Gary W. Moore

Fibrinogen estimates are influenced by methods of measurement and hemodilution with colloid plasma expanders

BACKGROUND: Measurement of plasma fibrinogen is often required in critically ill patients or massively bleeding patients being resuscitated with colloid plasma expander. This study aimed at evaluating different assays of plasma fibrinogen after in vitro dilution with commonly used plasma expanders and challenged the hypothesis that levels of fibrinogen are estimated significantly higher in plasma diluted with colloid plasma expander compared with isotonic saline.

Complement activation in patients with isolated antiphospholipid antibodies or primary antiphospholipid syndrome

The antiphospholipid syndrome (APS) is the association of thrombosis and recurrent pregnancy loss and/or pregnancy morbidity with persistent antiphospholipid antibodies (aPL). Increased complement activation has been implicated in the pathogenesis of APS in animal models. It was our objective to evaluate complement activation in patients with aPL or primary antiphospholipid syndrome (PAPS). We measured complement activation products, fragments Bb and C3a-desArg by ELISA in 186 aPL/PAPS patients and 30 healthy controls. All patients with aPL had significantly increased levels of complement activation products. Fragment Bb levels (mean, 95% CI); (thrombotic APS 0.54 units/ml, 0.31-0.83, obstetric APS 0.60 units/ml,0.39-1.02, isolated aPL 0.48 units/ml, 0.29-0.85, overall 0.39 units/ml, 0.33-0.47) and C3a-desArg levels (mean, 95% CI): (thrombotic APS 261 ng/ml, 219-311, obstetric APS 308 ng/ml, 243-391, isolated aPL 258 ng/ml, 193-337, overall 225 ng/ml, 202-251) were significantly higher compared to controls (fragment Bb 0.06 units/ml, 0.03-0.11, C3a-desArg 69 ng/ml, 50-92). There were correlations between Fragment Bb and C3a-desArg levels in all patients with aPL. Receiver operator characteristic (ROC) analysis showed increased fragment Bb and C3a-desArg levels had strong associations with the presence of persistent lupus anticoagulant (area under ROC: Bb 0.89, and C3a-desArg 0.90), dual and triple aPL positivity (Bb 0.71-0.82, C3a-desArg 0.71-0.80) but not with high titre anti-cardiolipin antibodies (Bb 0.62, C3a-desArg 0.65), or anti β2-glycoprotein 1 antibodies (Bb 0.66, C3a-desArg 0.67). Complement activation is present in all patient groups within this large cohort of patients aPL. This suggests it may have a major role in the pathogenesis of APS and merits further study.

Heterozygous factor XI deficiency associated with three novel mutations

To determine the utility of single‐stranded conformation polymorphism (SSCP) analysis for screening mutations in the factor XI (fXI) gene, we investigated three patients with heterozygous factor XI deficiency. DNA sequence analysis confirmed three novel mutations; a CGC → TGC (Arg308Cys) mutation in exon 9, a GCT→GTT (Ala412Val) mutation in exon 11 and an AGC → AGA (Ser576Arg) mutation in exon 15. We postulated on the structural implications of these missense mutations. Our results demonstrated that genotypic analysis is a useful tool for conclusive differentiation between heterozygous factor XI deficiency and normal subjects.

The Ecarin time is an improved confirmatory test for the Taipan snake venom time in warfarinized patients with lupus anticoagulants

&NA; The Taipan snake venom time using dilute phospholipid as a screening test with a platelet neutralization procedure as a confirmatory test has been shown to be a sensitive and specific approach to detection of lupus anticoagulants. Taipan venom is largely insensitive to the effects of ongoing warfarin anticoagulation and this can be useful in detection of lupus anticoagulants in patients receiving this treatment. This study compared the use of the platelet neutralization procedure with the Ecarin time as confirmatory tests for the Taipan snake venom time, the Ecarin venom fraction being insensitive to both lupus anticoagulants and the effects of oral anticoagulants. Screening and confirmatory test data were assessed for phospholipid dependence by three different mathematical methods and there was no advantage in using the Ecarin time in detection of ‘uncomplicated’ lupus anticoagulants. In lupus anticoagulant‐positive warfarinized patients, the Ecarin time achieved higher detection rates than the platelet neutralization procedure irrespective of the method used to assess correction. The Ecarin time confirmed lupus anticoagulants in all of those samples that generated elevated Taipan snake venom time ratios whereas the platelet neutralization procedure identified only 33%. Taipan snake venom time plus Ecarin time offers good diagnostic precision for lupus anticoagulant detection in a group of patients where lupus anticoagulant identification is difficult due to ongoing anticoagulation. Blood Coagul Fibrinolysis 14:307‐312

