Search

APS diagnosis is dependent on accurate detection and interpretation of antiphospholipid antibodies (aPL). Lupus anticoagulant (LA), anticardiolipin antibodies (aCL) and anti-beta2 glycoprotein I antibodies (aβ2GPI) remain the cornerstone of the laboratory part of APS diagnosis. In the 2023 ACR/EULAR APS classification criteria, the type of laboratory parameters remain essentially unchanged compared to the updated Sapporo classification criteria, and aCL and aβ2GPI antibody measurement is still restricted to enzyme-linked immunosorbent assays (ELISA) with moderate and high titer aPL thresholds defined as 40 and 80 units, respectively, and a cutoff calculated by the 99 th percentile has been abandoned. We must differentiate between classification criteria and assessment of aPL in clinical care. Classification criteria are strict and meant for participant inclusion in studies and trials to study homogeneous populations of patients. In contrast, laboratory detection for APS diagnosis in daily practice is broader meant to diagnose each APS patient to optimize their management. Nowadays, there is increasing use of measurement of aPL by methods other than ELISA, the semiquantitative reporting of titers is a matter of debate, as well as the role of the isotypes IgM and IgA, and the role of other aPL, such as antiphosphatidylserine/prothrombin antibodies. Patients diagnosed with the disease may or may not fulfill the classification criteria and inappropriate use of classification criteria may lead to mis(under)diagnosis. The aim of this guidance, based on literature and expert opinion, is to provide guidance recommendations for laboratory workers and clinicians, on routine diagnostic assessment of patients with suspected APS., (Copyright © 2024. Published by Elsevier Inc.)

Competing Interests: Declaration of competing interests There are no competing interests to disclose.

Antiphospholipid syndrome (APS) diagnosis hinges on identifying antiphospholipid antibodies (aPL). Currently, laboratory testing encompasses lupus anticoagulant (LA), anticardiolipin (aCL), and anti-β2-glycoprotein I antibodies (aβ2GPI) IgG or IgM, which are included in the APS classification criteria. All the assays needed to detect aPL antibodies have methodological concerns. LA testing remains challenging due to its complexity and susceptibility to interference from anticoagulant therapy. Solid phase assays for aCL and aβ2GPI exhibit discrepancies between different assays. Antibody profiles aid in identifying the patients at risk for thrombosis through integrated interpretation of all positive aPL tests. Antibodies targeting domain I of β2-glycoprotein and antiphosphatidylserine-prothrombin antibodies have been evaluated for their role in thrombotic APS but are not yet included in the APS criteria. Detecting these antibodies may help patients with incomplete antibody profiles and stratify the risk of APS patients. The added diagnostic value of other methodologies and measurements of other APS-associated antibodies are inconsistent. This manuscript describes laboratory parameters useful in the diagnosis of thrombotic APS and will concentrate on the laboratory aspects, clinical significance of assays, and interpretation of aPL results in the diagnosis of thrombotic APS., Competing Interests: None declared., (Thieme. All rights reserved.)

This paper focuses on the laboratory tests necessary for the diagnosis of antiphospholipid syndrome (APS). Diagnosis starts with the selection of patients suspicious of having APS. The timing related to the clinical event is important to avoid false classification of APS patients. For the interpretation of the test results of antiphospholipid antibodies (aPL) understanding of all pitfalls and interferences is necessary. Lupus anticoagulant (LA) measurement remains a complex procedure with many pitfalls and interferences. The effect of anticoagulant therapy, the main confounder of LA measurement, can be overcome by removal agents for direct oral anticoagulants (DOAC) or by considering assays as Taipan snake venom time / Ecarin clotting time not affected by antivitamin K therapy and anti-Xa DOAC. However, both procedures have limitations. Solid-phase assays for anticardiolipin antibodies (aCL) and anti-β2-glycoprotein 1 antibodies (aβ2GPI) show inter-assay differences. Diagnosis is based on the measurement of three groups of aPL: LA, aCL and aβ2GPI, of IgG and IgM isotype. This allows to make antibody profiles that helps in identifying patients at risk. Other aPL, such as antibodies against the domain I of β2GPI and anti-phosphatidylserine-prothrombin antibodies may be useful in risk stratification of APS patients and in some specific situations of patients with incomplete antibody profile, but are not needed for diagnosis. Laboratory diagnosis of APS remains challenging. To increase the diagnostic efficacy and reliability, an integrated interpretation of all results and an interpretative comment should be provided on the laboratory report. Therefore, a close interaction between clinical pathologists and clinicians is mandatory.

Background: Ticagrelor, used in acute coronary syndrome (ACS), can be administered via nasogastric tube when oral intake is impossible. We investigated platelet inhibition and pharmacokinetics in resuscitated ACS patients and those undergoing semi-urgent coronary artery bypass graft (CABG) surgery. Our study aimed to assess platelet inhibition with use of the Platelet Function Analyser (PFA) and measured plasma concentrations of ticagrelor and its active metabolite in these ACS patients., Methods: We included resuscitated cardiac arrest patients (STEMI/NSTEMI) and semi-urgent CABG patients. Crushed ticagrelor tablets were administered using a nasogastric tube. PFA closure time (CT) was determined with CT longer than 113 s as reference range. Plasma concentrations of ticagrelor and its active metabolite were measured after protein precipitation, by using liquid chromatography with mass spectrometry detection., Results: In 20 resuscitated patients, 89% showed platelet inhibition at 24 h and 92% at day 4. For semi-urgent CABG patients, 85% exhibited platelet inhibition at 24 h and 84% at day 4. For ticagrelor in resuscitated patients, the median time to peak plasma concentration (Tmax) was 100 h [8; 100] for a median maximal concentration (Cmax) of 615.5 ng/mL [217.5; 1385.0]. For AR-C124910XX median Tmax was 100 h [8; 100] for a Cmax of 131.0 ng/mL [52.1; 177.7]. Among 20 patients undergoing semi-urgent CABG, Tmax for ticagrelor was 100 h [100; 100] for a median Cmax of 857.0 ng/ml [496.8; 1157.5]. For AR-C124910XX, median Tmax was 100 h [43; 100] for a Cmax of 251.0 ng/ml [173.0; 396.5]., Conclusion: Crushed ticagrelor via nasogastric tube achieved targeted platelet inhibition. Pharmacokinetics aligned with previous studies.EudraCT number: 2013-004191-35; Study protocol code: AGO/2013/011; EC/2014/1061; ClinicalTrial.gov identifier: NCT02341729.

