Algorithm for Rapid Exclusion of Clinically Relevant Plasma Levels of Direct Oral Anticoagulants in Patients Using the DOAC Dipstick: An Expert Consensus Paper.

BACKGROUND: With the widespread use of direct oral anticoagulants (DOACs), there is an urgent need for a rapid assay to exclude clinically relevant plasma levels. Accurate and rapid determination of DOAC levels would guide medical decision-making to (1) determine the potential contribution of the DOAC to spontaneous or trauma-induced hemorrhage; (2) identify appropriate candidates for reversal, or (3) optimize the timing of urgent surgery or intervention. METHODS AND RESULTS: The DOAC Dipstick test uses a disposable strip to identify factor Xa- or thrombin inhibitors in a urine sample. Based on the results of a systematic literature search followed by an analysis of a simple pooling of five retrieved clinical studies, the test strip has a high sensitivity and an acceptably high negative predictive value when compared with levels measured with liquid chromatography tandem mass spectrometry or calibrated chromogenic assays to reliably exclude plasma DOAC concentrations ≥30 ng/mL. CONCLUSION: Based on these data, a simple algorithm is proposed to enhance medical decision-making in acute care indications useful primarily in hospitals not having readily available quantitative tests and 24/7. This algorithm not only determines DOAC exposure but also differentiates between factor Xa and thrombin inhibitors to better guide clinical management.

Lupus anticoagulant-hypoprothrombinemia syndrome with severe bleeding diathesis after coronavirus disease 2019: a case report.

Acquired antibodies against factor II (prothrombin) are rare and most commonly associated with severe liver disease or vitamin K antagonist treatment. In very rare cases, these antibodies and associated hypoprothrombinemia are found in patients with lupus anticoagulant (LAC), an antiphospholipid antibody that inhibits phospholipid-dependent coagulation tests. This uncommon entity, called lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS), may cause both severe, life-threatening bleeding and a predisposition to thrombosis. Coronavirus disease 2019 (COVID-19) is associated with a variety of coagulation abnormalities and an increased risk of thrombosis. Bleeding may occur, but it is less common than thromboembolism and has mostly been described in association with the severity of the disease and anticoagulation treatment in hospitalized patients, rarely in the post-acute phase of the disease. We report on a case of an 80-year-old man who developed LAHPS with prothrombin antibodies and severe bleeding after COVID-19.

DOAC Dipstick Testing Can Reliably Exclude the Presence of Clinically Relevant DOAC Concentrations in Circulation.

In certain clinical situations, it is necessary to determine whether clinically relevant plasma levels of direct oral anticoagulants (DOACs) are present. We examined whether qualitative testing of DOACs in urine samples can exclude DOAC plasma concentrations of ≥30 ng/mL. This prospective single-center cohort study included consecutive patients treated with an oral direct factor Xa inhibitor (DXI) (apixaban, n = 31, rivaroxaban, n = 53) and direct thrombin inhibitor (DTI) (dabigatran, n = 44). We aimed to define the negative predictive value (NPV) and other statistical parameters of detecting DXIs and DTIs by DOAC Dipstick at plasma concentrations of ≥30 ng/mL. We also determined the best-fit threshold plasma levels using chromogenic substrate assays by logistic regression analysis. Between July 2020 and July 2021, 128 eligible patients (mean age 66 years, 55 females) were included into the study. The NPVs and sensitivities for DXI and DTI of DOAC Dipstick were 100% at ≥30 ng/mL plasma, for specificities 6 and 21% and for positive predictive values 62 and 72%, respectively. All diagnostic statistical tests improved to values between 86 and 100% at best-fitting plasma thresholds of ≥14 ng/mL for DXI and ≥19 ng/mL for DTI. Visual analysis using the DOAC Dipstick was 100% in agreement with that of the optoelectronic DOASENSE Reader for all the three DOACs.DOAC Dipstick testing can reliably exclude the presence of DOACs in urine samples at best-fitting thresholds of >14 and >19 ng/mL in plasma. The performance of the DOAC Dipstick at detecting lower DOAC concentrations in plasma requires confirmation.