Clinically significant differences between point-of-care analysers and a standard analyser for monitoring the international Normalized Ratio in oral anticoagulant therapy : a multi-instrument evaluation in a hospital outpatient setting

The increasing number of patients requiring oral anticoagulant therapy has lead to an expansion in the use of point-of-care test (POCT) analysers for measuring the International Normalized Ratio (INR) for monitoring purposes. Availability of new technology inevitably leads to comparisons with standard methodologies, and studies to date have reached varying conclusions about the comparability of POCT INRs with conventional testing. We compared the performance of five POCT instruments (Hemochron Junior Signature, ProTime, CoaguChek S, INRatio and TAS) against Innovin thromboplastin on a Sysmex CA1500 automated analyser. The Hemochron Junior Signature, ProTime and CoaguChek S demonstrated strong correlation with the laboratory method (R2 > 0.94). These three analysers demonstrated higher percentages of paired results within 0.5 INR units (81.5, 92.0 and 74.0%, respectively); the INRatio and TAS demonstrated 54.2 and 62.2%, respectively. Within INR ranges of up to 2.0, 2.1–3.0, 3.1–4.0 and above 4.0, none of the POCT analysers demonstrated significant agreement with the standard method in every range. All POCT instruments showed a degree of bias and greater variation from the standard method at INR values above 3.0. These data indicate the potential for POCT analysers to generate INR values sufficiently different from conventional methods that they may impact on clinical decision-making.

Combining Taipan snake venom time/Ecarin time screening with the mixing studies of conventional assays increases detection rates of lupus anticoagulants in orally anticoagulated patients

BackgroundOral anticoagulation compromises conventional lupus anticoagulant (LA) screening assays. Mixing studies can counteract the oral anticoagulant effect but the dilution reduces sensitivity and can generate false negative results. A firm diagnosis can be made from mixing studies when an elevated screen ratio is accompanied by a confirm ratio that generates significant correction to demonstrate phospholipid dependence, but also returns into the reference range, indicating complete normalisation of the oral anticoagulant effect. Taipan snake venom time (TSVT) with Ecarin time (ET) as a confirmatory test comprises an oral anticoagulant insensitive LA detection system and this study investigates the potential impact on detection rates when coupled with mixing studies on standard assays.MethodsEighty patients known to have LA who were receiving oral anticoagulation were tested with TSVT/ET and 1:1 mixing studies with normal plasma by dilute Russells viper venom time (DRVVT) and dilute activated partial thromboplastin time (DAPTT) to assess detection rates by single and multiple assays.ResultsThirty three of the 80 samples from known LA positive patients were positive in all three assays and 15 were positive in combinations of DRVVT, DAPTT or TSVT/ET. The remainder were positive in only one assay; 12 by DRVVT, 4 by DAPTT and 16 by TSVT/ET. Although all DRVVT and DAPTT positive mixing studies generated significant correction of the screen ratio by the confirm ratio, not all confirm ratios corrected back into the reference range. This was the case for 87.5% of the DRVVT results, 44.7% of the DAPTT results and 13.3% of the TSVT/ET positive mixing tests.ConclusionAddition of TSVT/ET screening for LA in orally anticoagulated patients could increase diagnostic efficacy either by detecting antibodies diluted in the mixing tests of conventional assays or those that do not react in DRVVT or DAPTT. Additionally, TSVT/ET can affirm the presence of a LA where conventional assay mixing tests may not have fully counteracted the oral anticoagulant effect but confirmatory test correction suggests the presence of a LA.

Lupus anticoagulant detection: out of control?

Expert guidelines indicate that normalised ratios are preferred to clotting times for lupus anticoagulant (LA) assays to mitigate analytical variation. We investigated the effects of deriving normalised ratios from the reference interval (RI) mean or different normal pooled plasmas (NPP).