Background: Cardiac catheterizations in horses are mainly performed in the right heart, as access to the left heart traditionally requires an arterial approach. Transseptal puncture (TSP) has been adapted for horses but data on follow-up and closure of the iatrogenic atrial septal defect (iASD) are lacking., Hypothesis/objectives: To perform TSP and assess postoperative complications and iASD closure over a minimum of 4 weeks., Animals: Eleven healthy adult horses., Methods: Transseptal puncture was performed under general anesthesia. Serum cardiac troponin I concentrations were measured before and after puncture. Weekly, iASD closure was monitored using transthoracic and intracardiac echocardiography. Relationship between activated clotting time and anti-factor Xa activity during postoperative enoxaparin treatment was assessed in vitro and in vivo., Results: Transseptal puncture was successfully achieved in all horses within a median duration of 22 (range, 10-104) minutes. Balloon dilatation of the puncture site for sheath advancement was needed in 4 horses. Atrial arrhythmias occurred in 9/11 horses, including atrial premature depolarizations (N = 1), atrial tachycardia (N = 5), and fibrillation (N = 3). Serum cardiac troponin I concentrations increased after TSP, but remained under the reference value in 10/11 horses. Median time to iASD closure was 14 (1-35) days. Activated clotting time correlated with anti-factor Xa activity in vitro but not in vivo., Conclusions and Clinical Importance: Transseptal puncture was successfully performed in all horses. The technique was safe and spontaneous iASD closure occurred in all horses. Clinical application of TSP will allow characterization and treatment of left-sided arrhythmias in horses., (© 2024 The Author(s). Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Objective: The 2023 American College of Rheumatology/EULAR antiphospholipid syndrome (APS) classification criteria development, which aimed to identify patients with high likelihood of APS for research, employed a four-phase methodology. Phase I and II resulted in 27 proposed candidate criteria, which are organized into laboratory and clinical domains. Here, we summarize the last stage of phase III efforts, employing a consensus-based multicriteria decision analysis (MCDA) to weigh candidate criteria and identify an APS classification threshold score., Methods: We evaluated 192 unique, international real-world patients referred for "suspected APS" with a wide range of APS manifestations. Using proposed candidate criteria, subcommittee members rank ordered 20 representative patients from highly unlikely to highly likely to have APS. During an in-person meeting, the subcommittee refined definitions and participated in an MCDA exercise to identify relative weights of candidate criteria. Using consensus decisions and pairwise criteria comparisons, 1000Minds software assigned criteria weights, and we rank ordered 192 patients by their additive scores. A consensus-based threshold score for APS classification was set., Results: Premeeting evaluation of 20 representative patients demonstrated variability in APS assessment. MCDA resolved 81 pairwise decisions; relative weights identified domain item hierarchy. After assessing 192 patients by weights and additive scores, the Steering Committee reached consensus that APS classification should require separate clinical and laboratory scores, rather than a single-aggregate score, to ensure high specificity., Conclusion: Using MCDA, candidate criteria preliminary weights were determined. Unlike other disease classification systems using a single-aggregate threshold score, separate clinical and laboratory domain thresholds were incorporated into the new APS classification criteria., (© 2024 American College of Rheumatology.)

Background: Improving harmonization of the clinical interpretation of anticardiolipin (aCL) and anti-β2-glycoprotein I (aβ2GPI) antibodies immunoglobulin G (IgG)/immunoglobulin M (IgM) in the diagnosis of antiphospholipid syndrome (APS) is desirable. Likelihood ratios (LRs) with corresponding test-result intervals can identify the power of a test to discriminate between a diseased and nondiseased patient and may be useful for the semiquantitative interpretation of aCL/aβ2GPI results., Objectives: To determine moderate and high thresholds for aCL and aβ2GPI IgG/IgM measured with chemiluminescent immunoassay, enzyme-linked immunosorbent assay, fluorescence enzyme immunoassay, and multiplex flow immunoassay., Methods: aCL and aβ2GPI antibodies IgG/IgM were determined with 4 solid-phase systems in a case-control study population including 381 APS patients and 727 controls. Interval-specific LRs (IS-LR) were calculated for ranges determined by prespecified specificity and sensitivity levels. Three methods were used for determining thresholds that separated low, moderate, and high positive antibody levels. Interassay agreement was checked with Cohen's kappa statistics., Results: Assay- and antibody-specific thresholds demonstrated increasing IS-LR, reflecting different clinical significance for low, moderate, and high levels, especially for IgG aCL and aβ2GPI and in thrombotic APS. IS-LRs per antibody and unit range were comparable across solid-phase platforms resulting in enhanced harmonization of result interpretation. Agreement between assays for identifying high levels was improved by semiquantitative interpretation compared with that by quantitative reporting., Conclusion: aCL and aβ2GPI IgG/IgM moderate and high thresholds were determined for 4 analytical platforms. Thresholds improve harmonized interpretation of aCL/aβ2GPI levels across platforms. The proposed thresholds should be verified in an independent case-control study to check interlaboratory transferability., Competing Interests: Declaration of competing interests G.W.M. was employed at Guy’s and St. Thomas’ Hospitals at the time of sample collection and reports consultancy fees from Technoclone. The other authors have no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombotic manifestations and/or obstetric complications in patients with persistently positive antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies, but only lupus anticoagulant, anticardiolipin (aCL), and antibeta2-glycoprotein I antibodies (aβ2GPI) IgG or IgM are included as laboratory classification criteria. Seronegative APS patients are usually defined as patients with the clinical symptoms of APS but who test negative for aPL. The negativity to classic aPL criteria does not exclude the presence of other aPL. Several noncriteria aPL have been identified. Some noncriteria aPL are well studied, such as IgA aCL and aβ2GPI, the antiphosphatidylserine-prothrombin (aPS/PT) antibodies, and the antibodies against the domain I of beta2-glycoprotein I (aDI), both latter groups receiving more attention for their role in thrombotic events and pregnancy complications. Other noncriteria aPL that have been studied are antibodies against annexin V, prothrombin, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, vimentin-cardiolipin complex, anti-protein S/protein C. Measurement of some of these noncriteria aPL (aPS/PT, aDI) is useful in the laboratory work-out of APS in specific situations. We have to differentiate between patients who are positive for noncriteria aPL only, and patients who have both criteria and noncriteria aPL to enable us to study their role in the diagnosis or risk stratification of APS. The research on noncriteria aPL is continually developing as the clinical relevance of these antibodies is not yet fully clarified., (© 2024 John Wiley & Sons Ltd.)