Direct oral anticoagulants (DOACs): From the laboratory point of view.

Direct oral anticoagulants (DOACs) represent a new generation of drugs that have been increasingly used in the prevention and treatment of thromboembolic states. According to the mechanism of anticoagulant action, DOACs are divided into two groups: direct inhibitors of thrombin (dabigatran) and direct inhibitors of activated factor X (FXa) (rivaroxaban, apixaban, edoxaban, betrixaban). Compared to the vitamin K antagonists, DOACs are superior in terms of onset of action, pharmacokinetic and pharmacodynamics properties and fixed daily dose without the need for routine coagulation monitoring. Despite these advantages, there are clinical conditions in which laboratory measurement of DOACs should be performed. Although DOACs have an impact on screening haemostasis assays (prothrombin time, PT; activated partial thromboplastin time, aPTT; and thrombin time, TT), these tests are not appropriate for quantifying drug levels. Therefore, specific quantitative methods (LC-MS/MS as a gold standard method for all DOACs, coagulometric and chromogenic assays for dabigatran, and chromogenic anti-Xa assays with drug-specific calibrators for inhibitors of FXa) should only be used for determination of DOACs concentration. The aim of this review is to present all aspects of laboratory assessment of DOACs, including pre-analytical, analytical and post-analytical factors in the overall testing process with a special accent on the available specific quantitative methods for measurement of DOACs in circulation.

Reporting of activated partial thromboplastin time (aPTT): Could we achieve better comparability of the results?

INTRODUCTION: Activated partial thromboplastin time (aPTT) is determined and reported as clotting time in seconds aPTT(s), but it is presumed that reporting results as patient-to-normal clotting time ratio, aPTT(r), could minimize within-laboratory variability. The aim of study was to investigate differences in reporting aPTT results that can affect comparability of the results among Croatian laboratories and suggest further steps for its harmonization. MATERIALS AND METHODS: The questionnaire on aPTT reporting practice was distributed to 83 laboratories through Survey Monkey application in March 2019 as the part of the first regular round of Croatian Centre for Quality Assessment in Laboratory Medicine proficiency testing. RESULTS: The survey response rate was 0.49. Majority of laboratories report aPTT results as both, seconds and ratio. Participants reported use of 23 different aPTT(s) reference intervals along with 17 different combinations of reagent/coagulometer and 25 aPTT(s) denominators of different origin for aPTT(r) calculation. Despite the same aPTT(s) results, the use of different denominators caused a dispersion of aPTT(r) results that can lead to exceeding external quality assessment performance criteria of 7%, particularly when results were compared for the same reagent group only. By applying aPTT(s) reference interval mean as denominator for calculation of aPTT(r) reference interval better concordance to harmonized one was obtained (17 vs. 27; χ2 = 3.972; P = 0.046). CONCLUSION: In order to improve comparability of the results, laboratories are advised to use mean of aPTT(s) reference interval as denominator for aPTT(r) calculation. Type of coagulometer need to be considered when evaluating aPTT proficiency test results and its currently acceptable limit of performance evaluated accordingly.

Analytical verification and comparison of chromogenic assays for dabigatran, rivaroxaban and apixaban determination on BCSXP and STA Compact Max analysers.

