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Summary Introduction A new prothrombin time reagent (Revohem™ PT) based on recombinant human tissue factor produced by the silkworm-baculovirus expression system was tested. The aim of this study was to compare the performance of the new PT reagent with two widely used routine PT reagents. Methods All testing was performed on a Sysmex CS-5100 coagulometer. Revohem™ PT was tested for imprecision and stability using normal and abnormal lyophilized commercial control plasmas. Comparability was assessed with two widely used reagents: one containing recombinant human tissue factor (Reagent A) and the other a human placental thromboplastin (Reagent B) using a wide range of normal and abnormal plasmas and analyser-specific ISI values. Results Excellent between-day imprecision was obtained for Revohem™ PT (CV

Essentials Complement activation has a pathogenic role in thrombotic antiphospholipid syndrome (APS). Coagulation proteases such as factor Xa can activate complement proteins. Complement activation markers were elevated in anticoagulated thrombotic APS patients. Complement activation decreased in APS patients switching from warfarin to rivaroxaban. SummaryBackground Complement activation may play a major role in the pathogenesis of thrombotic antiphospholipid syndrome (APS). Coagulation proteases such as factor Xa can activate complement proteins. Aims To establish whether rivaroxaban, a direct factor Xa inhibitor, limits complement activation compared with warfarin in APS patients with previous venous thromboembolism (VTE). Methods A total of 111 APS patients with previous VTE, on warfarin target INR 2.5, had blood samples taken at baseline and at day 42 after randomization in the RAPS (Rivaroxaban in Antiphospholipid Syndrome) trial. Fifty-six patients remained on warfarin and 55 switched to rivaroxaban. Fifty-five normal controls (NC) were also studied. Markers of complement activation (C3a, C5a, terminal complement complex [SC5b-9] and Bb fragment) were assessed. Results APS patients had significantly higher complement activation markers compared with NC at both time-points irrespective of the anticoagulant. There were no differences between the two patient groups at baseline, or patients remaining on warfarin at day 42. In 55 patients randomized to rivaroxaban, C3a, C5a and SC5b-9 were lower at day 42 (median (ng mL−1) [confidence interval] 64 [29–125] vs. 83 [35–147], 9 [2–15] vs. 12 [4–18] and 171 [56–245] vs. 201 [66–350], respectively) but levels of Bb fragment were unchanged. There were no correlations between rivaroxaban levels and complement activation markers. Conclusions APS patients with previous VTE on warfarin exhibit increased complement activation, which is likely to occur via the classical pathway and is decreased by rivaroxaban administration. Rivaroxaban may therefore potentially provide an additional benefit to its anticoagulant effect in this patient group by limiting complement activation.

Background: ADAMTS13 mutations play a role in thrombotic thrombocytopenic purpura (TTP) pathogenesis. Objectives: To establish a phenotype-genotype correlation in a cohort of congenital TTP patients. Patients/Methods: Clinical history and ADAMTS13 activity, antigen and anti-ADAMTS13 antibody assays were used to diagnose congenital TTP, and DNA sequencing and in vitro expression were performed to identify the functional effects of the ADAMTS13 mutations responsible. Results: Seventeen (11 novel) ADAMTS13 mutations were identified in 17 congenital TTP patients. All had severely reduced ADAMTS13 activity and antigen levels at presentation. Six patients with pregnancy-associated TTP and six patients with childhood TTP were homozygous or compound heterozygous for ADAMTS13 mutations located in the metalloprotease (MP), cysteine-rich, spacer and/or distal thrombospondin type 1 domains. The adults had TTP precipitated by pregnancy, and had overall higher antigen levels (median, 30ngmL-1; range

We report here a young female who underwent a successful deceased donor liver transplant for hepatic vein thrombosis. Five years after transplantation she developed postpartum atypical hemolytic uremic syndrome (aHUS). She did not recover renal function. Mutation screening of complement genes in her DNA did not show any abnormality. Mutation screening of DNA available from the donor showed a nonsense CFH mutation leading to factor H deficiency. Genotyping of the patient showed that she was homozygous for an aHUS CD46 at-risk haplotype. In this individual, the development of aHUS has been facilitated by the combination of a trigger (pregnancy), an acquired rare genetic variant (CFH mutation) and a common susceptibility factor (CD46 haplotype).

A persistently elevated level of factor VIII (FVIII) is an independent risk factor for venous thromboembolism (VTE). Although the pathophysiology of VTE is unclear, the involvement of thrombin generation (TG) has been postulated. Consequently this study was designed to (i) investigate the relationships between FVIII, Thrombin generation test (TGT) parameters and D-dimer in VTE patients, (ii) determine whether elevated levels of FVIII and increased TG in these patients are transient or sustained.After an initial period of anticoagulation had been completed 91 VTE patients and 52 healthy controls were recruited. FVIII levels were determined by one-stage clotting (FVIII:C) and chromogenic (FVIII:Ch) assays. The potential to generate thrombin was measured using the Calibrated Automated Thrombogram (CAT) and D-Dimer was by immuno-turbidometric assay.Patients' FVIII:C levels and FVIII:Ch, exhibited good agreement (rs=0.94; p0.0001), although FVIII:C exhibited a mean bias of -6%. FVIII:Ch show a significant correlation with TGT Peak Thrombin (rs=0.30; p=0.004) and Peak Thrombin was found to be significantly higher (p=0.04) in patients with FVIII200 iu/dL. Furthermore elevated levels of FVIII and increased thrombin generation parameters appeared to be consistent over time.Our data suggests that high FVIII leading to increased TG confers a significant risk of recurrent VTE and therefore we speculate that these patients may benefit from prolonged anticoagulation therapy.

