Chris Gardiner

Patient self-testing is a reliable and acceptable alternative to laboratory INR monitoring

An ageing population and the continuing expansion of clinical indications for coumadin therapy have increased pressure on hospital anticoagulant clinics. One solution is patient self‐testing (PST) of the international normalized ratio (INR) using capillary blood samples on point‐of‐care coagulation monitors at home. We conducted a prospective study to determine whether patients can achieve accurate INR values through PST, using the CoaguChek S (Roche Diagnostics, Lewes, UK). The main outcome measurements were: comparability of INR values obtained by PST and the hospital laboratory, patient acceptability as assessed by a questionnaire and anticoagulant control. Eighty‐four patients [53 men, 31 women; median age 59 years (range 26–83)], receiving long‐term oral anticoagulation (warfarin), were recruited from our Anticoagulation Clinic. Patients were randomized to weekly self‐testing or continuing 4‐weekly hospital laboratory monitoring of INR. Comparison of INRs (n = 234) showed no significant differences between the CoaguChek (median INR 3·02) and laboratory testing (median INR 3·07). There was excellent correlation between the two methods (r = 0·95), with 85% of CoaguChek results within 0·5 INR units of the laboratory method. On four occasions, differences of >1 unit INR were obtained, but in each case the patients anticoagulation was unstable (INR >4·5 by both methods) and the differences in INR would not have altered patient management. 87% of patients found self‐testing straightforward, 87% were confident in the result they obtained and 77% preferred self‐testing. We conclude that PST is a reliable alternative to hospital clinic attendance and is acceptable to the majority of suitably trained patients.

Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey

Extracellular vesicles (EVs) represent an important mode of intercellular communication. Research in this field has grown rapidly in the last few years, and there is a plethora of techniques for the isolation and characterization of EVs, many of which are poorly standardized. EVs are heterogeneous in size, origin and molecular constituents, with considerable overlap in size and phenotype between different populations of EVs. Little is known about current practices for the isolation, purification and characterization of EVs. We report here the first large, detailed survey of current worldwide practices for the isolation and characterization of EVs. Conditioned cell culture media was the most widely used material (83%). Ultracentrifugation remains the most commonly used isolation method (81%) with 59% of respondents use a combination of methods. Only 9% of respondents used only 1 characterization method, with others using 2 or more methods. Sample volume, sample type and downstream application all influenced the isolation and characterization techniques employed.

An evidence‐based review and guidelines for patient self‐testing and management of oral anticoagulation

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.

A randomised control trial of patient self-management of oral anticoagulation compared with patient self-testing.

Several studies suggest that patient self‐management (PSM) may improve the quality of oral anticoagulation therapy as measured by time spent within the international normalised ratio (INR) target range. We performed a prospective randomised control trial to determine whether the improvement in quality of treatment afforded by PSM is greater than that achieved by patient self‐testing (PST) alone. A total of 104 of 800 eligible patients aged 22–88 years (median = 59·8), attending our hospital anticoagulant clinic and receiving long‐term warfarin for >8 months agreed to participate. Patients were randomised to PSM (n = 55) or PST (n = 49). Both groups measured their INR using the CoaguChek S every 2 weeks or more frequently if required, for a period of 6 months. Seventy‐seven of 104 (74%) patients completed the study (PSM = 41 and PST = 36). The ‘drop out’ rates for both groups were similar. There was no significant difference between the percentage time in target therapeutic range for PSM (69·9%) and PST (71·8%). Both groups combined showed a significant improvement over the previous 6 months (71·0% vs. 62·5%; P = 0·04). Changes in time within the therapeutic range in individual patients (+5·86) also showed a significant difference. The quality of warfarin control in both PST and PSM may be superior to that achieved by conventional management in a specialised hospital anticoagulation clinic.

An evaluation of rapid D-dimer assays for the exclusion of deep vein thrombosis

We evaluated the performance of eight d‐assays for the exclusion of deep vein thrombosis (DVT); Biopool AutoDimer, Biopool MiniQuant, bioMèrieux MDA d‐Dimer, VIDAS, Dade Behring d‐Dimer Plus, Trinity Biotech AMAX, NycoCard d‐dimer and IL Test d‐Dimer. The assays were evaluated both as stand‐alone tests, and in combination with pretest probability (PTP). d‐dimer assays and PTP assessment were performed on 410 patients presenting to the emergency department with suspected acute DVT. DVT was diagnosed in 76 of 410 patients (18·5%) by compression ultrasound or other imaging techniques, as required. Receiver operator characteristics analysis established optimum cut‐off values and these were compared with manufacturers cut‐off values where provided. As stand‐alone tests, the assays varied immensely regarding cut‐off value, negative predictive value (NPV 93–100%) and specificity (0–67%). At least one patient with confirmed DVT had a low d‐dimer level by each method: to achieve 100% sensitivity it would be necessary to reduce cut‐off values to levels below clinical usefulness. When low d‐dimer was used in combination with PTP, six of eight methods achieved ≥98% NPV, with a diagnosis of DVT excluded in 16–44% of patients without the requirement for diagnostic imaging. The highly variable diagnostic performance of these d‐dimer assays means that some assays are unsuitable for certain diagnostic strategies. However, our data suggest that the combination of sensitive d‐dimer assays with an assessment of PTP may be used to exclude a diagnosis of DVT.