INTRODUCTION: The aim of the study was to perform analytical verification and comparison of chromogenic assays for determination of dabigatran, rivaroxaban and apixaban concentration on BCSXP and STA Compact Max analysers. MATERIALS AND METHODS: Precision, linearity, measurement uncertainty estimation and determination of limit of blank, limit of determination and limit of quantification were calculated. Analytical performance specifications were set according to manufacturer specifications and literature data on between laboratory variability. Comparison of the methods was done using Bland-Altman and Passing-Bablok regression analysis. RESULTS: Obtained results have shown acceptable precision on STA Compact Max only for dabigatran (CV = 3.5%) at lower concentration level comparing to manufacturer declaration (CV = 3.6%). On BCSXP, the highest coefficient of variation has been shown for apixaban (6.1%) at lower concentration level. Within laboratory precision was not met on STA Compact Max for all assays. Bland-Altman analysis has shown statistically significant bias for dabigatran (23.2%, 95%CI 11.2 - 35.3; P < 0.001) and apixaban (8.4%, 95%CI 1.2 - 15.6; P = 0.023). Passing-Bablok regression analysis has shown systematic and proportional deviation between methods for rivaroxaban (y = 6.52 (2.94 to 11.83) + 0.84 (0.80 to 0.89) x. CONCLUSION: Chromogenic assays for dabigatran, rivaroxaban and apixaban on BCSXP and STA Compact Max analysers are shown as methods with satisfactory long-term analytical performance specifications for determination of direct oral anticoagulants in clinical laboratories. However, we cannot recommend interchangeable use because of the significant bias between assays.

Interference of M-protein on Thrombin Time Test: A Case Report.

OBJECTIVE: A case of interference of monoclonal protein (M-protein) on thrombin time (TT) test in a 39-year-old Caucasian male patient is presented. METHODS: Coagulation screening tests were performed where altered results only for TT result (>150 seconds) and activated partial thromboplastin time (aPTT) result (36 seconds) were measured. Further specific coagulation testing included measurement of individual coagulation factors FII, FV, FVII, FVIII, FIX, FX, FXI, and FXII. Diagnostic steps in detection and identification of monoclonal protein included serum protein electrophoresis and immunofixation (both serum and urine specimen). RESULTS: Monoclonal protein immunoglobulin G kappa detection and identification in serum and urine clarified the situation. CONCLUSION: Unexpectedly altered results of screening coagulation tests without any appropriate clinical signs and symptoms in a patient without any anticoagulant therapy needs to be critically considered in the context of extended next diagnostic steps in order to clarify the cause of pathological test results.

Chromogenic anti-FXa assay calibrated with low molecular weight heparin in patients treated with rivaroxaban and apixaban: possibilities and limitations.

INTRODUCTION: Clinical application of rivaroxaban and apixaban does not require therapeutic monitoring. Commercial anti-activated factor X (anti-FXa) inhibition methods for all anti-FXa drugs are based on the same principle, so there are attempts to evaluate potential clinical application of heparin-calibrated anti-FXa assay as an alternative method for direct FXa inhibitors. We aimed to evaluate relationship between anti-FXa methods calibrated with low molecular weight heparin (LMWH) and with drug specific calibrators, and to determine whether commercial LMWH anti-FXa assay can be used to exclude the presence of clinically relevant concentrations of rivaroxaban and apixaban. MATERIALS AND METHODS: Low molecular weight heparin calibrated reagent (Siemens Healthineers, Marburg, Germany) was used for anti-FXa activity measurement. Innovance heparin (Siemens Healthineers, Marburg, Germany) calibrated with rivaroxaban and apixaban calibrators (Hyphen BioMed, Neuville-sur-Oise, France) was used for quantitative determination of FXa inhibitors. RESULTS: Analysis showed good agreement between LMWH calibrated and rivaroxaban calibrated activity (κ = 0.76) and very good agreement with apixaban calibrated anti-Xa activity (κ = 0.82), respectively. Low molecular weight heparin anti-FXa activity cut-off values of 0.05 IU/mL and 0.1 IU/mL are suitable for excluding the presence of clinically relevant concentrations (< 30 ng/mL) of rivaroxaban and apixaban, respectively. Concentrations above 300 ng/mL exceeded upper measurement range for LMWH anti-FXa assay and cannot be determined by this method. CONCLUSION: Low molecular weight heparin anti-FXa assay can be used in emergency clinical conditions for ruling out the presence of clinically relevant concentrations of rivaroxaban and apixaban. However, use of LMWH anti-FXa assay is not appropriate for their quantitative determination as an interchangeable method.

Croatian Society of Medical Biochemistry and Laboratory Medicine: National recommendations for blood collection, processing, performance and reporting of results for coagulation screening assays prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and D-dimer.

