Evaluation of a new silica clotting time in the diagnosis of lupus anticoagulants

INTRODUCTION: A new commercial silica clotting time (SCT), the HemosIL SCT assay (Instrumentation Laboratory, Milan, Italy) was evaluated in the laboratory diagnosis of lupus anticoagulants (LAC). This integrated test system for screening and confirmation was compared with the frequently used aPTT-based PTT-LA and Staclot-LA (Diagnostica Stago, Asnières, France) in a patient population investigated for LAC and in a subpopulation who met the clinical criteria for antiphospholipid syndrome (APS). MATERIALS AND METHODS: 201 samples were analysed with the HemosIL SCT assay. Own reference values were calculated. Results are expressed as measured clotting times in seconds or as normalised ratios. RESULTS: SCT screen and PTT-LA had a sensitivity of, 61.1% and 63.8%, respectively. Normalising the results gained sensitivity up to 72.2% and 90%, respectively. The confirmation SCT and the Staclot-LA had a sensitivity of 30.6% and 63.9% with a specificity of 86.7% and 100%, respectively. Sensitivity of SCT for detecting LAC in clinical criteria positive patients was lower compared to aPTT and dRVVT (45.8% versus 66.7% and 65%). Combination of SCT/dRVVT and aPTT/dRVVT gave a sensitivity of 51.2% and 63.6%, with a specificity of 50.0% and 52.3%, respectively. CONCLUSIONS: In comparison with PTT-LA as screening test, the SCT screen shows an acceptable sensitivity. However, the HemosIL SCT assay including the confirmation step, has a much lower sensitivity in the diagnosis of LAC in comparison with the Staclot-LA test. Combining the HemosIL SCT assay with dRVVT results in a better sensitivity, although lower than the combined aPTT/dRVVT based method as usually performed in our lab.     

Thrombophilic screening in young patients (< 40 years) with idiopathic ischemic stroke: a controlled study

Introduction

Despite extensive clinical and laboratory investigations, the etiology of ischemic stroke remains unknown in approximately one third of patients.

Materials and Methods

Thirty-four consecutive patients less than 40 years old (Males 13, Females 21, mean age 26.6 years, range 2-39) with documented ischemic stroke underwent, one year after the acute event, laboratory evaluation of antithrombin, protein C, free and total protein S, activated protein C resistance, fibrinogen, factor VII:C, homocysteine levels and antiphospholipid antibodies (APA). Moreover, prevalence of F5 R506Q, F2 G2021A and homozygosis for thermolabile variant C677T of the methylenetetrahydrofolate reductase (MTHFR) were also evaluated and compared to the results obtained in 120 normal controls.

Results

Antithrombin and protein C levels resulted normal in all cases. One patient (2.9%) showed free protein S deficiency and 3 patients (8.8%) had activated protein C resistance. Homocysteine levels above 15 μmol/L were found in one patient (2.9%). APA were found in 21 patients (61.7%) and in only 2 out of 120 (1.66%) controls (OR = 95.31; 95% C.I.: 18.22-667.81). The multivariate analysis selected that the presence of APA was significantly associated with an increased risk of stroke (OR = 156.60; 95% C.I.: 25.99-943.47) in this cohort of patients. The combination between APA and cardiovascular risk factors determined a risk of 29-fold (OR = 29.31; 95% CI: 3.28-261.69).

Discussion

Our data suggest that the presence of APA is associated with an increased risk of idiopathic ischemic stroke in young patients. Furthermore, also the combination of APA and cardiovascular risk factors is significantly associated with development of idiopathic ischemic stroke.     

Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulants

INTRODUCTION: Mixing studies are part of the laboratory diagnosis of lupus anticoagulants (LA). They are used to determine the evidence of an inhibitor by demonstrating persistence of an abnormal clotting time by mixing patient plasma with normal plasma. Since there is no standardised interpretation of results of mixing studies, percent correction formulas are proposed. The sensitivity of mixing studies strongly depends on the interpretation of the results. MATERIALS AND METHODS: A retrospective analysis was performed on 361 samples, 75 LA-positive and 286 LA-negative samples. For all the LA-positive samples and for 181/286 LA-negative samples mixing tests on one or more screening tests were performed. A percent correction formula and the Rosner index were calculated for all mixing tests on aPTT and dPT. RESULTS: The <70% correction formula for the mixing tests on aPTT showed the highest sensitivity (95%). The Rosner index had a sensitivity (93%) comparable with the <70% correction formula. dPT is shown to be less sensitive in the detection for LA and, even when the screening test is prolonged, interpretation of the mixing test by the percent correction formula misclassifies many samples. Rosner index in the interpretation of mixing tests for dPT is more sensitive than the percent correction formula, 49% and 57%, respectively. CONCLUSIONS: This study demonstrates that the Rosner index and the percent correction formulas for interpretation of mixing studies are complementary and can help to reduce misclassification of LA-positive or LA-negative samples.     

Automated mixing studies and pattern recognition for the laboratory diagnosis of a prolonged activated partial thromboplastin time using an automated coagulation analyzer

Introduction

The objective of this study was to explore whether an automated coagulation analyzer could be applied to normal plasma mixing studies for the assessment of blood samples showing a prolonged activated partial thromboplastin time (APTT).

Materials and methods

Ten laboratory staff members performed normal plasma mixing studies and evaluated plasma samples using 3 different methods: (1) manual dilution and analysis, (2) manual dilution and automatic analysis with STA-R^®, and (3) automatic dilution and analysis with the Coapresta^® 2000 (CP2000). The time from the start of the analysis to the generation of the result plots and the plasma volumes required were determined. We analyzed patient plasma samples showing a prolonged APTT using the CP2000, and the result plots were categorized into 3 curve patterns based on the area ratio values: the inhibitor type (convex pattern), deficiency type (concave pattern), and suspicious inhibitor type (approximately straight pattern).

Results

When pooled patient plasma was used, the same patterns were obtained from normal plasma mixing studies using the 3 different methods. The time required to complete the mixing studies and the plasma volumes required were 28.2 ± 2.4 min and 350 μL for manual analysis, 23.2 ± 2.1 min and 875 μL for STA-R^®, and 8.5 ± 0.1 min and 175 μL for CP2000, respectively. Of 31 patient samples, 9 were categorized into the inhibitor type, 15 were categorized into the deficiency type, and 7 were categorized into the suspicious inhibitor type.

Conclusions

The CP2000 analyzer is applicable to the laboratory diagnosis of a prolonged APTT using pattern recognition, as it requires a shorter time to complete mixing studies and a smaller plasma volume in comparison with manual analysis.     

Laboratory identification of factor inhibitors: an update

Coagulation factor inhibitors comprise antibodies that bind to and then neutralise specific pro-coagulant plasma proteins. Coagulation factor inhibitors can develop against any coagulation factor, although the most common are against factor VIII (FVIII). These can develop in individuals with inherited haemophilia A (HA) as an immune response to factor replacement therapy, or as auto-antibodies leading to the condition of acquired HA. Clinical suspicion for inhibitors may arise when individuals present with bleeding symptoms without any prior bleeding diathesis, or when a patient with known mild haemophilia presents with a bleeding diathesis more extreme to their usual presentation, or when there is failure of factor replacement therapy to arrest bleeding in a known haemophiliac. The laboratory identification of factor inhibitors requires a careful and systematic approach that excludes other possible causes of prolonged screening tests, most commonly the activated partial thromboplastin time (APTT), and sometimes prothrombin time (PT). Coagulation factor inhibitor studies, including the Bethesda assay, are then undertaken to measure inhibitor titre, which guides treatment. This paper overviews the laboratory investigation of factor inhibitors, and also briefly reviews recent cross-laboratory inhibitor studies and the most recent evidence related to differential inhibitor formation according to type of therapy.     