Background: No guidelines for administering and monitoring anticoagulants intraprocedurally are currently available in dogs, despite the prevalence of procedures necessitating systemic anticoagulation with heparin., Objectives: To evaluate an activated clotting time (ACT)-based heparin dose-response (HDR) test to predict the individual required heparin dose in dogs during intravascular procedures, and to investigate both the in vitro heparin - ACT and in vitro heparin - factor anti-Xa activity (anti-Xa) relationships in dogs., Methods: Blood was collected from eight healthy beagles undergoing a cardiac procedure and utilised to establish baseline ACT and for in vitro evaluation. Subsequently, 100 IU/kg heparin was administered intravenously (IV) and ACT was remeasured (HDR test). The required heparin dose for an ACT target response ≥300 s was calculated for each individual and ACT was remeasured after administration of this dose. For in vitro testing, a serial heparin blood dilution (0-0.5-1-2-4 international unit (IU)/mL) was prepared and ACT and anti-Xa were determined using whole blood and frozen plasma, respectively., Results: The HDR test overestimated the required heparin dose in 3/7 dogs. In vitro, ACT and anti-Xa increased significantly with increasing blood heparin concentration. Heparin - ACT was nonlinear in 4/8 dogs at heparin concentrations >2 IU/mL, whereas heparin - anti-Xa remained linear throughout the tested range., Conclusions: The HDR test poorly estimated the required heparin dose in dogs. This is most likely attributed to a nonlinear heparin - ACT relationship, as observed in vitro. Anti-Xa is a promising alternative for ACT; however, unavailability as a point-of-care test and lack of in vivo target values restrict its current use., (© 2023 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Abstract: Thrombosis is an important manifestation of the antiphospholipid syndrome (APS). The thrombin generation (TG) test is a global hemostasis assay, and increased TG is associated with thrombosis. APS is currently diagnosed based on clinical and laboratory criteria, the latter defined as anti-cardiolipin, anti-β2-glycoprotein I antibodies, or lupus anticoagulant (LA). APS testing is often performed after a thrombotic episode and subsequent administration of anticoagulation, which might hamper the interpretation of clotting assays used for LA testing. We set out to develop an artificial neural network (NN) that can diagnose APS in patients who underwent vitamin K antagonist (VKA) treatment, based on TG test results. Five NNs were trained to diagnose APS in 48 VKA-treated patients with APS and 64 VKA-treated controls, using TG and thrombin dynamics parameters as inputs. The 2 best-performing NNs were selected (accuracy, 96%; sensitivity, 96%-98%; and specificity, 95%-97%) and further validated in an independent cohort of VKA-anticoagulated patients with APS (n = 33) and controls (n = 62). Independent clinical validation favored 1 of the 2 selected NNs, with a sensitivity of 88% and a specificity of 94% for the diagnosis of APS. In conclusion, the combined use of TG and NN methodology allowed for us to develop an NN that diagnoses APS with an accuracy of 92% in individuals with VKA anticoagulation (n = 95). After further clinical validation, the NN could serve as a screening and diagnostic tool for patients with thrombosis, especially because there is no need to interrupt anticoagulant therapy., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: The optimal strategy for diagnosis and antithrombotic treatment of patients with antiphospholipid syndrome (APS)-associated acute ischemic stroke (AIS), transient ischemic attack (TIA), or other brain ischemic injury is poorly defined., Objectives: The survey goal was to capture variations in diagnosis and antithrombotic treatment of APS-associated ischemic stroke and related disorders to inform guidance and clinical trials to define optimal management., Methods: Professional colleagues, including key opinion leaders, were invited to complete a REDCap survey questionnaire initiated by the International Society on Thrombosis and Haemostasis Scientific and Standardisation Committee Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies. The survey data were tallied using simple descriptive statistics., Results: There was generally good agreement on several aspects, including which patients to test for antiphospholipid antibodies (aPL), use of a lifelong vitamin K antagonist for AIS or recurrent TIA, and formal cognitive assessment for suspected cognitive impairment. There was less agreement on other aspects, including aPL testing for brain ischemic injury other than AIS/TIA or if an alternative cause for AIS or TIA exists; choice of aPL tests, their timing, and age cutoff; the aPL phenotype to trigger antithrombotic treatment; management for patent foramen ovale; antithrombotic treatment for first TIA or white matter hyperintensities; head magnetic resonance imaging specifications; and low-molecular-weight heparin dosing/anti-Xa monitoring in pregnancy. The survey highlighted that approximately 25% practice at dedicated APS clinics and <50% have a multidisciplinary team structure for patients with APS., Conclusion: Much of the variation in practice reflects the lack of evidence-based recommendations. The survey results should inform the development of a more uniform multidisciplinary consensus approach to diagnosis and antithrombotic treatment., Competing Interests: Declaration of competing interests H.C. reports lecture fees from Technoclone, paid to University College London Hospitals Charity. M.C. reports consulting for Bayer for products other than rivaroxaban. M.K. reports being GSK Global Medical Expert (Lupus), Dubai, United Arab Emirates. The other authors and remaining study group members have no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization., Objectives: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results., Methods: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied., Results: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine., Conclusion: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents., Competing Interests: Declaration of competing interests M.L. has received speaker fees from Bayer, participated in industry-funded trials from Idorsia, served on advisory boards for Servier and JAMP/Orimed Pharma, and received in-kind support for investigator-initiated grants from Fujimori Kogyo. M.C.A. has received fees from Novo Nordisk, participated in industry-funded trials from Agrobio, participated in PIA-funded project in collaboration with Stago and Agrobio, and served on advisory boards for Novo Nordisk. U.J.S. has received research grants from Octapharma; consulting fees from Bayer, SOBI, CSL Behring, and Pfizer; and travel support from Bayer, SOBI, CSL Behring, Biotest, Takeda, and Leo Pharma. D.F. has received honorarium and travel support from Viatris. S.V. has received travel support from Stago. F.M. has received speaker fees from Fresenius, Technoclone, and Werfen. P.F. has received travel support from SOBI and Novo Nordisk. A.G. reports personal fees from Aspen, Bayer Vital, Chromatec, Instrumentation Laboratory, Portola, Sanofi-Aventis, Roche, and GTH e.V.; grants from Ergomed, Boehringer Ingelheim, Rovi, Sagent, Biokit, Fa. Blau Farmaceutics, Prosensa/Biomarin, DRK-BSD Baden-Würtemberg/Hessen, Deutsche Forschungsgemeinschaft, Robert-Koch-Institut, Dilaflor, and GIZ Else-Körner-Stiftung; grants and personal fees from Macopharma; grants and other fee from DRK-BSD NSTOB; and nonfinancial support from Veralox, Vakzine Projekt Management GmbH, AstraZeneca, and Janssen Vaccines & Prevention B.V. outside the submitted work. In addition, A.G. has a patent—screening methods for transfusion-related acute lung injury (TRALI)—with royalties paid to EP2321644, 18.05.2011. M.C. has received research grant from Agios Pharmaceuticals, Genentech Inc, and Novartis Inc; consulting fees from Novo Nordisk; and honoraria for board participation with Novo Nordisk, Takeda Inc, Genentech Inc, BPL Inc, CSL Behring Inc, and Genzyme Corp Agios Pharmaceuticals. C.P. has received research grant from Stago. I.D. has received travel support from Sobi, Takeda, and Novonordisk and speaker fees from Novonordisk. A.B. has received fees from Aguettant, Alexion, and Viatris. J.R. has received support from Instituto de Salud Carlos (PI20/00926 [ISCIII&Feder], PMP21/00052 [ISCIII&NG EU], and CB15/00055) and Sociedad Española de Trombosis y Hemostasia (Ayuda a Grupo Español de Alteraciones Plaquetarias Congénitas). P.G. has received speaker’s fees from Sanofi and Roche, and honoraria for board participation with Viatris. M.R.C., M.C., C.L.-B., M.I.K., L.G., B.H., A.S., C.C., A.N.S., M.B., M.F., K.D., W.H., M.F.H., A.N.M., J.P.F., P.S., F.P., A.C., N.B., J.R., and D.L. have no conflict of interest to declare., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: The added value of antiphosphatidylserine/prothrombin antibodies (aPS/PT) in the diagnostic workup of antiphospholipid syndrome (APS) is unclear. Currently, diagnosis of thrombotic APS (TAPS) and obstetric APS (OAPS) requires persistent presence of lupus anticoagulant (LAC), anticardiolipin (aCL) immunoglobulin (Ig) G/IgM, or anti-β2-glycoprotein I (aβ2GPI) IgG/IgM antibodies., Objectives: To evaluate the role of aPS/PT IgG and IgM in OAPS., Methods: aPS/PT IgG/IgM, aCL IgG/IgM, aβ2GPI IgG/IgM, and LAC were determined in 653 patients (OAPS, TAPS, and controls). In-house aPS/PT cut-off values were calculated, titers and prevalence were compared between OAPS, TAPS, and controls and type of pregnancy morbidity. Sensitivity, specificity, likelihood ratios, and odds ratios (OR) with 95% CI were calculated., Results: In OAPS, aPS/PT IgG and IgM showed an OR of 4.32 (95% CI, 2.54-7.36) and 3.37 (95% CI, 1.93-5.89), respectively, but the association was not independent of LAC. Prevalence and titers of aPS/PT IgG and IgM were lower in OAPS than in patients with TAPS. aPS/PT were more prevalent and showed higher titers in patients with late pregnancy loss than in patients with early pregnancy loss with a positivity of 86.4% and 39.3%, respectively. Higher aPS/PT titers did not increase the likelihood of having OAPS., Conclusion: The added value of aPS/PT testing in the current diagnostic workup of OAPS seems limited compared with LAC, aCL, and aβ2GPI. aPS/PT might be useful in specific subsets of patients with OAPS. However, future multicentric studies are needed to elucidate the risk of less frequent and most severe obstetrical manifestations., Competing Interests: Declaration of competing interests G.W.M. was employed at Guy’s & St. Thomas’ Hospitals at the time of sample collection and reports consultancy fees from Technoclone, D.W. reports personal fees, outside the submitted work, from GlaxoSmithKline. The other authors state that there are no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) took place in person in Montréal, Canada, from June 24-28, 2023. The conference, held annually, highlighted cutting-edge advances in basic, translational, population and clinical sciences relevant to the Society. As for all ISTH congresses, we offered a special, congress-specific scientific theme; this year, the special theme was immunothrombosis. Certainly, over the last few years, COVID-19 infection and its related thrombotic and other complications have renewed interest in the concepts of thromboinflammation and immunothrombosis; namely, the relationship between inflammation, infection and clotting. Other main scientific themes of the Congress included Arterial Thromboembolism, Coagulation and Natural Anticoagulants, Diagnostics and Omics, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostatic System in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. Among other sessions, the program included 28 State-of-the-Art (SOA) sessions with a total of 84 talks given by internationally recognized leaders in the field. SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the Congress., (© 2023 Published by Elsevier Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Background: The antiphospholipid syndrome (APS) is classified by the presence of antiphospholipid antibodies (aPL) and thrombotic and/or adverse obstetric outcomes. The diagnosis and risk assessment of APS is challenging. This systematic review investigated if the thrombin generation (TG) assay could be helpful for APS diagnosis and risk assessment., Methods: A systemic review was performed by searching two databases (MEDLINE and Embase) until March 31, 2022, using a search strategy with two concepts: APS and TG, and related keywords. Two reviewers independently screened the articles based on predefined inclusion and exclusion criteria. Data extraction and quality assessment with the Newcastle-Ottawa Scale (NOS) were performed independently. Synthesis Without Meta-analysis guidelines were followed for data synthesis reporting., Results: Fourteen studies with 677 APS and 1,349 control subjects were included with variable quality according to the NOS. Twelve studies measured TG via the calibrated automated thrombogram (CAT) method using a fluorogenic substrate, whereas two used a chromogenic substrate-based TG assay. One study compared the CAT assay to the fully-automated ST Genesia® (Stago, France). Two studies initiated TG using platelet-rich plasma, whereas the rest of the studies used platelet-poor plasma. Resistance to activated protein C (aPC) was examined in ten studies. They reported a significant increase in aPC-resistance in APS patients compared to healthy controls, aPL-carriers, and thrombotic controls. Based on two studies, the prevalence of aPC-resistance was higher in APS patients compared to healthy controls and thrombotic controls with odds ratios of 5.9 and 6.8-12.8, respectively ( p  < 0.05). In contrast, no significant difference in aPC-resistance was found between APS patients and autoimmune disease controls. Furthermore, 7/14 studies reported TG-parameters including peak height, endogenous thrombin potential, lag time, and time to peak, but these outcomes were highly variable between studies. Furthermore, TG methodology between studies differed greatly, impacting the comparability of the studies., Conclusion: aPC-resistance measured with TG was increased in APS patients compared to healthy and thrombotic controls, but the diagnostic and prognostic value is unclear compared to current diagnostic strategies. Studies of other TG-parameters were heterogeneous and more research is needed to identify their potential added value in APS diagnosis., Systematic Review Registration: https://www.PROSPERO/, identifier: CRD42022308363., Competing Interests: BDL and RG are employees of Synapse Research Institute, which is a part of the Stago group that markets the Calibrated Automated Thrombography and ST Genesia®. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Gehlen, Vandevelde, de Laat and Devreese.)

