormal before therapy, improved during treatment. The intracellular free calcium concentration in platelets after thrombin stimulation also increased significantly during erythropoietin administration. We hypothesize that the effect of erythropoietin on platelet aggregability and on blood pressure may be due to an increase in the intracellular free calcium mobilisation in platelets and possibly in smooth muscle cells respectively.

Detection of lupus-like anticoagulants by an enzyme-linked immunosorbent assay using a partial thromboplastin as antigen; a comparative study.

Clotting assays allow qualitative rather than quantitative detection of the lupus anticoagulant. We have therefore studied the usefulness of an ELISA using a commercial partial thromboplastin, Throombofax, as antigen; the results obtained on 146 selected patient plasmas were compared to the results of coagulation tests (kaolin clotting time, tissue thromboplastin inhibition test, activated partial thromboplastin time) and of ELISAs using cardiolipin or phosphatidylserine as antigen. While satisfactory agreement was found within the group of coagulation tests or that of ELISAs, only a moderate agreement was obtained between clotting tests and ELISAs, the best being with the partial thromboplastin ELISA using low plasma dilutions. The study further indicates that ELISA techniques cannot entirely replace coagulation tests for the detection of a lupus anticoagulant, even when a partial thromboplastin is used as antigen. On the other hand, coagulation tests are less sensitive than ELISAs for the detection of antiphospholipid antibodies.

Haemostatic effects of recombinant human erythropoietin in chronic haemodialysis patients.

Recombinant human erythropoietin was administered for up to 40 weeks to nine patients on chronic haemodialysis. From the third week of administration onwards, not only haemoglobin and haematocrit but also the platelet count rose, the latter, however, transiently. Subnormal platelet aggregation before therapy also improved transiently and in parallel with the erythropoietin dosage. The bleeding time normalized in almost all patients. There were no major side-effects. We conclude that recombinant erythropoietin improves haemostasis in chronic haemodialysis patients by increasing the haematocrit and in addition transiently enhances platelet number and function.