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.

von Willebrand's disease (VWD) is regarded as the most common congenital bleeding disorder, and although not available in all laboratories von Willebrand factor (VWF) activity is most frequently assessed as ristocetin cofactor (VWF:RCo). This test can be technically challenging, is subject to poor sensitivity (∼20 IU dL(-1) VWF:RCo) and has a high degree of inter- and intra-assay imprecision [coefficient of variation (cv) > 25%]. We studied an automated assay using a combined fixed platelet/ristocetin reagent (BC von Willebrand reagent, Siemens Healthcare Diagnostics) on the CS-2000i analyser (Sysmex UK Ltd). Initially inter- and intra-assay imprecision was assessed. The automated method showed good day-to-day reproducibility and linearity of standard curves. This technique, also gave low intra- and inter-assay imprecision using commercial normal (cv < 4.5%) and pathological (cv < 8.1%) control plasmas. We then compared automated technique results from 30 healthy normal subjects and 39 VWD patients to those obtained using standard aggregometry (Bio/Data, PAP4) with lyophilised fixed platelets (Helena BioSciences) and ristocetin (American Biochemical and Pharmaceutical Ltd). The automated method had a sensitivity limit of approximately 10 IU dL(-1) vs. 20 IU dL(-1) for aggregometry. Samples giving results within the aggregometry measurable range (n = 50) exhibited good correlation with the automated technique (median 70 IU dL(-1), range 7-184 IU dL(-1); and 64 IU dL(-1), 6-138 IU dL(-1) respectively; R(2) = 0.85). We subsequently compared 3 different batches of BC von Willebrand reagent, using a second group of normal subjects and VWD patients (n = 35, 55-139 IU dL(-1) and n = 30

Summary. Background: Increasingly, patients with acute, idiopathic, antibody mediated thrombotic thrombocytopenic purpura (TTP) are being treated with rituximab to achieve a durable remission, however, there is the potential that it is removed by plasma exchange (PEX). Objectives: To look at the pharmacokinetics and pharmacodynamics of rituximab in patients with acute idiopathic TTP undergoing PEX. Patients and methods: Patients who received rituximab for acute idiopathic TTP (group 1, n = 30) and a control group (group 2, n = 3) of TTP patients in remission receiving rituximab electively as maintenance were included. Rituximab levels were measured before/after each infusion, before/after PEX and in follow-up. ADAMTS-13 activity, anti-ADAMTS-13 IgG and CD19% were measured to assess response. Results: The median number of PEX to remission after rituximab was 10 (range 4–25). In group 1 there was no significant incremental rise in the peak serum rituximab level until dose 4. Trough levels were lower in patients who had had PEX since their last rituximab infusion. In the control group, there was an incremental rise in the peak serum rituximab level and all patients had detectable trough levels. The median fall in rituximab per PEX was 65%. All patients achieved CD19 < 1%. In group 1, the median time to undetectable rituximab was 5 months (range 0–12 months) and to B cell return was 7 months (range 3–24 months). ADAMTS-13 increased and anti-ADAMTS-13 fell after therapy. There were three deaths and two relapses in group 1. Relapse was not temporally related to B cell return.

Background Octapharma PPGmbH has recently modified its manufacturing process for solvent/detergent-treated plasma to incorporate a prion reduction step, in which a 3 log reduction has been demonstrated. The current study was undertaken to assess the impact of this procedure on haemostatic variables in the new product OctaplasLG in comparison with standard Octaplas. Methods Production batches of standard Octaplas (n = 4) and OctaplasLG (n = 16) were assessed for levels of coagulation factors, physiological protease inhibitors, markers of activation and procoagulant microparticles. Global haemostasis was assessed by a thrombin generation test (TGT) and rotational thromboelastometry (ROTEM). Results Mean levels of factors: II, V, VII, IX, X, XI, XII and XIII, VWF:Ag, antithrombin, protein C and free protein S were all > 75 u/dl. ADAMTS-13 activity levels were normal. Factor VIII and VWF:RCo were > 55 u/dl. TGT and ROTEM were similar in both preparations, and microparticles were present at negligible levels. Two units of OctaplasLG had slightly elevated levels of Prothrombin Fragments 1 + 2, but D-Dimer and thrombin-antithrombin complexes were normal in all batches. Conclusion These studies indicate that the affinity chromatography procedure used in OctaplasLG does not appear to adversely affect the proven haemostatic quality of Octaplas, while offering a selective reduction in the concentration of pathological prion proteins.

Summary. Prostacyclin (Epoprostenol) or a stable prostacyclin analogue (ZK 36, 374) were added to platelet concentrates prior to cryopreservation. This resulted in significantly better preserved function of the thawed platelet concentrate, assessed by platelet aggregation to various concentrations of ADP, collagen and ristocetin, compared to control cryopreserved platelet concentrates. The use of prostacyclin or one of its stable analogues should be considered to reduce platelet activation and subsequent loss of function during the various manipulative procedures when preparing standard and cryopreserved platelet concentrates.

Background Factor VIII (FVIII) levels are used as a quality marker of fresh-frozen plasma (FFP); however, other clotting factors are not routinely measured. Methods We assessed additional haemostatic parameters and the dynamics of coagulation using Thrombelastography (TEG®) and a thrombin generation test (TGT). FFP was prepared on the day of donation (Day 0) or after overnight hold at 4°C (Day 1). Results Factor VIII in Day 1 FFP was 18% lower than in Day 0. TEG® parameters in Day 1 FFP were consistent with increased coagulability and did not correlate with altered levels of clotting factors, but were consistent with the increased levels of microparticles seen in the Day 1 samples. TGT studies exhibited increased lag time, time to peak and reduced peak thrombin generation, but no change in endogenous thrombin potential (ETP) on Day 1. There was a weak association between FVIII level and both ETP and peak thrombin (ETP rs≥ 0·22, P≤ 0·003; peak thrombin rs≥ 0·48, P≤ 0·0001), which was influenced by ABO group, with the lowest levels in group O. Conclusion We conclude that levels of FVIII do not predict the haemostatic potential of FFP and that there may be a role for alternative technologies in monitoring the quality of FFP.

Summary. Thrombin-induced platelet aggregation was inhibited in vitro by washed human neutrophils. Aggregation was inhibited in a neutrophil concentration dependent manner but glutaraldehyde fixed neutrophils had no significant effect on platelet aggregation. The neutrophil-derived inhibitory factor had the pharmacological profile of nitric oxide. Its action was potentiated by both superoxide dismutase and M&B22, 948, a selective cyclic guanosine mono-phosphate (cyclic GMP) phosphodiesterase inhibitor. Haemoglobin lessened this inhibitory action of neutrophils. L-Arginine, the substrate for nitric oxide formation, enhanced inhibition, whereas, manavanine, a structural analogue of L-arginine, prevented it. Nitric oxide release by neutrophils antagonized platelet ATP secretion and thromboxane B2 release. Inhibition was mediated by nitric oxide activation of guanylate cyclase with a subsequent rise in cyclic GMP. When neutrophils were stimulated with formyl-met-leuphe, there was a further increase in platelet cyclic GMP. This was enhanced by superoxide dismutase, but lessened by haemoglobin. Leukotriene B4 stimulation of neutrophils promoted inhibition of platelet aggregation. Leukotriene B4 alone had no direct effect on thrombin-induced aggregation of platelets. Platelets, when incubated with neutrophils and stimulated with calcium ionophore A23187, increased leukotriene B4 production by neutrophils in a platelet concentration dependent manner. Platelets alone were unable to release leukotriene B4. The action of platelets in haemostasis is modified as they come into contact with neutrophils. This may be an important physiological mechanism.