Since the discovery that antiphospholipid antibodies (aPL) bind to a cofactor at the phospholipid membrane, the proteins beta-2-glycoprotein I (β2GPI) and prothrombin seemed to be the antigens of importance in the antiphospholipid syndrome (APS). Anti-β2GPI antibodies (aβ2GPI) were soon included in the classification criteria, while anti-prothrombin antibodies (aPT) are still regarded as "non-criteria" aPL. Evidence is accumulating that antibodies against prothrombin are clinically relevant and closely associated with APS and the presence of lupus anticoagulant (LA). Among the non-criteria aPL, anti-phosphatidylserine/prothrombin antibodies (aPS/PT) are one of the most frequently studied aPL. More and more studies illustrate the evidence of the pathogenic capacity of these antibodies. aPS/PT IgG and IgM are associated with arterial and venous thrombosis, show an overlap with LA presence, and are frequently present in triple-positive patients, regarded as patients at highest risk for APS-related clinical symptoms. Moreover, the association of aPS/PT with thrombosis increases with higher titers, confirming that presence of aPS/PT consolidates the risk. So far, the added value of aPS/PT on top of the criteria aPL to diagnose APS is not clear with opposing findings in literature. Described in this chapter is the procedure for detecting these antibodies with a commercial ELISA, which can be used to determine the presence of IgG and IgM aPS/PT in human samples. Additionally, general guidelines that will facilitate optimal performance of the aPS/PT assay will be provided., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Anti-β2GPI antibodies (aβ2GPI) are one of the laboratory criteria for antiphospholipid syndrome (APS), along with lupus anticoagulant (LA) and anticardiolipin antibodies (aCL). A subset of the aβ2GPI are the antibodies directed toward the domain I of the β2GPI (aDI). The aDI are regarded as non-criteria aPL and are among the most studied non-criteria aPL. Antibodies directed against a specific epitope in the domain I (G40-R43) of β2GPI were shown to be strongly correlated with thrombotic and obstetric events in APS. Many studies illustrated the pathogenic capacity of these antibodies, although with various results, depending on the assay used. The first studies were performed with an in-house ELISA with high specificity for aDI toward the G40-R43 epitope. More recently, a commercial chemiluminescence immunoassay for aDI IgG became obtainable for diagnostic laboratories. Although the added value of aDI on top of the criteria aPL is not clear, with opposing findings in literature, the assay might help in the diagnosis of APS, identifying the patients at risk since aDI are frequently present with high titers in triple-positive patients (positive for LA, aβ2GPI, and aCL). aDI can be used as a confirmatory test and is useful for proving the specificity of the aβ2GPI antibodies. In this chapter, the procedure for detecting these antibodies is outlined, using an automated chemiluminescence assay which can be used to determine the presence of IgG aDI in human samples. General guidelines that will facilitate optimal performance of the aDI assay are also provided., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The diagnosis of antiphospholipid syndrome (APS) relies on the detection of circulating antiphospholipid antibodies (aPL). Currently, lupus anticoagulant (LA), anticardiolipin (aCL), and anti-β2-glycoprotein I antibodies (aβ2GPI) IgG or IgM are the laboratory criteria if persistently present over time. As aCL and aβ2GPI are two out of the three laboratory criteria, the detection of aPL by solid phase assays is an essential step in the diagnosis of APS. Advancement has been made to resolve some of the methodological challenges of aCL and aβ2GPI assays by providing guidelines how to measure aPL, as well as to gain a better understanding of their diagnostic role. However, solid phase assays for aCL and aβ2GPI still show substantive inter-assay differences, resulting in disagreement concerning positive/negative results, but also differences in titer of antibodies. This hampers the semiquantitative classification into low-medium-high positivity. The non-criteria aPL, such as antibodies against the domain one of β2GPI and anti-phosphatidylserine/prothrombin antibodies (aPS/PT) have roles in confirming the risk in APS, and can be useful, especially in patients with incomplete antibody profiles., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Diagnosis of antiphospholipid syndrome (APS) requires persistent presence of lupus anticoagulant (LAC), anticardiolipin (aCL) IgG/IgM, or anti-β2 glycoprotein I (aβ2GPI) IgG/IgM antibodies. Other antiphospholipid antibodies (aPL) such as antiphosphatidylserine/prothrombin antibodies (aPS/PT) are promising in assessment of thrombotic APS (TAPS)., Aim: To evaluate the added value of aPS/PT IgG and IgM in TAPS., Material and Methods: aPS/PT IgG/IgM, aCL IgG/IgM, aβ2GPI IgG/IgM, and LAC were determined in 757 patients (TAPS and controls). aPS/PT cut-off values were calculated, and aPS/PT titers and positivity were compared between TAPS and controls, type of thrombosis, and antibody profiles. Likelihood ratios (LR), odds ratios (OR), and aPL score were determined., Results: aPS/PT IgG and IgM were associated with TAPS and triple positivity. In-house calculated cut-offs were higher for IgM (43 units), compared to manufacturer's cut-off (30 units). Thresholds of 90 (IgG) and 200 (IgM) units were determined as high-titer cut-off. Higher aPS/PT titers were observed in triple positive patients and showed higher LR and OR for TAPS. aPS/PT was independently associated with TAPS when adjusted for aCL/aβ2GPI, but not when adjusted for LAC. In isolated LAC positive patients, aPS/PT was positive in 27.1% TAPS patients and in 77.3% patients with autoimmune disease. Diagnostic value of aPL score did not differ with and without including aPS/PT., Conclusion: aPS/PT positivity, especially with high antibody titer, is associated with TAPS diagnosis. Analysis on top of current laboratory criteria is not essential in TAPS diagnosis, but aPS/PT could be useful in patients with thrombosis and a double positive aPL profile (aCL+/aβ2GPI+)., (© 2022 International Society on Thrombosis and Haemostasis.)

