A. S. Lawrie

Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis

Publications known to the writing group were supplemented with additional papers identified by searching Medline/PubMed for publications in the last 12 years using keywords: coagulation assays, amidolytic assays, haemostasis assays, and thrombophilia testing, in core clinical journals and English language. Additional relevant articles were identified by screening reference lists and by publications known to the writing group. The writing group produced the draft guideline which was subsequently revised by consensus by members

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.u2002 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.

Monitoring of oral anticoagulant therapy in lupus anticoagulant positive patients with the anti‐phospholipid syndrome

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 <1.40. When using the thromboplastin manufacturers ISI there was a significant difference (ANOVA, P<0.0001) between INR results obtained with the eight reagents for both APS (average CV = 12.4%) and non‐APS (average CV = 12.5%) patient groups. Variation using the eight thromboplastins was assessed by calculating the CV for each sample; these values were then pooled for each patient group to give the average CV for all samples with all reagents for the two patient groups. Results for both patient groups exhibited markedly reduced variation (APS group average CV = 6.5%, non‐APS group average CV = 5.8%) when locally assigned ISI values were employed in the calculation of INRs. Our data does not support the suggestion that the INR may not reflect the true level of anticoagulation in the long‐term warfarin‐treated patient, in whom lupus anticoagulant was detected. However, there was strong evidence that thromboplastin use should be restricted to those clot detection systems for which the reagents manufacturer has assigned an ISI, or local ISI assignment must be undertaken. The inappropriate use of a generic (i.e. optical or mechanical clot detection system without regard to specific analyser type) ISI value can lead to ambiguous results.

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

Backgroundu2002 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.

Factor XIII - an under diagnosed deficiency - are we using the right assays?

Summary.u2002 Background:u2002 The clot solubility test is the most widely used method for detection of factor (F)XIII deficiency. However, it will only detect severe deficiencies; consequently mild deficiencies and heterozygous states are probably under diagnosed. Objective: As an alternative first‐line screening test, we assessed an automated quantitative ammonia release assay (QARA). Patients/methods: Inter‐assay imprecision was evaluated with commercial normal and pathological control plasmas (10 replicates on each of 5u2003days). Using the QARA and other commercial assays a comparative assessment of congenital (FXIII range <u20031–70u2003uu2003dL−1, nu2003=u20039) and acquired (nu2003=u200343) deficiencies was made. We also investigated the prevalence of acquired deficiencies in hospitalized patients using residual samples from adult patients (nu2003=u20031004) and from a paediatric intensive care unit (ICU, nu2003=u200356). Results: Assay imprecision was acceptably low (normal control: mean 86.6u2003uu2003dL−1; cvu2003=u20032.0%; pathological control: mean 27.5u2003uu2003dL−1; cvu2003=u20033.8%). Using an iodoacetamide blanking procedure, the QARA results (FXIII range <u20031–70u2003uu2003dL−1) exhibited close agreement with those from an immuno‐turbidometric FXIII A‐subunit (FXIII‐A) method. There was also good correlation (R2u2003≥u20030.89) between the QARA (range 20–180u2003uu2003dL−1), a second chromogenic assay, the FXIII‐A and FXIII A+B‐subunit ELISA. We found that 21% of samples from adult patients had FXIII levels <u200370u2003uu2003dL−1 (mean normalu2003±u20032u2003SD 73–161u2003uu2003dL−1) with 6% <u200350u2003uu2003dL−1. Within the paediatric ICU samples, 52% were <u200370u2003uu2003dL−1, with 21% <u200350u2003uu2003dL−1. Conclusions: Our data demonstrates that the automated assay is sensitive, highly reproducible and the results from clinical samples suggest that acquired FXIII deficiency is a relatively common phenomenon in hospital patients after surgery and in ICU.

A comparative evaluation of a new automated assay for von Willebrand factor activity

The ristocetin cofactor assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity in the diagnosis of von Willebrands Disease (VWD). However, the assay suffers from poor reproducibility and sensitivity at low levels of VWF and is labour intensive. We have undertaken an evaluation of a new immunoturbidimetric VWF activity (VWF:Ac) assay (INNOVANCE® VWF Ac. Siemens Healthcare Diagnostics, Marburg, Germany) relative to an established platelet‐based VWF:RCo method. Samples from 50 healthy normal subjects, 80 patients with VWD and 50 samples that exhibited ‘HIL’ (i.e. Haemolysis, Icterus or Lipaemia) were studied. VWF:Ac, VWF:RCo and VWF:Ag were performed on a CS–analyser (Sysmex UK Ltd, Milton Keynes, UK), all reagents were from Siemens Healthcare Diagnostics. The VWF:Ac assay, gave low intra‐ and inter‐assay imprecision (over a 31‐day period, n = 200 replicate readings) using commercial normal (Mean 96.2 IU dL−1, CV < 3.0%) and pathological (Mean 36.1 IU dL−1, CV < 3.5%) control plasmas. The normal and clinical samples exhibited good correlation between VWF:RCo (range 3–753 IU dL−1) and VWF:Ac (rs = 0.97, P < 0.0001), with a mean bias of 5.6 IU dL−1. Ratios of VWF:Ac and VWF:RCo to VWF:Ag in the VWD samples were comparable, although VWF:Ac had a superior lower level of detection to that of VWF:RCo (3% and 5% respectively). A subset (n = 97) of VWD and HIL samples were analysed for VWF:Ac at two different dilutions to assess the effect on relative potency, no significant difference was observed (P = 0.111). The INNOVANCE® VWF Ac assay was shown to be reliable and precise.