A modern diagnostic laboratory offers wide spectrum of coagulation assays utilized in the diagnosis and management of patients with haemostatic disorders, preoperative screening and anticoagulation therapy monitoring. The recent survey conducted among Croatian medical biochemistry and transfusion laboratories showed the existence of different practice policies in particular phases of laboratory process during coagulation testing and highlighted areas that need improvement. Lack of assay standardization together with non-harmonized test results between different measurement methods, can potentially lead to incorrect decisions in patient's treatment. Consequently, patient safety could be compromised. Therefore, recommended procedures related to preanalytical, analytical and postanalytical phases of prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and D-dimer testing are provided in this review, aiming to help laboratories to generate accurate and reliable test results.

Policies and practices in haemostasis testing among laboratories in Croatia: a survey on behalf of a Working Group for Laboratory Coagulation of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

INTRODUCTION: The objective of this survey was to assess current policies and practice in haemostasis testing among both hospital and outpatient laboratories in Republic of Croatia. MATERIALS AND METHODS: A questionnaire with seventy questions divided into nine sections was created in May 2015. Participants were asked about their practice related to test request form, sample collection, prothrombin time (PT) and activated partial thromboplastin time assays, other individual haemostasis assays, point-of-care testing (POCT), reporting of coagulation tests results and quality assurance of procedures, the personnel and other laboratory resources, as well as on issues related to education and implementation of additional coagulation assays in their laboratory. The survey was administered and data were collected between June and September 2015. RESULTS: A total survey response rate was 104/170 (61.2%). Most respondents were faced with incomplete information on prescribed therapy and diagnosis on the test request or inappropriate samples withdrawn on distant locations, but also do not have protocols for handling samples with high haematocrit values. Reporting of PT-INR and D-dimer results was different between laboratories. Although almost all laboratories developed a critical value reporting system, reporting a value to general practitioners is still a problem. Result on coagulation POCT testing showed that not all devices were supervised by laboratories, which is not in compliance with Croatian Chamber of Medical Biochemistry acts. CONCLUSION: Obtained results highlighted areas that need improvement and different practice patterns in particular field of haemostasis testing among laboratories. A harmonization of the overall process of haemostasis testing at national level should be considered and undertaken.

Interference of M-protein on prothrombin time test - case report.

The aim of this report was to present a case of interference on prothrombin time (PT) test that directed further laboratory diagnostics and resulted with final detection of monoclonal gammopathy in an 88-year old man. Routine coagulation testing during medical examination at Emergency Department revealed unmeasurable PT (< 7% activity) and activated partial thromboplastin time (aPTT) within reference range. After repeated sampling for coagulation testing, PT was unmeasurable again, as well as fibrinogen level (< 0.8 g/L), thrombin time (TT) was significantly prolonged (107 seconds) and aPTT was within reference range. In both plasma samples refrigerated at 4 ˚C overnight, white gelatinous precipitate was visible between the cell and plasma layers and the presence of monoclonal protein (M-protein) was suggested in our patient. Further laboratory diagnostics revealed total serum proteins at concentration of 123 g/L and the presence of M-protein IgG lambda (λ) at concentration of 47.1 g/L. These results suggested monoclonal gammopathy as an underlying pathophysiological condition in our patient. Activities of coagulation factors II, V, VII and X were within reference ranges or increased. These results and correction of unmeasurable PT result to 67% in mixing test with commercial normal plasma suggest in vitro rather than in vivo interference of M-protein on PT result. In contrast, significantly prolonged TT results in all analysed samples suggest impact of M-protein on this global coagulation test due to possible effect on fibrin polymerization.

Inflammation and haemostasis.