Introduction: Global coagulation assays may be of added value to the anti-Xa assay for monitoring heparin therapy. Unlike most testing methods, the thrombin generation assay (TGA) has the ability to assess the overall function of the hemostatic system, which provides information on the anticoagulation status of patients. We compared the TGA, measured with ST Genesia ® STG-DrugScreen ® reagent, with the anti-Xa assay for monitoring heparin therapy in inflammatory and non-inflammatory patients. We also determined reference values for STG-DrugScreen ® thrombin generation (TG) parameters., Methods: Reference values were determined on 120 healthy donors. Furthermore, a spiking experiment with unfractionated heparin (UFH) and low molecular weight heparin (LMWH) was performed, and samples of patients receiving UFH or LMWH were analyzed with ST Genesia ® and the anti-Xa assay., Results: High discrepancy between TG parameters and anti-Xa activity was observed for low LMWH anti-Xa levels. TG parameters were affected in 36/46 (time to peak) to 42/46 (peak height) patients during UFH therapy with sub-target anti-Xa activity levels., Conclusion: TGA seems insufficiently sensitive for low concentrations of LMWH. There may be an added value of the TGA for monitoring UFH in so-called heparin-resistant patients. Therefore, the TGA has the potential to be introduced as an additional tool for monitoring heparin therapy., (© 2022 John Wiley & Sons Ltd.)

Diagnosis of antiphospholipid syndrome (APS) requires the presence of a clinical criterion (thrombosis and/or pregnancy morbidity), combined with persistently circulating antiphospholipid antibodies (aPL). Currently, laboratory criteria aPL consist of lupus anticoagulant (LAC), anticardiolipin antibodies (aCL) IgG/IgM, and anti-β2 glycoprotein I antibodies (aβ2GPI) IgG/IgM. Diagnosis and risk stratification of APS are complex and efforts to standardize and optimize laboratory tests have been ongoing since the initial description of the syndrome. LAC detection is based on functional coagulation assays, while aCL and aβ2GPI are measured with immunological solid-phase assays. LAC assays are especially prone to interference by anticoagulation therapy, but strategies to circumvent this interference are promising. Alternative techniques such as thrombin generation for LAC detection and to estimate LAC pathogenicity have been suggested, but are not applicable yet in routine setting. For aCL and aβ2GPI, a lot of different assays and detection techniques such as enzyme-linked immunosorbent and chemiluminescent assays are available. Furthermore, a lack of universal calibrators or standards results in high variability between the different solid-phase assays. Other non-criteria aPL such as anti-domain I β2 glycoprotein I and antiphosphatidylserine/prothrombin antibodies have been suggested for risk stratification purposes in APS, while their added value to diagnostic criteria seems limited. In this review, we will describe laboratory assays for diagnostic and risk evaluation in APS, integrating applicable guidelines and classification criteria. Current insights and hindrances are addressed with respect to both laboratory and clinical implications.