Background: Thrombotic thrombocytopenic purpura (TTP) is most commonly associated with deficiency or inhibition of von Willebrand factor-cleaving protease (ADAMTS-13) activity. ADAMTS-13 mutations and polymorphisms have been reported in childhood congenital TTP, but their significance in adult onset TTP remains unclear. Objectives: We sought to identify common ADAMTS-13 mutations in adults with late onset TTP and to investigate whether they may predispose acute clinical episodes of the disorder in adulthood. Patients/Methods/Results: We detected a missense mutation (C3178T) in exon 24 of ADAMTS-13 in 6/53 (11.3%) adult onset TTP patients, but no normal controls (n = 100). Three of the patients had pregnancy-associated TTP; three had chronic relapsing acute idiopathic TTP. C3178T encodes an arginine to tryptophan (R1060W) substitution in the TSP1-7 domain of ADAMTS-13. In vitro expression of mutant and wild-type ADAMTS-13 demonstrated that R1060W caused severe intracellular retention of ADAMTS-13 (

We report a feasibility study of patient self-testing telemedicine for anticoagulant management using TOPCARE (Telematic Homecare Platform in Cooperative Health Care Provider Networks, developed by the European Commission for a user-friendly information society). TOPCARE comprises a Home Telematic Box (TOPCARE BOX), a central database held on a remote server and secure computer terminals in the anticoagulant clinic. The TOPCARE BOX transmits encrypted International Normalized Ratio (INR) results from the CoaguChek S monitor (Roche Diagnostics) via the patient's telephone line to the database, which is accessed by health-care professionals via the Internet. The database displays the patient's anticoagulant record, highlighting out of range results, overdue tests and quality control results. The database can also send information back to the TOPCARE box, although currently only the next test date can be transmitted. 23 patients, on long-term oral anticoagulation were recruited from the hospital anticoagulant clinic. After completing a nurse-led training course, patients tested their INR weekly on a CoaguChek S and transmitted their results via the TOPCARE BOX. The nurse specialist accessed the patient's results, electronically entered the date of the next test. Changes in warfarin dose were telephoned. Four patients dropped out early in the study and 19 patients received TOPCARE BOXs of which nine were fully functional. Unintentional software misconfiguration meant that remaining 10 TOPCARE BOXs were nonfunctional (these were later reconfigured but not reintroduced into the study). Patients successfully transmitted 222 results over a 5-month period using the TOPCARE system. Early server problems were resolved, but intermittent problems with database access persisted and five results were not received by the server. Although concerns were raised regarding technical problems, feedback from patients and staff was favourable and the system thought to be user-friendly. In conclusion, this pilot study showed that telemedicine is a feasible option for anticoagulant management, but that the technology requires thorough testing prior to installation.

There is a limited evidence base for self-testing and -management for oral anticoagulation management. Available data suggest that these are credible models for a significant minority of patients if underpinned by structured training and follow-up. The guidelines presented are necessarily consensual and outline procedures for patient selection, training, product procurement, product maintenance, quality assurance procedures, dosage adjustment and clinical supervision. The cost-effectiveness of these models remains to be elucidated within the UK. Further data on both health economic and clinical outcomes are required from UK based studies before widespread implementation of self-testing and management can be recommended on a wider scale.

Summary. Background: Cellular prion protein (PrPC) is a naturally occurring protein in normal individuals which adopts an abnormal conformation, termed scrapie prion protein (PrPSc) that is associated with disease. There is great concern that clinically asymptomatic variant Creutzfeldt–Jacob disease (vCJD) may transmit PrPSc in blood transfusion products. PrPC is widely expressed and has been found in human blood. The majority of cellular borne PrPC is associated with platelets (84%). Although PrPC mRNA has been demonstrated in platelets, the quantity is unknown and may not reflect the total PrPC present. Objective: To investigate the expression of PrPC in the megakaryocyte lineage. Methods: The expression of PrPC was studied in CD34+ cells, cultured megakaryocytes and platelets using electron microscopy, flow cytometry, semi-quantitative RT-PCR and immunofluorescence confocal microscopy. Results and conclusions: The expression of PrPC appeared to increase with differentiation and polyploidization in the megakaryocyte lineage. PrPC was located within platelet α-granules and its source is likely to be from megakaryocyte precursors. If PrPSc has a similar distribution, these results have implications for the selection of blood donors and preparation of cell-depleted blood products.

The importance of testing for anticardiolipin antibodies (aCL) in the diagnosis of antiphospholipid syndrome (APS) in patients with thrombosis has recently been challenged (ISTH SSC meeting, Boston 2002). We have analyzed the antiphospholipid serology of 123 patients with persistent antiphospholipid antibodies (aPL) attending our hematology department. The cohort was tested for anti-beta(2)-glycoprotein I (beta(2)-GPI) antibodies and aCL of IgG and IgM class and for lupus anticoagulant (LA). Ninety-six of these patients fulfilled Sapporo clinical criteria for APS and 70 of these patients had venous and/or arterial thrombosis. Patients with LA plus anti-beta(2)-GPI antibodies had significantly higher levels of IgG aCL and anti-beta(2)-GPI antibodies than those exhibiting positivity for only LA or anti-beta(2)-GPI antibodies (P0.05). Patients with aCL IgG levels over 60 GPLU were found in all cases to be positive for LA and anti-beta(2)-GPI antibodies; 25.2% (31/123) of all patients and 26.04% (25/96) of patients fulfilling Sapporo clinical criteria for APS were positive for aCL only. The mean IgG aCL level in the Sapporo clinical criteria positive patients who had aCL only was 11.5 GPLU (normal5 GPLU). These data indicate that omission of aCL testing from the clinical investigation of APS could lead to a failure to diagnose the syndrome in a proportion of patients.