Improved detection of lupus anticoagulants by the dilute Russell's Viper venom time

Accurate and timely detection of lupus anticoagulants (LAs) is of diagnostic and prognostic importance due to the association of persistent LAs with thrombotic disease. A review of LA screening results by kaolin clotting time and dilute Russells Viper venom time (dRVVT) on 2843 patient samples demonstrated that only 40.7% (417 of 1024) of elevated dRVVT ratios could be interpreted as consistent with the presence of an LA by confirmatory procedures. Apart from those due to the effects of anticoagulant therapy, the remainder generated inconclusive interpretations, necessitating significant numbers of costly repeat investigations. Manipulation of dRVVT assay conditions by increasing confirmatory reagent concentration, and altering venom concentration to maintain analytical parity with the standard assay, revealed LAs not fully neutralized by confirmatory tests at standard concentrations. Further experiments were performed using Russells Viper venom reagents from five different manufacturers to demonstrate that the findings were not a reagent-specific phenomenon. Higher detection rates were achieved using multiple conventional assays but samples remained that required a modified confirmatory test to demonstrate LA activity. A previously unreported group of LAs was identified with raised dRVVT ratios that failed to correct with any of the dRVVT assays but demonstrated significant correction with all reagents in the modified confirmatory test. Use of modified confirmatory tests enhances sensitivity and specificity, and doubles LA detection rates by dRVVT. Adoption of the technique will significantly increase cost-effectiveness of LA detection in clinical practice.

Mixing test specific cut-off is more sensitive at detecting lupus anticoagulants than index of circulating anticoagulant

INTRODUCTION Recent guidelines for lupus anticoagulant (LA) detection recommend mixing test interpretation with either a mixing test-specific cut-off (MTC) or index of circulating anticoagulant (ICA). Few studies directly compare efficacy of these approaches. We retrospectively applied MTC and ICA assessment to raw data of 350 LA-positive plasmas from non-anticoagulated patients to compare detection rates of inhibition. MATERIALS AND METHODS Screen and confirm dRVVT and dilute APTT assays were performed on undiluted plasma and 1:1 mixtures with normal pooled plasma. Samples were considered LA-positive if one or both screening test ratios were elevated and corrected by ≥10% with the confirmatory test. Mixing tests were assessed against locally derived cut-offs for MTC (dRVVT >1.13, dAPTT >1.15) and ICA (dRVVT >11.9%, dAPTT >13.2%). RESULTS 105 of 350 (30%) were positive in dRVVT and dAPTT, 109/350 (31.1%) were dRVVT positive only and 136/350 were dAPTT positive only (38.9%), from undiluted plasma results. Of the 214 dRVVT positive plasmas, 53 (24.8%) were negative for inhibition by MTC and 65 (30.4%) negative by ICA. Of the 241 dAPTT positive plasmas, 48 (19.2%) were negative by MTC and 97 (40.2%) negative by ICA. CONCLUSION Whilst integrated testing often detects LA without mixing tests they are diagnostically useful in certain circumstances. Thus, it is valuable to maximise mixing test interpretation as the dilution can lead to false-negative results. These data on a large cohort of LA-positive plasmas reveal that, with the reagents and equipment employed, MTC is superior to ICA in detecting the in vitro inhibition of LA.

The Activated Seven Lupus Anticoagulant (ASLA) assay: a new test for lupus anticoagulants (LAs). Evidence that some LAs are detectable only in extrinsic pathway-based assays.

Accurate and timely detection of lupus anticoagulants (LAs) is of diagnostic and prognostic importance due to the association of persistent LAs with thrombotic disease. In the present study, a sensitive and specific assay for LAs has been developed using recombinant activated factor VII to initiate in vitro coagulation. The Activated Seven Lupus Anticoagulant (ASLA) assay uses dilute brain-derived phospholipid in the screening test and a platelet neutralization procedure (PNP) in the confirmatory test. Tests are reported as ratios of patient clotting time to normal control clotting time and percentage correction by PNP assessed for abnormal ratios. Evaluation with 70 known LA-positive plasmas demonstrated higher detection rates than with individual assays from a wide range of commonly employed LA tests. The ASLA assay identified 61 of 70 (87%) known LAs, compared with 65.7% with the most sensitive of the other assays. The various LA assays were used to test 110 plasma samples from patients with thrombotic disease previously negative for LA. These experiments demonstrated that 18 of 110 (16.4%) contained LAs detectable only in extrinsic pathway-based assays, 10 of these by ASLA testing alone. ASLA testing showed high diagnostic precision and has the potential to make a significant contribution to LA detection.