Background: DOAC Filter (DF) is a new device to overcome interference in lupus anticoagulant (LAC) testing by direct oral anticoagulants (DOACs)., Objectives: We evaluated DOAC removal from plasma and elimination of DOAC interference in LAC testing by DF, and impact of DF on LAC assays in a representative patient cohort, including a comparison with DOAC-Stop (DS)., Methods: Normal pooled plasma (NPP) was spiked with increasing concentrations of apixaban, rivaroxaban, edoxaban, and dabigatran. DOAC and LAC was measured on untreated, DF-treated, and DS-treated spiked samples. Coagulation parameters and thrombin generation were measured on patient samples (n = 20) before and after DF. Patients treated with DOAC, vitamin K antagonist, or heparin and nonanticoagulated patient samples (n = 139) were tested for LAC before and after DF., Results: In spiked NPP, levels were below the lower limit of quantification (LLoQ) after DF/DS treatment for all DOAC concentrations. Following DF, levels were below LLoQ for 53 of 56 DOAC-containing patient samples. Twenty-eight of 33 LAC-positive DOAC-containing samples became negative after filtration, whereas 5 remained LAC-positive (1/5 from a patient with antiphospholipid syndrome [APS]). Four LAC-positive DOAC-containing samples (from patients without APS), became negative after filtration, whereas they remained LAC positive after DS. In the non-DOAC patient groups following DF, LAC changed from positive to negative in 8 (due to a procoagulant effect) and vice versa in 2 cases., Conclusion: DF reduces DOAC interference in LAC testing. As incomplete DOAC removal may occur, DOAC measurements should be performed after filtration. A procoagulant effect after filtration may lead to erroneous LAC results in non-DOAC-containing samples. Therefore, using DF should be restricted to DOAC-containing samples., (© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Background: Antiβ2glycoprotein I (aβ2GPI) and anticardiolipin (aCL) IgG/IgM show differences in positive/negative agreement and titers between solid phase platforms. Method-specific semiquantitative categorization of titers could improve and harmonize the interpretation across platforms., Aim: To evaluate the traditional 40/80-unit thresholds used for aCL and aβ2GPI for categorization into moderate/high positivity with different analytical systems, and to compare with alternative thresholds., Material and Methods: aCL and aβ2GPI thresholds were calculated for two automated systems (chemiluminescent immunoassay [CLIA] and multiplex flow immunoassay [MFI]) by receiver operating characteristic curve analysis on 1108 patient samples, including patients with and without antiphospholipid syndrome (APS), and confirmed on a second population (n = 279). Alternatively, regression analysis on diluted standard material was applied to identify thresholds. Thresholds were compared to 40/80 threshold measured by an enzyme-linked immunosorbent assay (ELISA). Additionally, likelihood ratios (LR) were calculated., Results: Threshold levels of 40/80 units show poor agreement between ELISA and automated platforms for classification into low/moderate/high positivity, especially for aCL/aβ2GPI IgG. Agreement for semiquantitative interpretation of antiphospholipid antibodies (aPL) IgG between ELISA and CLIA/MFI improves with alternative thresholds. LR for aPL IgG increase for thrombotic and obstetric APS based on 40/80 thresholds for ELISA and adapted thresholds for the other systems, but not for IgM., Conclusion: Use of 40/80 units as medium/high thresholds is acceptable for aCL/aβ2GPI IgG ELISA, but not for CLIA and MFI. Alternative semiquantitative thresholds for non-ELISA platforms can be determined by a clinical approach or by using monoclonal antibodies. Semiquantitative reporting of aPL IgM has less impact on increasing probability for APS., (© 2021 International Society on Thrombosis and Haemostasis.)

Background: Lupus anticoagulant (LA) assays are compromised in anticoagulated patients, and existing strategies to overcome the interferences have limitations. The prothrombin-activating Taipan snake venom time (TSVT) screening test and ecarin time (ET) confirmatory test are innately insensitive to vitamin K antagonists (VKA) and direct factor Xa inhibitors (DFXaI)., Objectives: Validate standardized TSVT/ET reagents for LA detection, in a multicenter, multiplatform study., Patients/methods: Six centers from four countries analyzed samples with TSVT/ET from 81 nonanticoagulated patients with LA, patients with established antiphospholipid syndrome (APS), and proven persistent LA who were either not anticoagulated (n = 120) or were anticoagulated with VKAs (n = 180) or DFXaIs (n = 71). Additionally, 339 nonanticoagulated LA-negative patients, and 575 anticoagulated non-APS patients (172 VKA, 403 DFXaI) were tested. Anticoagulant spiking experiments were performed and 112 samples containing potential interferences (i.e., direct thrombin inhibitors) were tested. Results were evaluated against locally derived cutoffs. Imprecision was evaluated., Results: Cutoffs were remarkably similar despite use of different analyzers and donor populations. Cutoffs for TSVT ratio, ET ratio, percent correction, and normalized TSVT ratio/ET ratio ranged between 1.08 and 1.10, 1.09 and 1.12, 9.3% and 14.8%, and 1.10 and 1.15, respectively. Coefficients of variation for TSVT and ET ratios were ≤5.0%. TSVT/ET exhibited sensitivity, specificity, and negative and positive predictive values of 78.2%/95.0%/86.3%/91.5%, respectively, with established APS as the LA-positive population, and 86.9%/95.0%/76.8%/97.4%, respectively, with triple-positive APS. Interference was seen with direct thrombin inhibitors, unfractionated heparin, and low molecular weight heparins, but not VKAs or DFXaIs., Conclusions: TSVT/ET are validated for LA detection in nonanticoagulated patients and those on VKAs or DFXaIs., (© 2021 International Society on Thrombosis and Haemostasis.)

The diagnosis of antiphospholipid syndrome (APS) relies on the detection of antiphospholipid antibodies (aPL). Currently, lupus anticoagulant (LA), anticardiolipin (aCL), and antibeta2-glycoprotein I antibodies (aβ2GPI) IgG or IgM are included as laboratory criteria, if persistently present. LAC measurement remains a complicated procedure with many pitfalls and interfered by anticoagulant therapy. Solid-phase assays for aCL and aβ2GPI show interassay differences. These methodological issues make the laboratory diagnosis of APS challenging. In the interpretation of aPL results, antibody profiles help in identifying patients at risk. Other aPL, such as antibodies against the domain I of beta2-glycoprotein (aDI) and antiphosphatidylserine-prothrombin (aPS/PT) antibodies have been studied in the last years and may be useful in risk stratification of APS patients. Because of the methodological shortcomings of immunological and clotting assays, these non-criteria aPL may be useful in patients with incomplete antibody profiles to confirm or exclude the increased risk profile. This manuscript will focus on the laboratory aspects, the clinical relevance of assays and interpretation of aPL results in the diagnosis of APS., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Introduction: To perform comparability of instruments, the laboratory can select patient samples spanning the reportable range or can use plasma sets from commercial suppliers. We evaluated ExpertCor Routine (ECR) plasma set (Stago), a set of frozen plasmas enabling to verify the agreement between different coagulation analysers. Additionally, we evaluated whether the concept of transference of the reference range is acceptable between instruments, once comparability between the instruments is approved., Methods: Patient samples and the ECR plasma set were evaluated for method comparison for prothrombin time, activated partial thromboplastin time and fibrinogen on five instruments. Results of one instrument were compared to the mean of all analysers by Passing-Bablok regression and Bland-Altman analysis. Reference ranges were checked on all instruments., Results: The %mean difference was ≤5% and ≤3.7% for all analyser/parameter combinations, for ECR and patient sample data sets, respectively. All predefined criteria to fulfil good comparability between instruments were met. The between-instrument comparison with the ECR plasma set and the patient samples was equal for PT, INR and fibrinogen. After demonstrating comparability between instruments by either of the two plasma sample sets, reference ranges can be used interchangeably between identical instruments., Conclusion: Instrument-to-instrument reproducibility showed comparable results using a data set obtained with patient samples or a commercial plasma set. Once comparability between instruments is confirmed, defined reference ranges can be transferred from one instrument to the other instrument without additional testing. The ECR plasma set is a good alternative to the use of local patient samples to evaluate instrument comparability., (© 2021 John Wiley & Sons Ltd.)