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.

A comparative evaluation of four commercial coagulation test kits for screening the protein C pathway kits was performed at two centres. The tests were Acticlot® V-OUT (V-OUT), the PCA test (PCA), the GradiThrom PCP test (PCP) and ProC© Global (ProC). Reference ranges were established in 40 normal subjects and, with the exception of V-OUT and ProC, significant differences were observed between males and females. Consequently, sex-specific normal cut-off values (fifth percentile) were used that led to greatly improved sensitivity when compared with the manufacturers' recommended cut-off values. Plasma from patients with factor V Leiden (n = 23), congenital protein S deficiency (n = 19), congenital protein C deficiency (n = 11), lupus anticoagulant (n = 17) and thrombophilia with no demonstrable protein C pathway defect (n = 20) were tested. All kits showed 100% sensitivity to factor V Leiden, but sensitivity was variable for protein C deficiency (27-73%), and poor for protein S deficiency (29-35%). The lupus anticoagulant affected all kits to some degree, with 29-35% giving values below the fifth percentile of normal, whereas all kits gave 1/20 unexpected abnormal results in the thrombophilia group, with the same sample accounting for the abnormal results in three of the four kits. Overall sensitivity and specificity, respectively, for defects in the protein C pathway were: V-OUT, 60 and 91%; PCA, 70 and 86%; PCP 75 and 94%; and ProC, 66 and 88%. We conclude that all four kits lack the sensitivity and specificity required for routine laboratory screening for defects in the protein C pathway and cannot replace assays for the individual proteins of this system.

SummaryA significant proportion of patients with Essential Thrombocythaemia (ET) have thrombotic complications which have an important impact upon the quality, and duration of their life. We performed a retrospective cross sectional study of the prevalence of antiphospholipid antibodies (APA) in 68 ET patients. Compared to 200 “elderly” controls (> 50 years) there was a significant increase in anticardiolipin IgM (p < 0.0001) and anti β2 glycoprotein I (anti-β2GPI) IgM (p < 0.0001) antibodies in ET. Thrombosis occurred in 10/20 with APA and 12/48 without, p = 0.04, relative risk 2.0 (95% confidence intervals 1.03–3.86); these patients did not differ in terms of other clinical features. The prevalence of thrombosis in patients with dual APA (6/7) was significant when compared to those with single APA (p = 0.02) and the remaining patients (p < 0.0002). Also anti-β2GP1 IgM antibodies either alone, or in combination with another APA, were associated with thrombosis (p = 0.02). These results suggest that the prevalence of APA in ET and their influence upon thrombotic risk merit investigation in a larger study.

Rivaroxaban is non-inferior to warfarin for the treatment of venous thromboembolism, with regard to clinical efficacy and safety. The ex-vivo effects of warfarin versus therapeutic dose rivaroxaban on in-vivo markers of coagulation activation and thrombin generation remain undefined. The aim of this study was to compare the effects of warfarin and therapeutic dose rivaroxaban on ex-vivo thrombin generation (TG), and the in-vivo markers of coagulation activation, prothrombin fragment 1.2 (F1.2), thrombin-antithrombin complex (TAT), and D-dimer.Eighty-five patients with venous thromboembolism were studied, 45 on warfarin, target INR 2.5 and 40 on rivaroxaban 20mg once daily.Anticoagulation was in therapeutic range in 71% (32/45) warfarin and 65% (26/40) rivaroxaban treated patients. 8 patients on warfarin and 9 patients on rivaroxaban had subtherapeutic INR and rivaroxaban levels respectively. Both rivaroxaban and warfarin reduced endogenous thrombin potential (ETP) and peak thrombin, and prolonged lag time and time to peak, compared to normal controls (p0.0001). The lag time and time to peak TG were longer, and peak thrombin was lower in patients receiving rivaroxaban (p0.0001) compared with warfarin, although warfarin-treated patients had lower ETP (p=0.0008). In-vivo coagulation activation markers were within the normal ranges in all rivaroxaban-treated patients (including those with levels considered to be subtherapeutic) and in 37/45 warfarin-treated patients who had an INR≥2.0. The warfarin-treated patients with subtherapeutic INRs exhibited slightly raised F1.2 and/or TAT.In conclusion, both rivaroxaban and warfarin provided effective anticoagulation, as assessed by inhibition of TG and makers of in-vivo coagulation activation.

Summary Background Antiphospholipid antibodies may interfere with the anticoagulant activity of activated protein C (APC) to induce acquired APC resistance (APCr). Aims To investigate the frequency and characteristics of APCr by using recombinant human APC (rhAPC) and endogenous protein C activation in antiphospholipid syndrome (APS). Methods APCr was assessed in APS and non-APS venous thromboembolism (VTE) patients on warfarin and normal controls with rhAPC or Protac by thrombin generation. IgG anti-protein C and anti-protein S antibodies and avidity were assessed by ELISA. Results APS patients showed greater resistance to both rhAPC and Protac than non-APS patients and normal controls (median normalized endogenous thrombin potential inhibition): APS patients with rhAPC, 81.3% (95% confidence interval [CI] 75.2–88.3%; non-APS patients with rhAPC, 97.7% (95% CI 93.6–101.8%; APS patients with Protac, 66.0% (95% CI 59.5–72.6%); and non-APS patients with Protac, 80.7 (95% CI 74.2–87.2%). APS patients also had a higher frequency and higher levels of anti-protein C antibodies, with 60% (15/25) high-avidity antibodies. High-avidity anti-protein C antibodies were associated with greater APCr and with a severe thrombotic phenotype (defined as the development of recurrent VTE while patients were receiving therapeutic anticoagulation or both venous and arterial thrombosis). Twelve of 15 (80%) patients with high-avidity anti-protein C antibodies were classified as APS category I. Conclusion Thrombotic APS patients showed greater APCr to both rhAPC and activation of endogenous protein C by Protac. High-avidity anti-protein C antibodies, associated with greater APCr, may provide a marker for a severe thrombotic phenotype in APS. However, in patients with category I APS, it remains to be established whether anti-protein C or anti-β2-glycoprotein I antibodies are responsible for APCr.

