Essentials Between-lab variations of cut-off values in lupus anticoagulant detection are unknown. Cut-off values were calculated in 11 labs each testing plasma from 120 donors with 3 platforms. Major ...variation was observed even within the same platform. Cut-off values determined in different labs are not interchangeable.
Background Cut-off values for interpretation of lupus anticoagulant (LA) detection are poorly investigated. Aims (i) To assess whether results from healthy donors were normally distributed and (ii) the between-laboratories differences in cut-off values for screening, mixing and LA confirmation when calculated as 99th or 95th centiles, and (iii) to assess their impact on the detection rate for LA. Methods Each of 11 laboratories using one of the three widely used commercial platforms for LA detection was asked to collect plasmas from 120 healthy donors and to perform screening, mixing and LA confirmation with two methods (activated partial thromboplastin time APTT and dilute Russell viper venom dRVV). A common set of LA-positive or LA-negative freeze-dried plasmas was used to assess the LA detection rate. Results were centralized (Milano) for statistical analysis. Results and conclusions (i) Clotting times or ratios for healthy subjects were not normally distributed in the majority of cases. The take-home message is that cut-off values should be determined preferably by the non-parametric method based on centiles. (ii) There were relatively large inter-laboratory cut-off variations even within the same platform and the variability was marginally attenuated when results were expressed as ratios (test-to-normal pooled plasma). The take-home message is that cut-off values should be determined locally. (iii) There were differences between cut-off values calculated as 99th or 95th centiles that translate into a different LA detection rate (the lower the centile the greater the detection rate). The take-home message is that cut-off values determined as the 95th centile allow a better LA detection rate.
Factor XI (FXI) deficiency is a rare congenital hemostatic disorder associated with increased bleeding tendency in trauma, surgery or when other hemostatic defects are present. Perioperative ...hemostatic management of a patient with a severe FXI deficiency undergoing major oncological liver and colorectal surgery with therapeutic plasma exchange (TPE) with fresh frozen plasma (FFP) is reported.
A 54-year-old male with severe FXI deficiency was scheduled for resection of synchronous rectal cancer and multiple liver metastases. Baseline prothrombin time
(PT) was 97 %, activated partial thromboplastin time (aPTT) 89 s(s) and FXI levels <1 IU/dL. The rotational thromboelastometry (ROTEM™) presented a prolonged INTEM clotting time (CT) = 443 s (RV 100−240 s) and a clot formation time (CFT) = 110 s (RV 30–100 s). TPE with FFP was carried out achieving FXI levels up to 46 IU/dL and an aPTT of 33 s, normalizing thromboelastometry parameters to an INTEM CT = 152 s and a CFT = 86 s before the procedure. After surgery, the patient received daily FFP to maintain FXI levels above 30 IU/dL until discharge on the eighth day. A total of 30 FFP units were transfused during hospital stay. No significant bleeding events neither transfusion related complications were observed during the perioperative period.
Given the lack of correlation between FXI levels and bleeding risk, a multidisciplinary approach based on daily FXI levels monitoring, close clinical assessment and factor supplementation is mandatory. In conclusion, TPE with FFP is an efficacious alternative strategy to correct severe FXI deficiency in patients undergoing major surgery.
Summary
Introduction
Oral direct thrombin and Xa inhibitors are worldwide distributed for prevention and treatment of thrombosis. It is important to recognize their effects on lupus anticoagulant ...(LA) testing. The aim of the study is to describe the rate of false‐positive results of LA tests on plasmas of patients with previous negative LA tests results that receive dabigatran etexilate (DAB) 110 mg/twice a day, rivaroxaban (RIV) 10 mg/day or 15 mg/twice a day, or enoxaparin 40 mg/day.
Methods
Blood was taken between 1.5 and 4 h post administration. Tests evaluated are as follows: prothrombin time, APTT, dilute Russell viper venom time (DRVVT) screen, APTT, and DRVVT mixing studies, index of circulating anticoagulant (ICA) with normal plasma, screen/confirm normalized ratio (NR) for DRVVT and silica clotting time (SCT).
Results
Plasmas from patients taking DAB (n = 22) presented 100% prolonged APTT and DRVVT with ICA above the cutoff point and 81.8% positive screen/confirm NR, 100% prolonged SCT screen, but 4.5% positive confirmatory NR. All patients receiving RIV at 15 mg/twice a day (n = 4) presented positive DRVVT screen, mixing, and confirmatory tests, 75% and 100% prolonged APTT and SCT screen, with negative screen/confirm NR. Those taking RIV 10 mg/day (n = 22) showed 81.8% prolonged DRVVT screen, 82.3% and 76.5% of them with positive mixing and confirmatory studies. Patients receiving enoxaparin also presented high prevalence of APTT and DRVVT false‐positive results.
Conclusion
Dabigatran etexilate, RIV, and enoxaparin affect tests for LA not only in screening and mixing, but also in confirmatory studies. We considered that LA testing should not to be performed when patients are taken these drugs, particularly if blood is collected at peak, in order to avoid false‐positive results.