The VWF/ADAMTS13 axis in the antiphospholipid syndrome: ADAMTS13 antibodies and ADAMTS13 dysfunction

Autoantibodies to ADAMTS13 (a disintegrin‐like and metalloprotease with thrombospondin type I motif, member 13) play an important role in the development of microthrombosis in thrombotic thrombocytopenic purpura (TTP). In severe cases of antiphospholipid syndrome (APS), microthrombosis can occur similar to that seen in TTP, suggesting possible mutual pathogenic factors. However, the role of ADAMTS13 in APS is unknown. We hypothesised that aberrations in ADAMTS13 may occur in APS and evaluated ADAMTS13 and von Willebrand factor (VWF) in 68 patients with antiphospholipid antibodies (aPA) including 52 with APS. Thirty‐three (49%) had IgG anti‐ADAMTS13 with 12 of these patients having reduced ADAMTS13 activity, suggesting neutralising antibodies. Low ADAMTS13 activity (median 34%) was demonstrated in 22/68 (33%), all with normal ADAMTS13 antigen levels consistent with dysfunctional ADAMTS13. Reduced ADAMTS13 activity was not secondary to elevated von Willebrand factor (VWF), or increased VWF secretion (normal VWF propeptide), although a reduced VWF clearance was noted in APS. Analysis found no associations between the ADAMTS13 abnormalities and any aPA profile or thrombotic/obstetric complications, although this study was not adequately powered to address clinical associations. Nevertheless, these findings highlight that ADAMTS13 autoantibodies and ADAMTS13 dysfunction can occur in APS, and although the clinical significance remains undetermined, ADAMTS13 dysfunction may be contributory to thrombogenesis in autoimmune conditions other than TTP.

A two-centre comparative evaluation of new automated assays for von Willebrand factor ristocetin cofactor activity and antigen

von Willebrand disease (VWD) is caused by a quantitative and/or qualitative deficiency of the von Willebrand factor (VWF). The laboratory diagnosis of VWD is dependent on the measurement of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo). The aim of this study was to undertake a two‐centre evaluation of two new automated VWF:Ag and VWF:RCo assays systems from Instrumentation Laboratory (Bedford, USA). Using the two new analytical systems that operated with different detection principles: immunoturbidimetric (TOP500 analyser) and chemiluminescent (AcuStar analyser), VWF:Ag and VWF:RCo levels were determined in samples from 171 healthy normal subjects, 80 VWD patients (16 type 1, 58 type 2 and 6 type 3) and 7 acquired von Willebrand syndrome patients. With commercial lyophilized normal and pathological plasmas VWF: Ag and VWF:RCo assays performed on both analysers exhibited low levels of inter‐assay imprecision (AcuStar: CV% range 3.3–6.9; TOP500: CV% range 2.6–6.3). Samples from normal healthy subjects (range: VWF:Ag 44.6–173.9 IU dL−1; VWF:RCo 43.1–191.5 IU dL−1) and patients (range: VWF:Ag <0.3–115.1 IU dL−1; VWF:RCo <0.5–57.2 IU dL−1) showed a good correlation between the two VWF:Ag and VWF:RCo methods (rs = 0.92 and 0.82 respectively), with only a few inconsistent cases among the patients samples evaluated. The chemiluminescent assays had a lower limit of detection for both VWF:Ag and VWF:RCo compared to immunoturbidimetric tests (0.3 IU dL−1 vs. 2.2 IU dL−1 and 0.5 IU dL−1 vs. 4.4 IU dL−1 respectively). The TOP500 and AcuStar VWF:Ag and VWF:RCo assays were precise and compare well between centres, making these systems suitable for the diagnosis of VWD in non‐specialized and reference laboratories.

The effect of prion reduction in solvent/detergent-treated plasma on haemostatic variables.

Backgroundu2002 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.

The clinical significance of differences between point-of-care and laboratory INR methods in over-anticoagulated patients

INTRODUCTIONnPatients receiving warfarin are at increased risk of bleeding when their International Normalised Ratio (INR) >4.5. Although not standardised above 4.5 the INR is measured in over-anticoagulated patients, consequently we have examined the reliability of INR results ≥4.5. We assessed: the relationship between different prothrombin time systems for INRs >4.5; the relationships between the INR and levels of vitamin K-dependent coagulation factors (VKD-CF) and thrombin generation test (TGT) parameters; and the impact that variation in results would have on warfarin dosing.nnnMETHODSnINRs were performed using a CoaguChek XS Plus point-of-care (POC) device (measuring range 0.6-8.0). For POC INRs ≥4.5, laboratory INRs were also measured using a recombinant tissue factor (rTF) and a rabbit brain (RBT) thromboplastin.nnnRESULTSnThere was good correlation between POC (INR ≥4.5, <8.0) and Lab INRs (rTF n=154, rs=0.87, p<0.0001; RBT n=102, rs=0.76, p<0.0001); and significant correlations between each of the VKD-CF and the INR, the strongest being with FVII (POC INR rs=-0.53 p<0.0001; Lab rTF-INR rs=-0.70 p<0.0001). TGT peak thrombin and ETP also showed good correlations with INR values (R(2)>0.71). Using POC and Lab rTF-INR, 109/154 (71%), or POC and Lab RBT-INR 75/102 (74%) results exhibited dosage concordance and/or were within 0.5 INR units. In the remaining patients variation in warfarin dosing was generally slight.nnnCONCLUSIONSnOur data suggest that CoaguChek XS Plus INRs >4.5 and <8.0 are comparable to laboratory INRs (both methods) and it is probably unnecessary to perform laboratory INRs for clinical management of patients with INRs >4.5 including those >8.0.