Several methods are now available for the laboratory assessment of activated protein C resistance (APCR). In this study, we evaluated two activated partial thromboplastin time-based assays [Coatest activated protein C (APC) and Diagen protein C activator (PCA)], with and without predilution of test plasma in factor V-deficient plasma (FVdp) and an amidolytic assay (Immuno Ltd, Vienna, Austria). Testing plasmas from normal volunteers who had received 1-deamino-8-D-arginine vasopressin (DDAVP) also assessed the effect of elevated factor VIII on APCR. In the unmodified clotting tests, the Coatest kit gave overlapping results for normal and heterozygous FV:Q506 samples; some FV:Q506 samples on oral anticoagulant therapy (OAT) were misclassified as normal, and some normal samples with high factor VIII levels would be classified as APC resistant. The unmodified Diagen kit correctly classified these three types of sample, but had the disadvantage that prolonged PCA clotting times gave serious problems with instrument end-point detection. Both kits modified by diluting the samples in FVdp correctly classified all the samples, as well as samples from patients with lupus anticoagulant (LA) and patients receiving heparin. The Immunochrom kit correctly classified the normal and FV:Q506 samples, but would have misclassified most normal persons on OAT as well as some patients with LA or receiving heparin therapy as APC resistant.

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 micro/dl, s.d. = 39.1, median = 122 micro/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.

Antiphospholipid antibodies (aPL) are associated with an increased incidence of fetal loss, but the pathophysiology remains unclear. One mechanism may involve the binding of aPL directly to the placenta where they may initiate placental thrombosis and infarction. We have developed an immunofiuorescent technique to detect human aPL binding to human placenta. Endogenous immunoglobulins were eluted by extensive washing and residual staining was prevented by incorporating multiple blocking steps. APL were affinity purified on both cardiolipin and phosphatidylserine liposomes from the sera of six patients with aPL (five antiphospholipid syndrome (APS) patients and one post bone marrow transplant patient). Heterogeneous binding to normal term placenta, involving either the trophoblast microvillous surface, stromal and perivascular regions was demonstrated by affinity purified aPL from five of six patients. Preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting studies have demonstrated that aPL bind a number of placental proteins. b2GPI was not the predominant protein bound by aPL using this technique. This study provides further evidence for the involvement of aPL in mediating placental damage.

Mast cells are thought to play an important role in atherogenesis and plaque rupture, but their role in the subsequent platelet activation and thrombus formation is unclear. Tryptase positive cells (KU812(T+)) were established from the KU812 cell line as an in vitro model of human mast cells and used to study the effect of mast cell activation on human platelets, Overnight incubation of KU812(T+) with IgE and subsequent challenge with anti-IgE caused the release of heparinoid substances which inhibited 1 mu g/ml collagen-induced platelet aggregation. KU812(T+) challenged with compound 48/80 produced a releasate that had no apparent heparinoid content but caused full platelet aggregation. These findings showed that, although activation of KU812(T+) via Fc epsilon R1 partially abrogated collagen-induced platelet aggregation, activation of the C5a receptor signalling pathway, by compound 48/80, caused the release of potent platelet-activating substances. This cell culture model offers a unique insight into the role of platelet-mast cell interactions in arterial thrombogenesis.

Objective To compare the frequency of maternal serum antiphospholipid antibodies (to cardiolipin, β2-glyco-protein I and prothrombin) in pregnancies presenting with bilateral abnormal uterine artery Doppler waveforms. Design Retrospective analysis of stored serum. Subjects Cases comprised 47 singleton pregnancies with bilateral abnormal uterine artery Doppler waveforms at 24 weeks of gestation, followed from 20 weeks, and controls were 100 healthy pregnancies with normal uterine artery Doppler waveforms. Methods Ultrasound examination utilized a 5-MHz curvilinear transabdominal transducer with pulsed and color Doppler facilities. Antiphospholipid antibodies were analyzed by ELISA methodology, and reference ranges were established using the geometric mean ± 2 SD of healthy non-pregnant adults. Human chorionic gonadotropin (hCG) levels were obtained from patient notes. Results Anticardiolipin antibodies were detected in 11 (23%) of the cases (IgG, n = 7; IgM, n = 6) compared with ten (10%) of the controls (p < 0.05). Low titer anticardiolipin IgG (range, 5.5–35.3; median, 6.3 GPL units) and anticardiolipin IgM (range, 3.4–14.7; median, 5.3 MPL units) were detected in cases. Amongst the cases, adverse perinatal outcomes were more common in the presence of raised levels of anticardiolipin antibodies. Anti-β2-glycoprotein I IgG was not detected in any of the cases. Antiprothrombin IgG was not detected, but antiprothrombin IgM occurred in 10.6% of cases compared with 2% of controls. Conclusions Women with persistent bilateral abnormal uterine artery Doppler waveforms in mid-gestation were more likely to express raised levels of anticardiolipin antibodies than healthy controls with normal uteroplacental perfusion. Anticardiolipin antibodies without anti-β2-glycoprotein I binding may be involved in the pathogenesis of uteroplacental ischemia in a proportion of high-risk pregnancies. Copyright © 1999 International Society of Ultrasound in Obstetrics and Gynecology

SummaryThis study was undertaken to appraise the application of those reagents most widely used in the UK for the detection and confirmation of lupus anticoagulant (LA) on an Amelung KC4A and a Sysmex CA-6000™ coagulometer. Five sets of dilute Russell’s viper venom time (DRVVT) reagents were assessed as well as the Textarin®-PL/ Ecarin ratio. Each DRVVT method comprised both LA detection and confirmation reagents provided by the same manufacturer. Samples were obtained from 20 normal healthy subjects, 10 LA-positive patients, 10 patients receiving oral anticoagulant therapy (OAT) who had previously been documented as LA-positive, a further 10 LA-negative patients receiving OAT and 10 LA-negative patients receiving unfractionated heparin therapy. The sensitivity and specificity of the reagents exhibited considerable variation not only between reagents, but also when the same reagent was used on the two analysers. Sensitivity ranged from 62 to 97% (all reagents both analysers), specificity went as low as 23% (Gradipore reagent on the CA-6000™) and as high as 100% (American Diagnostica Inc on both KC4A and CA-6000™). On the KC4A instrument, Unicorn Diagnostics’ lupus anticoagulant kit offered the best compromise of sensitivity and specificity (sensitivity 83% and specificity 81%). On the CA-6000™ the reagents supplied by American Diagnostica Inc exhibited optimal performance (sensitivity 90% and specificity 100%). The results indicate a need to optimise test reagents for specific analyser types, a procedure which can only be undertaken with preparations such as the proposed NIBSC reference plasmas for the detection of lupus anticoagulant.