The hemostatic process is tightly regulated by several antithrombotic mechanisms. Among them, protein Z (PZ)-dependent protease inhibitor (ZPI) potently inhibits factor (F)Xa in a manner dependent on ...calcium ions, phospholipids and PZ. Autoimmune antiphospholipid antibodies (aPL) are mainly directed against phospholipid-binding plasma proteins such as beta2-glycoprotein I (beta2GPI) and prothrombin, and are known to interfere with phospholipid-dependent hemostatic pathways. In this study, we investigated whether purified aPL are able to interfere with inhibition of FXa by PZ/ZPI. beta2GPI modestly delayed the FXa inactivation by PZ/ZPI and most isolated aPL-IgGs were found to further increase the inhibitory potential of beta2GPI on PZ/ZPI activity. Without beta2GPI, the PZ/ZPI activity was unaffected by the addition of aPL-IgG. As PZ deficiency is hypothesized to lead to a prothrombotic state, we performed a case-control study to measure plasma levels of PZ and ZPI in 66 patients with autoimmune aPL and 152 normal controls. The prevalence of low PZ levels (below the 5th percentile of controls) was significantly greater in the 37 patients with definite antiphospholipid syndrome (APS) (24.3%) but not in the 29 aPL patients not fulfilling the criteria for APS (10.3%) compared with the normal group (4.6%, P < 0.001 vs. APS). ZPI antigen levels were similar in patients with aPL and normal controls. Concomitant PZ deficiency increased by approximately sevenfold the risk of arterial thrombosis in aPL patients. Taken together, these data suggest that the PZ/ZPI system is commonly impaired in aPL patients thus probably increasing the thrombotic risk.
The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies (aPL) in patients with thromboembolic complications. In APS, most aPL are autoantibodies to ...β2-glycoprotein I and prothrombin, which play a major role in the APS pathogenesis. Nevertheless, antibodies with the same antigen specificity are also found in aPL patients with leprosy, in whom thromboembolic complications are uncommon. The in vivo upregulation of the tissue factor (TF) pathway and the imbalance of cytokines have been proposed as potential mechanisms of thrombosis in the APS. We measured the circulating levels of TF, interleukin 6 (IL-6), IL-6 receptor (sIL-6R), tumor necrosis factor (TNF-a) and interferon g (IFN-g) in 83 patients with autoimmune aPL (42 with and 41 without clinical features of definite primary APS), 48 leprosy patients (33 with aPL) and 48 normal controls. There was a trend (P = 0.06) to higher median sTF in patients with autoimmune aPL (139 pg/mL) compared with leprosy patients (103.5 pg/mL) and controls (123 pg/mL). In addition, the frequency of raised sTF levels (.187 pg/mL) was significantly higher in the group with autoimmune aPL 22.9% (APS 21.4%, non-APS 24.4%) but not in leprosy (10.4%) compared with controls (4.2%). Elevated levels of IL-6 and TNF-a and a trend to lower IFN-g were found in patients with definite APS. Leprosy patients with aPL, however, had increased TNF-a and IFN-g but normal IL-6 levels. Levels of sIL-6R did not differ between controls and either patients with autoimmune aPL or leprosy. The different cytokine profiles as well as differences in circulating levels of TF might contribute to the high thrombotic risk found in patients with autoimmune aPL but not in leprosy related aPL patients.
As the activity of the tissue factor pathway inhibitor (TFPI) may be impaired in patients with antiphospholipid antibodies (aPL), 162 aPL patients were evaluated for autoantibodies to recombinant ...TFPI (anti-TFPI) using an optimized ELISA. Anti-TFPI (>18 U mL(-1) for IgG and/or > 15 U mL-1 for IgM) were detected in 54 patients with aPL (33.3%) and in three out of 79 normal controls (3.8%, P < 0.0001). Among aPL patients, the prevalence of positive anti-TFPI was 38.3 and 28.4% in those with or without diagnosis of definite antiphospholipid syndrome (APS). Patients with definite APS had a significantly greater frequency of high titer (>50 U mL(-1)) anti-TFPI than aPL patients from the no definite APS group (18.5% vs. 6.2%, OR 3.7, P= 0.017). Most aPL recognized full-length TFPI, but not a truncated form of TFPI lacking the C-terminus of the molecule. Isolated IgGs from subjects with anti-TFPI impaired the dose-dependent inhibitory effect of TFPI on factor Xa activity in the presence, but not in the absence of phospholipid vesicles. Thus, aPL with high titer anti-TFPI limit TFPI action and are associated with the APS.
The diagnosis of antiphospholipid syndrome (APS) requires the presence of both clinical and biological features. Due to the heterogeneity of anti-phospholipid antibodies (aPL) the laboratory approach ...for their detection includes clotting-based tests for lupus anticoagulant (LA) as well as solidphase assays for anticardiolipin antibodies (aCL). In addition, as it has been shown that autoimmune aPL recognize epitopes on phospholipid (PL)-binding plasma proteins, assays detecting antibodies to β2-glycoprotein I (β2-GPI) or prothrombin have been developed. The association between venous or arterial thrombosis and recurrent fetal loss with the presence of conventional aPL (LA and/or aCL) has been confirmed by many studies. The LA and IgG aCL at moderate/high titre seem to exhibit the strongest association with clinical manifestations of the APS. Several reports indicate that LA is less sensitive but more specific than aCL for the APS. Assays against PLs other than CL as well as the use of mixtures of PLs have been proposed to improve the detection of APS-related aPL. Concerning antibodies to PL-binding proteins (detected in the absence of PLs), there is evidence that anti-β2-GPI are closely associated with thrombosis and other clinical features of the APS. Moreover, these antibodies may be more specific in the recognition of the APS and in some cases may be present in the absence of aPL detected by standard tests. Many issues are still under debate and are discussed in this review, such as the problems of standardization of anti-β2-GPI assays, detection of the IgA isotype of aCL and anti-β2-GPI, the coagulation profiles of LA in the recognition of the thrombotic risk and the association of particular markers with subsets of patients with APS.
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