Ij Mackie

Guidelines on the investigation and management of antiphospholipid syndrome

This guidance updates and replaces the previous guideline on the investigation and management of antiphospholipid syndrome (APS) published in 2000 (Greaves et al, 2000), though where there have not been changes we refer back to them when appropriate. The guidance is updated with reference to relevant publications since 2000. Publications known to the writing group were supplemented with additional papers identified by searching PubMed for publications in the last 11 years using the key words: lupus anticoagulant, anticardiolipin, antiphospholipid, b2–glycoprotein I, antiprothrombin and limits (clinical trial, randomized control trial, meta-analysis, humans, core clinical journals, English language). The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 50 UK haematologists, the Royal College of Obstetricians and Gynaecologists (RCOG), and the British Committee for Standards in Haematology (BCSH) Committee and comments incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found at http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMEN DATION/43_GRADE.html. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of patients with antiphospholipid syndrome though individual patient circumstances may dictate an alternative approach.

The use of an anti-β2-glycoprotein-I assay for discrimination between anticardiolipin antibodies associated with infection and increased risk of thrombosis

Summary. Antiphospholipid antibodies (aPAs), occurring in association with infection, are not generally associated with an increased risk of thrombosis. Anticardiolipin antibodies (aCL) from patients with infection, unlike those from patients with SLE, do not have the β2GPI cofactor requirements. Antibodies to β2GPI (αβ2GPI) are more closely associated with a previous history of thrombosis than aCL in patients with SLE. In the present study we have investigated the reactivity of the αβ2GPI assay for aPAs associated with infection. Serum from 114 patients with infections including syphilis (n= 11), tuberculosis (n= 63) and Klebsiella (n=42) were assayed for αβ2GPI and aCL antibodies. The incidence of aCL in serum of patients with tuberculosis, Klebsiella infection and syphilis was 6.0%. 5.0% and 64.0%. respectively, but all patients were negative for αβ2GPI. These results indicate that the αβ2GPI assay is negative in patients with transiently positive aCL assays associated with infection.

Comparison of von Willebrand factor antigen, von Willebrand factor‐cleaving protease and protein S in blood components used for treatment of thrombotic thrombocytopenic purpura

Summary.  Replacement of normal levels of von Willebrand factor‐cleaving protease (VWF:CP, ADAMTS13) activity from infused plasma is important in plasma exchange (PEX) for the treatment of thrombotic thrombocytopenic purpura (TTP) patients. We have studied the VWF:CP activity, VWF multimer distribution, VWF:Ag, protein S (PS) activity and free PS antigen levels in fresh frozen plasma (FFP), cryosupernatant (CSP) and virally inactivated components treated with methylene blue/light (MB) or solvent detergent (SD) processes. VWF:CP activity was normal in all components tested and was retained following overnight storage at room temperature. CSP and SD plasma contained reduced levels of the highest molecular weight VWF multimers. Protein S activity was reduced below the normal range in SD plasma, but within the normal range for the other components tested. Virally inactivated SD‐ and MB‐treated plasma may be an effective alternative to FFP and CSP in PEX for TTP. Reduced PS activity in SD plasma may predispose to venous thromboembolism, especially if infused in large volumes.

Cigarette smoking and platelet adhesion

Non‐abraded rabbit endothelium has been exposed to human blood taken from male non‐smoking volunteers before and after the smoking of two medium tar cigarettes, in an in vitro system using a Baumgartner chamber. In each case the blood was allowed to circulate for 10 min at a constant flow rate. Blood from 10 volunteers has been tested in this way. Scanning electron microscopy of the endothelial surfaces demonstrates large numbers of adherent platelets when ‘post‐smoking blood’ is used, but very few and in some cases none with the ‘pre‐smoking’ blood. As a further control to ensure that this phenomenon did not occur as a result from changes in the vessel related to the time during which it had been removed from its normal physiological environment, blood from further non‐smoking volunteers was passed over seven of the remaining pieces of vessel at the completion of these runs. Platelets were either absent or very few in number, as with the pre‐smoking samples.

Reduced factor XII levels in patients with the antiphospholipid syndrome are associated with antibodies to factor XII.

Antibodies to factor XII (FXII) have previously been identified in some patients who were lupus anti‐coagulant‐positive. The relationship between these antibodies and FXII levels appeared to be variable. The aim of the present study was to confirm the presence of antibodies to FXII in patients with well characterized antiphospholipid syndrome (APS) and to establish their potential effect on levels of FXII. Forty‐two patients with APS were studied; 21 patients were found to have either immunoglobulin (Ig)G or IgM antibodies to FXII by enzyme‐linked immunosorbent assay (ELISA) using a highly purified preparation of FXII (> 99% pure). Levels of FXII were statistically significantly lower (P = 0·02) in patients with antibodies to FXII when compared with patients without antibodies to FXII (median = 91 μ/dl, s.d. = 39·1, median = 122 μ/dl, s.d. = 41·1 respectively). Four of the 21 patients with antibodies to FXII were found to have FXII levels below the laboratory normal range. Antibodies to FXII are present in significant numbers of patients with APS and may lead to acquired FXII deficiency.