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.

SUMMARY Objective. To determine anti-b2 glycoprotein-I (anti-b2GPI ) and anti-prothrombin (anti-ProT ) antibody levels, and the IgG subclass distribution of anti-b2GPI antibodies, in serial samples from patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) having initial or recurrent thrombotic/neurological ( T/N ) events during the study period. To investigate the correlations between these antibodies and b2GPI antigen, anticardiolipin antibody (aCL), anti-doublestranded (ds) DNA, C3 levels and disease activity. Methods. Fifty serum samples were identified from seven patients with SLE who had had T/N events during the follow-up from a cohort under long-term follow-up. IgG anti-b2GPI, anti-ProT, aCL, IgG subclasses of anti-b2GPI and b2GPI antigen levels were determined by ELISA. Corresponding disease activity [British Isles Lupus Assessment Group (BILAG)], antidsDNA and C3 levels were compared. Results. IgG anti-b2GPI antibody levels were elevated in six of the patients before and after the T/N events with less marked fluctuations than aCL antibody levels. The predominant subclass of anti-b2GPI antibodies was IgG2 before and after the T/N events. IgG anti-ProT antibodies were negative in all cases. There was a significant but weak correlation between anti-b2GPI and aCL antibodies. No correlation was found between disease activity and IgG anti-b2GPI antibody and b2GPI antigen levels. There were fluctuations in b2GPI antigen levels and a trend to increase after T/N events was observed in some patients. Conclusion. Most of the patients with a T/N event during the study period had IgG anti-b2GPI, but not IgG anti-ProT antibodies. Many IgG aCL-negative samples were found to have IgG anti-b2GPI activity during the follow-up period. The predominant subclass of IgG anti-b2GPI was IgG2, which may have importance in the pathogenesis of APS. b2GPI antigen levels were found to be increased in some patients with SLE after T/N events. IgG anti-b2GPI antibodies may be used as an adjunctive marker of future T/N events in patients with SLE and APS with aCL antibodies and lupus anticoagulant.

AIM: To evaluate PT derived fibrinogen determinations with reference to the Clauss fibrinogen assay using a Sysmex CA-6000 random access coagulation analyser. METHODS: Samples were analysed from normal subjects (n = 20), patients with renal or liver dysfunction (n = 25), critically ill patients (n = 25), patients receiving oral anticoagulant treatment (n = 50), and patients with a haemoglobinopathy (n = 127). Prothrombin times were performed using two thromboplastins: one derived from rabbit brain (Dade: Thromboplastin IS) and the other from recombinant human tissue factor (Dade: Innovin). Fibrinogen was assayed by the Clauss method using a commercial kit (Dade: Fibrinogen). RESULTS: The relation between Clauss fibrinogen and PT derived fibrinogen was found to be dependent on the patient's clinical group and source of the thromboplastin used. When the data from the above sample groups were pooled there was still a significant difference (p < 0.001) between Clauss fibrinogen and PT derived fibrinogen, irrespective of thromboplastin used. CONCLUSIONS: It is unsafe to use the PT derived fibrinogen for patient monitoring owing to non-uniform variability in response to clinical status and reagent employed; however, it may prove to be a useful screening test in a research environment for estimating fibrinogen levels among defined patient groups.

Thromboembolism is a common occurrence in patients with the antiphospholipid syndrome (APS). The mechanism responsible for this phenomenon is unclear; there are several theories. One possibility is that a pathogenic interaction exists between antiphospholipid antibodies and platelets, leading to their activation. This study examined the expression of the platelet activation markers CD62 and CD63 by flow cytometry in 20 patients with the primary antiphospholipid syndrome (PAPS). Levels of soluble P-selectin were also assayed. Reticulated platelets were measured as an indicator of increased platelet production and/or turnover. Median CD63 expression was significantly increased in patients (14.3%) compared to a group of healthy controls (10.1%, P , 0.0008). There was no significant difference in median CD62 expression or percent reticulated platelets between the two groups. The median level of soluble P-selectin was significantly higher in PAPS patients (35.5 ng/ml) compared to controls (18.8 ng/ml, P , 0.0028). Patients receiving aspirin had lower median CD63 values (13.1%) when compared to those patients who were not (18.0%, P , 0.023). However, aspirin therapy did not prevent significant platelet activation occurring in some individual patients. Our data suggest that although not excessive, there is a degree of increased platelet activation in some PAPS patients, which is not always suppressed by antiplatelet therapy with aspirin.

SUMMARYIt is known that antiphospholipid antibodies (aPL) hamper the anticoagulant activity of the protein C system, but the mechanism is still obscure. In this study, we demonstrate that anticardiolipin antibodies (not anti-protein C autoantibodies) can bind protein C via β2-GPI, which bears their binding epitope, in a fashion dependent on negatively charged phospholipids. We studied the binding of IgG from aPL to protein C in the presence of β2-GPI by ELISA (anti-‘protein C’ antibody ELISA), and compared their binding with those obtained in the absence of β2-GPI. In the anti-‘protein C’ antibody ELISA system, 47% of 78 aPL+ patients had a positive titre in the presence of cardiolipin (CL) and β2-GPI, but binding was not found in the absence of β2-GPI. Highly significant correlations were found between the titre of anti-‘protein C’ antibody in the presence of β2-GPI and that of anti-β2-GPI antibody (r = 0·802, P = 0·0001). We further analysed the interaction between protein C, phospholipids, β2-GPI and human aCL MoAbs established from patients with antiphospholipid syndrome. In a first set of experiments, the binding of β2-GPI to protein C and its phospholipid dependency were investigated. β2-GPI bound to protein C in the presence of CL or phosphatidylserine, but not in the presence of phosphatidylcholine or phosphatidylethanolamine. In a second group of experiments, the binding of three human monoclonal aCL recognizing the cryptic epitope of β2-GPI (virtually anti-β2-GPI antibodies) was evaluated in the presence of cardiolipin and β2-GPI. All three human monoclonal aCL bound to protein C in the presence of CL and β2-GPI, whereas they did not in the absence of either β2-GPI or CL. These data suggest that protein C could be a target of aCL by making a complex with CL and β2-GPI, leading to protein C dysfunction.