Self-monitoring of oral anticoagulation: does it work outside trial conditions?

Background: Patient self-monitoring (PSM) of oral anticoagulation therapy (OAT) can improve anticoagulant control, but poor uptake and high dropout rates have prompted suggestions that PSM is suitable for only a minority of patients in the UK. Aims: To determine whether PSM could be a viable alternative to regular hospital anticoagulant clinic attendance, if offered from the start of treatment. Methods: 318 consecutive patients referred, for the first time, to an anticoagulation clinic were assessed for eligibility using established criteria. Patients electing for PSM attended training and, following successful assessment, performed a capillary blood INR every two weeks or more frequently if directed to do so by the anticoagulation clinic. Primary outcome measures were uptake of PSM and the percentage time in target therapeutic INR range (TIR) compared to patients electing for routine clinic care. Results: Of 318 patients referred for OAT, 188 were eligible for PSM. 84 (26%) elected to self-monitor, of whom 72 (23%) remained self-monitoring or had completed their course of treatment at the end of the audit. Self-monitoring patients had significantly better anticoagulant control than those receiving routine hospital anticoagulation clinic care (TIR 71% vs 60%, p = 0.003) and significantly less time outside critical limits, ie, INR <1.5 or >5.0 (0.45% vs 2.04%, p = 0.008). Conclusions: Patients offered PSM from the start of treatment show increased uptake compared to previous UK studies and a level of oral anticoagulation control comparable to that reported in previous clinical trials.

Detection of acquired resistance to activated protein C associated with antiphospholipid antibodies using a novel clotting assay.

Antiphospholipid antibodies (aPA) frequently interfere with the protein C pathway. This manifests as acquired activated protein C (APC) resistance in the absence of factor V Leiden and has been proposed as a putative mechanism for the pathogenesis of the antiphospholipid syndrome (APS). We have developed a Russells viper venom test, performed with and without activation of endogenous protein C, which is sensitive to aPA-associated APC resistance. Results were reported as the endogenous APC ratio (EAPCr); the ratio of the two clotting times normalized against pooled normal plasma. Forty-four patients with aPA, anticardiolipin and/or lupus anticoagulant, including 34 with a history of thrombosis or pregnancy morbidity; a control group of aPA-negative patients; and 26 healthy normals were studied. EAPCr (mean, SD) was significantly higher in APS patients (1.94, 0.58) than normals (0.98, 0.12) or controls (1.14, 0.19; P < 0.00001). Elevated EAPCr (> 1.22) occurred in 91% of aPA-positive patients, predominantly due to resistance to APC (87%) rather than prolonged basal clotting times alone (15%). Significant correlation was observed between the EAPCr value and dilute Russells viper venom time (rs = 0.44, P = 0.003), IgG anticardiolipin (rs = 0.54, P = 0.002), protein S (r = −0.46, P = 0.01) and activated partial thromboplastin time-based APC resistance (r = −0.61, P = 0.001). There was no significant relationship between EAPCr and protein C concentration, anti-β2-glycoprotein-I (anti-β2GPI) or IgM anticardiolipin. Purified aPA IgG caused a dose-dependent increase in APC resistance when added to normal plasma. We conclude that aPA-associated acquired APC resistance is a common feature of APS and may be independent of anti-β2GPI.

Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility.

An editorial describing “minimal experimental requirements for definition of extracellular vesicles (EVs)”, or more simply “minimal information for studies of EVs (MISEV)” was published in the Journal of Extracellular Vesicles in late 2014 [1]. Similar to guidelines in other scientific fields [2–4], “MISEV2014”, as we will call it here, provided recommendations on experimental methods and minimal information in reporting. Specifically, three key areas were addressed: EV isolation/purification, EV characterization and EV functional studies (see Text Box 1).

Pregnancy loss, tissue factor pathway inhibitor deficiency and resistance to activated protein C