Laboratory testing of anticoagulants: the present and the future

This review provides an update on laboratory testing and monitoring for existing and emerging anticoagulants, starting with an overview of haemostasis and the routine coagulation tests currently employed within most haemostasis laboratories, including the prothrombin time (PT)/international normalised ratio (INR) and the activated partial thromboplastin time (APTT). Current anticoagulant therapy and laboratory monitoring is then discussed in terms of benefits and limitations, followed by a similar brief discussion of the new and emerging anticoagulants. The main focus, however, is laboratory testing related to vitamin K antagonists, heparin, lepirudin and the new agents dabigatran etexilate and rivaroxaban. Although the newer agents do not require laboratory monitoring, laboratory testing will occasionally be required, and pathology laboratories should become proactive in developing appropriate strategies. The tests most likely to fulfill this role are the ecarin clotting time (or chromogenic alternatives), and the chromogenic anti-Xa assay. Nevertheless, the dilute Russell viper venom time (dRVVT) assay may provide another alternative, and existing routine tests are also likely to be utilised for the foreseeable future, potentially also for laboratory testing of the new anticoagulants, albeit perhaps in modified form.     

Prothrombin time, activated partial thromboplastin time and dilute Russell's Viper Venom times are not shorter in patients with the prothrombin G20210A mutation, and dilute Russell's Viper Venom time may be longer

Introduction

Prothrombin G20210A (PT20210) carriers have increased prothrombin levels and increased risk for venous thrombosis. We hypothesized PT20210 carriers would have decreased PT, aPTT, and dRVVT clotting times.

Methods

We reviewed 1186 thrombotic risk panels that included PT, aPTT, dRVVT, and PT20210 genotype with potential confounding variables, excluding samples consistent with anticoagulant therapy or lupus anticoagulant presence. We examined relationships of PT20210 with PT, aPTT, and dRVVT correcting for covariates using multivariate regression. We confirmed associations in 1876 separate panel results and a group of homozygotes for PT20210 and used general linear models to determine if associated tests predict PT20210 status.

Results

Neither PT, aPTT, nor dRVVT was shorter in PT20210 carriers. Contrary to our hypothesis, PT20210 was significantly associated with higher dRVVT (p = 0.001), but not PT or aPTT. dRVVT differences were significant in a replicate sample p = 0.035 and an additional sample of PT20210 homozygotes (p = 0.02). Of all variables available, only dRVVT predicted PT20210 carrier status (p = 0.0008, AUC = 0.64).

Conclusions

We observed an association between longer dRVVT and the prothrombin G20210A mutation in a retrospective observational study. These findings merit further study in large well-characterized clinical cohorts and laboratory research experiments.     

The effect of DOACs on laboratory tests and their removal by activated carbon to limit interference in functional assays

We aimed to review the interfering effect of DOACs on tests for haemostatic function and then to discuss overcoming these with activated carbon (AC) products, thereby eliminating DOAC issues from test plasmas. Recent relevant articles were reviewed and are discussed. Laboratory tests for DOACs, lupus anticoagulant, factor assays and APC Resistance were carried out in such publications with and without an AC product on various instruments using reagents approved for diagnostic use in well‐regulated clinical laboratories. All reports on this plasma pre‐treatment by AC products agree that they extract DOACs from plasma samples with minimal effect on underlying clotting tests. The specific extraction of DOACs significantly reduced false positive lupus anticoagulant detection and provided more reliable results in clotting factor assays, APC resistance and other thrombophilia tests. Dabigatran and edoxaban seem to be adsorbed more thoroughly by AC from plasmas than rivaroxaban and apixaban. In summary, most of the AC products reviewed here appear to remove DOACs from test plasmas without significantly affecting underlying clotting tests and permit correct diagnosis of various haemostatic conditions despite the initial presence of DOACs. The application of such agents as a sample pre‐treatment to overcome the effects of DOACs for routine coagulation testing is supported by the emerging literature.