Introduction of the International Normalized Ratio (INR) has improved the standardization of laboratory control of oral anticoagulant therapy (OAT). However, it has been reported that misleading INR results can be obtained from OAT patients with lupus anticoagulant (LA). To investigate this claim, we studied 35 OAT patients, 14 of whom had anti-phospholipid syndrome (APS) with a documented LA. Attainment of anticoagulation was confirmed by chromogenic assay of factor VII and factor X. Prothrombin times were performed using eight thromboplastins (five derived from rabbit brain, two recombinant human tissue factor and one made from human placenta) with an International Sensitivity Index (ISI) of

Lupus anticoagulants (LA) are associated with an increased risk of thrombosis and laboratory detection is of major importance. Various tests are available for LA screening and confirmation, but they differ in sensitivity and specificity, frequently lacking the ability to discriminate between the presence of LA, heparin and oral anticoagulants. We noticed that a patient with LA who had a prolonged activated partial thromboplastin time (APTT) by our routine method, gave a normal result with a different APTT reagent. This latter reagent, which contained soy bean phosphatides (SBP), was compared with a reagent containing rabbit brain phospholipids complexed with kaolin (RBK), for APTT measurement in a variety of patients. There was no significant difference in APTT ratio between the two reagents in plasma samples from healthy normal subjects. In LA samples, SBP gave consistently lower APTT ratios than RBK (mean +/- SEM, 1.04 +/- 0.05 and 2.08 +/- 0.19 for SBP and RBK respectively; P < 0.001). In LA patients receiving oral anticoagulants for antithrombotic prophylaxis or treatment, the APTT ratio was again significantly shorter with SBP (1.60 +/- 0.17 and 3.40 +/- 0.67; P < 0.05). In LA negative patients receiving oral anticoagulants, the relationship was reversed, and a higher APTT ratio was obtained with SBP than RBK (1.61 +/- 0.13 and 1.31 +/- 0.12; P < 0.001). In addition, there were no significant differences in APTT ratios for the two reagents when samples from patients receiving heparin therapy, or patients with acquired factor VIII deficiency or inherited deficiency of factor VIII or IX were studied. The use of the SBP reagent alongside a LA sensitive APTT reagent allows a rapid screening for LA, as well as a confirmation of the phospholipid dependency of the inhibitor.

Young or reticulated platelets contain some residual mRNA, which is rapidly degraded after platelet release into the circulation. They can be easily detected either with supravital dye staining (e.g. new methylene blue) on blood films, or more commonly with fluorescent dyes (e.g thiazole orange) and flow cytometry. Using the latter technique many different groups have demonstrated that the measurement of reticulated platelets has much clinical potential. It is apparent that the level of reticulated platelets gives a relatively simple and non-invasive measurement of the rate of thrombopoiesis in an analogous fashion to the red cell reticulocyte count. Many research groups are currently measuring reticulated platelets but with wide variation in data and methods. An international platelet panel has begun to develop protocols and between laboratory comparisons, which will result in the standardization of the procedure. Platelet reticulocyte analysis should thus become part of accepted haematological practice and provide useful clinical information for the investigation and monitoring of platelet production in various thrombocytopenic conditions. In particular, measurement of reticulated platelets will provide an excellent and simple means for monitoring the response of chemotherapy and transplant patients to growth factors (e.g. thrombopoietin) resulting in a decrease in the demand for platelet transfusion.

Summary XR5118, a diketopiperazine-based low molecular weight inhibitor of plasminogen activator inhibitor-1 (PAI-1) activity, was studied ex vivo and in vivo in the rat to determine whether inhibition of PAI-1 activity resulted in increased fibrinolysis and protection against thrombus formation. XR5118 reversed the inhibitory effects of human PAI-1 against tissue-type plasminogen activator (tPA), in an vitro amidolytic assay (S2251) with an IC50 value of 3.5 μM±0.19 μM (n=7). This activity was confirmed in in vitro fibrinolysis assays against both human and rat PAI-1 and, following intravenous administration to rats, XR5118 (1–5mg/kg) dose-dependently increased clot lysis in an ex vivo dilute blood clot lysis time (DBCLT) assay. At 5 mg/kg, XR5118 increased clot lysis by 41±1.6% (n=39, P

Pregnancy is associated with significant haemostatic changes, with a progressive rise in most clotting factors. There is limited data on the changes of factor XIII (FXIII) level during pregnancy. This study assesses changes in FXIII activity during normal pregnancy and establish FXIII reference range during each trimester of pregnancy and immediate postnatal period. This is a cross sectional study of 376 women with normal uneventful pregnancies. Plasma FXIII activity was measured during first (weeks 0-12, n = 116), second (weeks 13-28, n = 132), third trimester (weeks 29-42, n = 128) and postnatal (day 0-3; n = 30). Samples were also collected from non-pregnant women (n = 25) as a control group. FXIII was assayed on CS-5100 analyser using chromogenic reagent. The mean ± SD FXIII activity was 112 ± 29 IU dL(-1) during first trimester, 96 ± 26 IU dL(-1) during second trimester, 83 ± 21 IU dL(-1) during third trimester, 90 ± 19 IU dL(-1) during postnatal period, and 113 ± 26 IU dL(-1) in the control. The reference range was calculated during the first (55-169 IU dL(-1)), second (45-147 IU dL(-1)), third trimester (42-125 IU dL(-1)) and postnatal period (61-137 IU dL(-1)). There was a significant reduction in the mean FXIII activity during the second and third trimester compared to the first trimester and control group (P < 0.0001). During the immediate postnatal period, the mean FXIII activity was not statistically different compared to the third and second trimester levels but was significantly lower compared to the first trimester (P < 0.0001) level and the control group (P = 0.0002). This study establishes the reference range for FXIII activity during the three trimesters of normal pregnancy and immediate postnatal period. Women have a significantly decreased level of FXIII activity during a normal uneventful pregnancy.