Inflammation and haemostasis are interrelated pathophysiologic processes that considerably affect each other. In this bidirectional relationship, inflammation leads to activation of the haemostatic system that in turn also considerably influences inflammatory activity. Such, the haemostatic system acts in concert with the inflammatory cascade creating an inflammation-haemostasis cycle in which each activated process promotes the other and the two systems function in a positive feedback loop. The extensive crosstalk between immune and haemostatic systems occurs at level of all components of the haemostatic system including vascular endothelial cells, platelets, plasma coagulation cascade, physiologic anticoagulants and fibrinolytic activity. During inflammatory response, inflammatory mediators, in particular proinflammatory cytokines, play a central role in the effects on haemostatic system by triggering its disturbance in a number of mechanisms including endothelial cell dysfunction, increased platelet reactivity, activation of the plasma coagulation cascade, impaired function of physiologic anticoagulants and suppressed fibrinolytic activity. The two examples of pathophysiologic processes in which the tight interdependent relationship between inflammation and haemostasis considerably contribute to the pathogenesis and/or progression of disease are systemic inflammatory response to infection or sepsis and acute arterial thrombosis as a consequence of ruptured atherosclerotic plaque. Close links between inflammation and haemostasis help explain the prothrombotic tendency in these two clinical conditions in which inflammation shifts the haemostatic activity towards procoagulant state by the ability of proinflammatory mediators to activate coagulation system and to inhibit anticoagulant and fibrinolytic activities. This review summarizes the current knowledge of the complex interactions in the bidirectional relationship between inflammation and haemostasis.

Diagnostic algorithm for thrombophilia screening.

Thrombophilia screening is aimed at detecting the most frequent and well-defined causes of venous thrombosis, such as activated protein C resistance/factor V Leiden mutation, prothrombin G20210A gene mutation, deficiencies of natural anticoagulants, such as antithrombin, protein C and protein S, the presence of antiphospholipid antibodies, hyperhomocysteinemia and increased factor VIII activity. At this time, thrombophilia screening is not recommended for those possible congenital or acquired risk factors, whose association with increased risk of thrombosis has not been proven sufficiently. Laboratory investigations should include a step-wise approach to the diagnosis of thrombotic disorders with respect to the assays and methods of analysis that are used. The assays recommended for the first diagnostic step of screening should establish, whether the subject has one of the common causes of thrombophilia. If one or more abnormal results are obtained, the second diagnostic step includes the assays recommended for confirmation and/or characterization of the defect. When performing the investigation of thrombophilia, it is important to consider all pre-analytical and other variables that may affect the results of thrombophilia testing, including time of testing, age, gender, liver function, hormonal status, pregnancy or the acute phase response to inflammatory diseases. This is necessary, in order to avoid, any misinterpretation of the results. This review summarizes the current knowledge concerning thrombophilia investigations, with special focus on the diagnostic algorithm regarding patient selection, the assays and methods of analysis used and all the variables that should be considered when employing tests for the diagnosis of thrombophilia.

Soluble transferrin receptor and transferrin receptor-ferritin index in iron deficiency anemia and anemia in rheumatoid arthritis.

The aim of the study was to evaluate the clinical efficiency of soluble transferrin receptor and transferrin receptor-ferritin index (sTfR/logF) in the diagnosis of iron deficiency anemia, as well as the differential diagnosis of iron deficiency anemia and anemia in rheumatoid arthritis. The study included 96 patients with anemia and 61 healthy volunteers as a control group. In healthy subjects there were no significant sex and age differences in the parameters tested. The study results showed these parameters to be reliable in the diagnosis of iron deficiency anemia, as well as in the differential diagnosis of iron deficiency anemia and anemia of chronic disease. The results indicate that sTfR/logF could be used to help differentiate coexisting iron deficiency in patients with anemia of chronic disease. Receiver operating characteristic analysis showed a higher discriminating power of transferrin receptor-ferritin index vs. soluble transferrin receptor in the diagnosis of iron deficiency anemia, as well as in the differential diagnosis between iron deficiency anemia and anemia of chronic disease. In patients with anemia in rheumatoid arthritis, the parameters tested showed no significant differences with respect to C-reactive protein concentration. These results suggested that the parameters tested are not affected by acute or chronic inflammatory disease.