Prothrombotic changes in children with sickle cell disease: relationships to cerebrovascular disease and transfusion

Vascular occlusion has a central role in the pathophysiology of sickle cell disease (SCD) and, although there is little evidence that thrombosis alone is responsible, patients with sickle cell disease are known to have an ill‐defined but increased thrombotic risk. The most serious complication of this in childhood is stroke which occurs in 7–10% of children and a further 14% have asymptomatic cerebrovascular disease (CVD) on imaging. We have performed a comprehensive profile of coagulation inhibitors and markers of thrombin generation in 96 children (83 non‐transfused [NTx] and 13 transfused [Tx]) with steady‐state SCD and 18 healthy sibling controls. The levels of protein S (free and total) and heparin cofactor II were reduced in both the NTx and Tx groups compared to controls and protein C and APC resistance ratios were reduced in the NTx group only. Antithrombin levels were not different from controls. Thrombin–antithrombin complexes and prothrombin fragment F1+2 were increased in both patient groups. In the NTx subgroups with or without CVD there were no differences for any of the parameters measured except for lower haemoglobin levels and higher white cell counts in those with asymptomatic CVD. We conclude that children with SCD have a reduction in levels of the majority of the coagulation inhibitors and increased thrombin generation in the steady‐state and these are only partially reversed by transfusion. However, these abnormalities do not appear to play a primary role in the development of cerebrovascular disease.

Maintaining blood flow in the extracorporeal circuit: haemostasis and anticoagulation

ObjectivesTo review the methods and developments in maintaining extracorporeal circuits in critically ill patients.DesignThe review includes details of the pathophysiological processes of haemostasis and coagulation in critically ill patients, methods of maintaining blood flow in the extracorporeal circuit and methods of monitoring anticoagulation agents used.SettingInformation is relevant to the management of critically ill patients requiring extracorporeal renal and respiratory support and cardiopulmonary bypass.ConclusionsHeparin is the mainstay of anticoagulation for the extracorporeal circuit although the complex abnormalities of the coagulation system in critically ill patients are associated with a considerable risk of bleeding. Alternative therapeutic agents and physical strategies (prostacyclin, low molecular weight heparin, sodium citrate, regional anticoagulation, heparin bonding and attention to circuit design) may reduce the risk of bleeding but expense and difficulty in monitoring are disadvantages.

Continuous venovenous haemofiltration using polyacrylonitrile filters does not activate contact system and intrinsic coagulation pathways

AbstractObjectives: To investigate whether continuous venovenous haemofiltration using polyacrylonitrile filters causes activation of the contact system and intrinsic coagulation pathways and if this, and/or low plasma levels of endogenous anticoagulants, influences filter lifespan. Design: Observational study. Setting: University Teaching Hospital Intensive Care Unit. Patients: Twelve critically ill patients with acute renal failure receiving continuous venovenous haemofiltration. Interventions: Blood samples were taken before starting haemofiltration, at 15 min, 1 h, 3–4 h, 8–12 h, 24 h and at 24-h intervals thereafter until filter blockage occurred. Measurement was made of the contact and intrinsic coagulation system proteins factor XII, activated factor XII and prekallikrein and the protease inhibitors antithrombin III, heparin co-factor II, alpha2-macroglobulin and C1-esterase inhibitor. Thrombin-antithrombin complex levels were measured to provide evidence of thrombin generation. Results: (i) Factor XII, prekallikrein and contact system inhibitors were subnormal in 10/12 and activated factor XII raised in 11/12 patients at baseline, implying pre-existing contact pathway activation. (ii) No change occurred during haemofiltration in the intrinsic coagulation pathway factor or inhibitor levels. (iii) Clotting of the filter circuit within the first 24 h occurred in 5/12 and was associated with low baseline levels of antithrombin III and heparin co-factor II. Only in these patients did thrombin-antithrombin complex levels rise significantly. Conclusions: The contact system was not activated further by continuous venovenous haemofiltration using polyacrylonitrile filters in critically ill patients. Premature clotting of the haemofilter circuit was more common in patients with very low levels of antithrombin III and heparin co-factor II; although this was related to thrombin generation, the intrinsic coagulation pathway does not appear to be implicated.

Some antiphospholipid antibodies bind to β2-glycoprotein I in the absence of phospholipid

Summary. Some antiphospholipid antibodies (aPL) only bind to anionic phospholipids in the presence of a serum cofactor, β2‐glycoprotein I (β2GPI). Whether these aPL can bind to β2GPI in the absence of phospholipid is controversial. We have purified anticardiolipin antibodies (aCL) from the plasma of four patients and β2GPI from normal plasma by solid phase affinity methods. All four aCL bound to cardiolipin and phosphatidylserine in the presence of β2GPI but not in its absence. The binding of two of the antibodies to cardiolipin and phosphatidylserine at various concentrations of human β2GPI was compared with that obtained using 10% bovine serum. The two antibodies responded differently to increasing β2GPI concentrations, and binding to phosphatidylserine was relatively greater than to cardiolipin using human β2GPI. All four aCL bound to plastic plates coated with β2GPI in the absence of phospholipid, and β2GPI in the fluid phase had no effect on binding. Binding to β2GPI coated plates was increased equally when bovine serum or bovine albumin were used as the sample diluent in place of gelatine. These findings and those of others have important implications for the design of assays for antiphospholipid antibodies.

The characterization and impact of microparticles on haemostasis within fresh‐frozen plasma

Background  We have previously demonstrated that clot formation in fresh‐frozen plasma (FFP) is influenced by the presence of microparticles (MP). In this study, the cellular source(s), properties and influence of MPs on clot formation within FFP were further characterized.