Introduction The investigation of platelet function by aggregometry requires specialist equipment and is labour intensive. We have developed an automated platelet aggregation method on a routine coagulation analyser. Methods We used a CS-2000i (Sysmex) with prototype software to perform aggregation in platelet-rich plasma (PRP), using the following agonists: ADP (0.5–10 μm), epinephrine (0.5–10 μm), collagen (0.5–10 mg/μL), ristocetin (0.75–1.25 mg/mL) and arachidonic acid (0.12–1.0 mm). Platelet agonists were from Hyphen Biomed, and an AggRAM aggregometer (Helena Biosciences) was used as the reference instrument. Results CS-2000i reaction cuvette stirrer speed was found to influence reaction sensitivity and was optimized to 800 rpm. There were no clinically significant changes in aggregation response when the PRP platelet count was 150–480 x 109/L, but below this there were changes in the maximum amplitude (MA) and slope (rate). Dose response with each of the agonists was comparable between CS-2000i and an AggRAM aggregometer and normal subjects receiving antiplatelet drugs. Aggregation imprecision was similar on both the CS-2000i and AggRAM systems, with a cv for 2–5 μm ADP MA and slope varying between 3–12%. Conclusion Our preliminary studies indicated that optimal sensitivity using the CS-2000i was obtained with a reaction cuvette stirrer speed of 800 rpm and a PRP platelet count of 200–300 x 109/L; aggregation with a PRP count

SummaryIt is now well recognised that antiphospholipid antibodies are associated with thrombosis and recurrent fetal loss. Some antiphospholipid antibodies (aPAs) have been shown to require a cofactor, β2 glyco-protein-I (β2GPI), for binding to phospholipids, and recently β2GPI has been identified as the antigenic target for some aPAs. β2GPI possesses in vitro anticoagulant properties and modulation of β2GPI function may therefore result in altered haemostatic regulation. In the present study, the influence of plasma derived aPAs and β2GPI on factor XII activation on the surface of very low density lipoprotein (VLDL) was investigated. Factor XIIa generation was dependent on lipoprotein lipase treatment of VLDL and β2GPI inhibited the factor XIIa generation in a concentration dependent manner. No consistent effects on factor XIIa generation were demonstrated with the IgG fractions from patients with aPAs. Inhibition of the β2GPI activity was demonstrated by some antibodies, and study with cardiolipin affinity purified antibody indicated that antibody concentration is critical. These results suggest that perturbation of β2GPI function may contribute to the pathogenic mechanism for thrombosis in some patients with aPAs. .

Introduction The Clinical and Laboratory Standards Institute (CLSI) has produced a guideline detailing how to determine the activated partial thromboplastin time’s (APTT) sensitivity to clotting factor deficiencies, by mixing normal and deficient plasmas. Using the guideline, we determined the factor sensitivity of two APTT reagents. Methods APTTs were performed using Actin FS and Actin FSL on a Sysmex CS-5100 analyser. The quality of factor-deficient and reference plasmas from three commercial sources was assessed by assaying each of the clotting factors within the plasmas and by performing thrombin generation tests (TGT). Results Testing samples from 50 normal healthy subjects gave a two-standard deviation range of 21.8–29.2 s for Actin FS and 23.5–29.3 s for Actin FSL. The upper limits of these ranges were subsequently used to determine APTT factor sensitivity. Assay of factor levels within the deficient plasmas demonstrated that they were specifically deficient in a single factor, with most other factors in the range 50–150 iu/dL (Technoclone factor VII-deficient plasma has 26 iu/dL factor IX). APTTs performed on mixtures of normal and deficient plasmas gave diverse sensitivity to factor deficiencies dependent on the sources of deficient plasma. TGT studies on the deficient plasmas revealed that the potential to generate thrombin was not solely associated with the levels of their component clotting factors. Conclusion Determination of APTT factor sensitivity in accordance with the CLSI guideline can give inconsistent and misleading results.

beta 2 glycoprotein-I (beta 2 GPI) is a 50 kDa plasma protein which associates with a number of anionic plasma constituents and has been identified as a cofactor for the binding of some antiphospholipid antibodies (aPAs). beta 2 GPI antigen levels are increased in some patients with aPAs. In order to examine the distribution of beta 2 GPI in these patients, we developed a method for measurement of free beta 2 GPI. Forty-three patients with SLE, of whom 18 had laboratory evidence of aPAs, and 22 normal healthy subjects were studied. Plasma was filtered by centrifugation at 650 x g through a 100 kDa filter in order to allow separation of free and complexed beta 2 GPI, and beta 2 GPI levels were measured in the starting plasma and filtrate by a standardized ELISA. Total beta 2 GPI levels of the SLE aPA positive patients (253.3 mg/l) were significantly increased compared with the SLE aPA negative patients and normal controls (188.0, P < 0.001 and 194.9 mg/l, P < 0.01, respectively), but there were no significant differences between free beta 2 GPI levels of these groups (20.8, 24.0 and 20.5 mg/l, respectively). These results suggest that levels of complexed beta 2 GPI are increased in patients with aPAs, perhaps as a result of immune complex formation, or altered binding to other plasma constituents.

beta(2) Glycoprotein-I (beta(2)GPI), a plasma component with in vitro anticoagulant properties, has been identified as a cofactor for the binding of some antiphospholipid antibodies (aPAs). In order to determine whether beta(2)GPI changes were associated with the thromboembolic complications of aPAs, we measured beta(2)GPI antigen (beta(2)GPI:Ag), beta(2)GPI aPA cofactor activity (beta(2)GPI:Cof) and antibodies to beta(2)GPI (alpha beta(2)GPI) in 44 systemic lupus erythematosus (SLE) patients, of whom 19 had evidence of aPAs (SLE-aPA+) and 17 patients with primary antiphospholipid syndrome (PaPS). beta(2)GPI:Ag levels were significantly increased in SLE-aPA+ patients and PaPS patients compared with SLE-aPA- patients and normal healthy controls. The ratio of beta(2)GPI:Cof/Ag was significantly reduced in SLE-aPA+ patients compared with SLE-aPA- patients, indicating functional modification of beta(2)GPI in SLE-aPA+ patients. Eighty per cent of patients with anticardiolipin (aCL) IgG also had alpha beta(2)GPI, and 13% patients with no detectable aCL IgG had alpha beta(2)GPI. Increased beta(2)GPI:Ag and alpha beta(2)GPI were associated with a clinical history of thrombosis or recurrent fetal loss. The results of these investigations suggest that beta(2)GPI may play a role in the pathogenic mechanism of thrombosis associated with aPAs.