Recurrent pregnancy loss is a common problem, with two or more losses in up to 5%ofwomen, and recurrent (three ormore consecutive losses) affecting 1–2%ofwomen.Markers of coagulation activation (D-dimer and fibrinopeptide A) are known to increase preceding spontaneous abortion [1], and there is a strong association with antiphospholipid antibodies (aPA) [2] and some hereditary thrombophilia [3]. However, the majority of cases of unexplained pregnancy loss occur in the absence of aPA or hereditary thrombophilic defects. Activated protein C (APC) resistance during normal pregnancy is well documented [4] and acquired APC resistance (measured by a clotting test), independent of the factor V Leiden mutation (FVL), is a risk factor for pregnancy loss [5,6] and pre-eclampsia [7]. Tissue factor pathway inhibitor (TFPI) is known to be a major determinant of thrombin generation-based APC sensitivity [8], i.e. low TFPI levels are associated with APC resistance. We therefore studied the role of endogenous thrombin potential (ETP) and TFPI antigen levels in women with recurrent pregnancy loss. The study population comprised 22 women with a history of recurrent early pregnancy loss (three or more consecutive losses at < 24 weeks, n 1⁄4 17) or intrauterine fetal death (IUFD; n 1⁄4 5). Six of the women had the antiphospholipid syndrome (APS), while the other 16 had no detectable aPA. All samples were taken in the non-pregnant state, at least 6 weeks after the end of pregnancy. The following womenwere excluded from the study: those with protein C or S deficiency, FVL, or who were receiving oral anticoagulation, heparin or oral contraceptives at the time of testing. Thrombin generation was measured with and without exogenous human recombinant APC, using a method based on that of Rosing et al. [9]. Briefly, coagulation was triggered by the addition of 7 pM tissue factor (Innovin, Marburg, Germany), 20 lM phospholipid (Rossix, Mölndal, Sweden) and 16 mM CaCl2 to defibrinated plasma. The ETP, calculated from the area under the curve, was measured continuously by cleavage of a chromogenic substrate (Pefachrome TG, Pentapharm, Basel, Switzerland) using the ACL9000 (Instrumentation Laboratory, Milan, Italy). This was performed with and without the addition of 5 nM APC (Eli Lilly, Indianapolis, IN, USA). Results were expressed as ratios relative to pooled normal plasma (PNP): ETP 1⁄4 thrombin formed in patient plasma/thrombin formed in PNP, with no APC. ETP 1⁄4 thrombin formed in patient plasma/thrombin formed in PNP, with 5 nM APC. Total TFPI antigen was assayed using the IMUBIND Total TFPI ELISA kit (American Diagnostica Inc, Stamford, CT, USA) a quantitative sandwich ELISA, which recognizes full-length, truncated and conjugated forms of TFPI [10]. The Mann–WhitneyU-test was used to test the differences between the medians and statistical significance was defined as P < 0.05. Normal reference ranges were established in citrated plasma from 20 normal non-pregnant women: ETPmedian 0.91 (95% reference range 0.70–1.07), ETP 0.96 (0.76–1.07) TFPI antigen 89 ng mL (75–120 ng mL). Both ETP and ETP were significantly higher in the women with previous pregnancymorbidity than innormal subjects (medianETP1.07, P < 0.0001;medianETP 1.32,P < 0.0001). Themedian TFPIwas 75.4 ng mL (range 31.5–120 ng mL,P 1⁄4 0.007), with lowTFPIantigen levels in 10outof 22 (45%)of thewomen withpreviouspregnancymorbidity, all ofwhomhad raisedETP and/or ETP (Table 1). Although there was a tendency towards higher ETP and lower TFPI antigen levels in the women with APS, antiphospholipid antibody status had no statistically significant effect on ETP, ETP or TFPI antigen levels. Both ETP and ETP showed a negative correlation with TFPI antigen level (r 1⁄4 )0.48 and )0.24, respectively). Furthermore, the addition of increasing amounts of a polyclonal antibody (rabbit antihuman TFPI IgG; American Diagnostica Inc.), that blocked TFPI function, caused a dose-dependent increase in ETP of up to 50% in normal plasma and a modest increase in ETP (approximately 10%). The mechanisms responsible for the association between thrombophilia and pregnancy morbidity are unclear [11]. Our preliminary data suggest that low levels of plasma TFPI, increased thrombin generation and resistance to APCmay be a common finding in women with pregnancy loss/morbidity. We have demonstrated an association between TFPI levels and APC resistance, and have shown that blocking TFPI activity in vitro causes APC resistance. It is clear that the causes of Correspondence: Chris Gardiner, Department of Haematology, University College London, London, UK. Tel.: +44 845 1555000 ext. 8527; fax: +44 207 3809886; e-mail: chris.gardiner@uchevaluation.co.uk

Performance Evaluation of a New Small-Volume Coagulation Monitor The SmartCheck INR System

The SmartCheck INR (Unipath, Bedford, England) is a point-of-care device for professional and patient self-monitoring of oral anticoagulant therapy. It measures the international normalized ratio (INR) and assesses test strip integrity, temperature, and sample quality. No significant differences were found in SmartCheck INR results from 3 different instruments or in 3 test-strip lots. Within run imprecision was 0.89% and 6.36% for low and high control samples, respectively (mean INR, 0.99 and 4.08, respectively). Comparability was assessed in 68 patients receiving warfarin by using PTHS Plus (Instrumentation Laboratory, Lexington, MA) and Innovin (Dade Behring, Marburg, Germany) thromboplastins. Good correlations were observed between the methods (r = 0.89 and r = 0.90, respectively) with no significant differences in means: SmartCheck INR, 2.82; Innovin, 2.75; PTHS Plus, 2.74. Clinical agreement with laboratory methods was 88% for Innovin and 97% for PTHS Plus. The SmartCheck INR was easy to use with no mechanical failures during the evaluation and a low test-strip failure rate of 4.7%. The SmartCheck INR provides accurate and reproducible results and is suitable for routine monitoring of oral anticoagulant therapy in the majority of patients.