Objective: An international multidisciplinary initiative, jointly supported by the American College of Rheumatology and European Alliance of Associations for Rheumatology, is underway to develop new rigorous classification criteria to identify patients with high likelihood of antiphospholipid syndrome (APS) for research purposes. The present study was undertaken to apply an evidence- and consensus-based approach to identify candidate criteria and develop a hierarchical organization of criteria within domains., Methods: During phase I, the APS classification criteria steering committee used systematic literature reviews and surveys of international APS physician scientists to generate a comprehensive list of items related to APS. In phase II, we reviewed the literature, administered surveys, formed domain subcommittees, and used Delphi exercises and nominal group technique to reduce potential APS candidate criteria. Candidate criteria were hierarchically organized into clinical and laboratory domains., Results: Phase I generated 152 candidate criteria, expanded to 261 items with the addition of subgroups and candidate criteria with potential negative weights. Using iterative item reduction techniques in phase II, we initially reduced these items to 64 potential candidate criteria organized into 10 clinical and laboratory domains. Subsequent item reduction methods resulted in 27 candidate criteria, hierarchically organized into 6 additive domains (laboratory, macrovascular, microvascular, obstetric, cardiac, and hematologic) for APS classification., Conclusion: Using data- and consensus-driven methodology, we identified 27 APS candidate criteria in 6 clinical or laboratory domains. In the next phase, the proposed candidate criteria will be used for real-world case collection and further refined, organized, and weighted to determine an aggregate score and threshold for APS classification., (© 2020, American College of Rheumatology.)

Background: Patients positive for all three types of antiphospholipid antibodies (aPLs; triple positivity) have been identified for having a high risk for thrombotic events. However, the clinical significance of isolated lupus anticoagulant (LAC) positivity is debated., Objectives: To investigate the clinical relevance of isolated LAC., Methods: A total of 456 patients were enrolled in this study; 66 antiphospholipid syndrome patients and 390 control patients. The control group consisted of autoimmune patients ( n  = 91), patients with thrombosis but without aPLs ( n  = 127), and normal controls ( n  = 172). LAC, anticardiolipin (anti-CL), and anti-β2 glycoprotein I (anti-β2GPI) immunoglobulin G (IgG) and immunoglobulin M (IgM) were determined according to the International Society on Thrombosis and Haemostasis (ISTH) guidelines. Anti-CL and anti-β2GPI were measured by four different solid-phase platforms to overcome variability between test systems. The noncriteria IgA anti-CL and anti-β2GPI, antidomain I of β2GPI IgG, and antiphosphatidylserine/prothrombin antibodies (anti-PS/PT) IgG and IgM were detected according to the ISTH guidelines for solid-phase assays., Results: In total, 70 patients were positive for LAC, of which 44 were negative for both anti-β2GPI and anti-CL antibodies. We found that isolated LAC proved to be strongly associated with vascular thrombosis (odds ratio [OR]: 7.3; 95% confidence interval [CI]: 3.3-16.1), even better than triple-positive samples (OR: 4.3; 95% CI: 1.6-12.2). The titers of the anti-PS/PT IgG and IgM were significantly higher in triple-positivity samples compared with samples with isolated LAC positivity. The majority of single LAC positives were anti-PS/PT-negative. We observed that LAC positivity was weaker in isolated LAC-positive patients compared with LAC activity in triple-positive patients., Conclusion: Isolated LAC was highly associated with thrombosis. The presence of anti-PS/PT antibodies could not explain LAC positivity in isolated LAC. Isolated LAC showed a weaker LAC activity compared with triple-positive patients., Competing Interests: None declared., (Thieme. All rights reserved.)

Introduction: Light transmission aggregometry (LTA), used to detect clopidogrel resistance in patients under antiplatelet therapy, is prone to multiple variables potentially influencing results and interpretation. Currently, no attention is given to type of aggregometer or reagent used. The aim of this study was to evaluate the interassay variability between two aggregometers (Chronolog700 and TA-8V), using two different ADP reagents (Chrono-Par ® and Agro-Bio ADP) in patients under clopidogrel therapy. Additionally, LTA results were correlated to CYP2C19-polymorphism., Methods: Light transmission aggregometry was performed in 20 healthy individuals and 30 patients using both aggregometers, and applying two different reagents (2.5 and 5 µmol/L). The maximum platelet aggregation (ADP max ), the platelet aggregation at 6 minutes (ADP 6min ), and the percentage of disaggregation at 6 min (ADP %disaggr ) were compared between four applied combinations. Additionally, 23 clopidogrel-resistant patients according to Chronolog700-Chrono-par ® ADP reagent analysis were tested for CYP2C19*2 polymorphism., Results: Comparison of the LTA of healthy individuals revealed a significant lower ADP max , lower ADP 6min , and higher ADP %disaggr with the TA-8V aggregometer compared to Chronolog700, regardless of the reagent. In contrast, LTA results in patients are depending on the reagent, with significant higher ADP max and ADP 6min and lower ADP %disaggr using Chrono-Par ® compared to Agro-Bio ADP reagent. All intermediate clopidogrel metabolizers (CYP2C19*2 carriers) were correctly classified as clopidogrel resistant using Chrono-Par ® , in contrast to the Agro-Bio ADP reagent., Conclusion: Light transmission aggregometry in clopidogrel-treated patients is mainly depending on the type of ADP reagent. Comparison of LTA with genotype reveals that the choice of instrument seems less influencing. In contrast, in the healthy individuals, differences could be attributed to the instrument., (© 2021 John Wiley & Sons Ltd.)

The alterations in the hemostatic balance in COVID-19 patients are strongly disturbed and contribute to a high prothrombotic status. The high rate of venous thromboembolism in COVID-19 patients goes along with derangements in coagulation laboratory parameters. Hemostasis testing has an important role in diagnosed COVID-19 patients. Elevated D-dimer levels were found to be a crucial laboratory marker in the risk assessment of thrombosis in COVID-19 patients. The diagnostic approach also includes prothrombin time and platelet count. Fibrinogen might give an indication for worsening coagulopathy. Other markers (activated partial thromboplastin time (aPTT), fibrinolysis parameters, coagulation factors, natural anticoagulants, antiphospholipid antibodies and parameters obtained by thromboelastography or thrombin generation assays) have been described as being deranged. These may help to understand the pathophysiology of thrombosis in COVID-19 patients but have currently no place in diagnosis or management in COVID-19 patients. For monitoring the heparin anticoagulant therapy, the anti-Xa assay is suggested, because the severe acute-phase reaction (high fibrinogen and high factor VIII) shortens the aPTT., (© 2021 John Wiley & Sons Ltd.)

The antiphospholipid syndrome (APS) is diagnosed by the presence of lupus anticoagulant and/or antibodies against cardiolipin or β 2 -glycoprotein-1 and the occurrence of thrombosis or pregnancy morbidity. The assessment of overall coagulation is known to differ in APS patients compared to normal subjects. The accelerated production of key factor thrombin causes a prothrombotic state in APS patients, and the reduced efficacy of the activated protein C pathway promotes this effect. Even though significant differences exist in the coagulation profile between normal controls and APS patients, it is not possible to rely on a single test result to diagnose APS. A neural network is a computing system inspired by the human brain that can be trained to distinguish between healthy subjects and patients based on subject specific data. In a first cohort of patients, we developed a neural networking that diagnoses APS. We clinically validated this neural network in a separate cohort consisting of APS patients, normal controls, controls visiting the hospital for other indications and two diseased control groups (thrombosis patients and auto-immune disease patients). The positive predictive value ranged from 62% in the hospital controls to 91% in normal controls and the negative predictive value of the neural network ranged from 86% in the thrombosis control group to 95% in the hospital controls. The sensitivity of the neural network was higher than 90% in all control groups. In conclusion, we developed a neural network that accurately diagnoses APS in the validation cohort. After further clinical validation in newly diagnosed patients, this neural network could possibly be clinically implemented to diagnose APS based on thrombin generation data., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Arterial and venous thrombosis are both common in antiphospholipid syndrome (APS). Recent studies have shown that anti-factor Xa (FXa) therapy in APS patients leads to a greater number of patients with arterial thrombosis than with warfarin. We hypothesize that this may be due to the lowering of prothrombin levels by warfarin., Objectives: To investigate whether antiprothrombin antibodies induce platelet aggregation and to identify the platelet receptors involved. A second aim was to investigate the effect of reduced prothrombin levels on antiprothrombin antibody-induced platelet aggregation., Methods: Enzyme-linked immunosorbent assays were performed to measure binding of antiprothrombin antibodies to prothrombin fragment 1+2 and prothrombin. Platelet aggregation assays in washed platelets were performed. FcγRIIA was immunoprecipitated and tyrosine-phosphorylated FcγRIIA was measured by western blot., Results: The antiprothrombin antibodies 28F4 and 3B1 had lupus anticoagulant (LAC) activity and caused platelet aggregation in the presence of Ca 2+ and prothrombin. Antiprothrombin antibodies without LAC activity did not activate platelets. Inhibition of Syk and Src kinases and FcγRIIA blocked platelet aggregation. Fab and F(ab') 2 fragments of 28F4 were unable to induce platelet aggregation. Immunoprecipitations showed that whole 28F4 immunoglobulin G induced tyrosine phosphorylation of FcγRIIA. Platelet aggregation was significantly reduced when prothrombin levels were reduced from 1 µM to 0.2 µM., Conclusions: Antiprothrombin antibodies with LAC activity are able to activate platelets via FcγRIIA. Decreased prothrombin levels resulted in less antiprothrombin antibody-mediated platelet aggregation. This may explain the lower incidence of arterial thrombosis in patients treated with warfarin than with anti-FXa therapy., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Mild to moderate bleeding disorders are a diagnostic challenge. Many patients remain undiagnosed despite thorough and repeated laboratory testing. Thrombin generation (TG) is an overall assay measuring the functionality of the hemostatic system and may be a useful tool in diagnosing patients with bleeding tendency., Objectives: We examined the added value of TG in patients with mild bleeding tendency with and without diagnosis after classical laboratory testing. Further, we investigated the role of different expressions of results, between-method variation, and reference ranges., Methods: TG of patients and controls was measured in parallel by two TG platforms (ST Genesia and calibrated automated thrombogram [CAT]). All TG parameters in patient and control groups were compared by statistical analysis (Mann-Whitney U tests) including visual representation with box-and-whisker plots. Results were expressed as normalized ratios (ST Genesia and CAT) or corrected values (ST Genesia). Reference intervals were calculated to which patient results were compared. We studied lot-to-lot reagent variability for both platforms., Results: In 62.7% (ST Genesia) to 69.5% (CAT) of patients undiagnosed with a traditional laboratory work-up, abnormal TG parameters (lag time and endogenous thrombin potential expressed as normalized ratio and/or corrected value) were detected. In the group of previously diagnosed patients, abnormal parameters were found in 58.1% of patients for both TG assays. No relevant lot-to-lot reagent variability was observed., Conclusions: Adding TG helps with diagnosing patients with mild bleeding disorder. TG seems a promising tool in diagnosis of bleeding tendency, but further evaluation is necessary before application in diagnostic laboratory testing., (© 2021 International Society on Thrombosis and Haemostasis.)

Thrombin generation (TG) assay is an overall assay to assess the functionality of the hemostatic system and may be a useful tool in diagnosing patients with hyper- and hypocoagulability. Lack of standardization in performing the assays contributes largely to poor correlation between assays and study results. The current lack of standardization remains a major issue in the setting of TG, as illustrated in a recent survey of the ISTH/SSC indicating differences in pre-, analytical, and post-analytical factors among users. These factors may considerably affect the between-laboratory reproducibility of results. Based on the results of the survey and a current review of the literature, along with insights and strong consensus of key investigators in the field, we present guidance for measurement of TG in a clinical setting. Recommendations on blood drawing, handling, processing, and sample storage; reagent concentration and source; analytical conditions on dilution of samples and temperature; calibration and replicate testing; calculation and interpretation of results; and reference values are addressed to help in reducing interlaboratory variation. These recommendations aim at harmonization between methods and laboratories to support the application of TG in patient diagnosis and management., (© 2021 International Society on Thrombosis and Haemostasis.)

Anticoagulation is central to the management of thrombotic antiphospholipid syndrome (APS). The standard anticoagulant treatment for thrombotic APS is life-long warfarin or an alternative vitamin K antagonist. The role of direct oral anticoagulants for thrombotic APS is not established due to the lack of definitive evidence and has recently been addressed in international guidance. Other anticoagulant options include low molecular weight heparin, unfractionated heparin, and fondaparinux. In APS patients, lupus anticoagulant can affect phospholipid-dependent coagulation monitoring tests, so that they may not reflect true anticoagulation intensity. Accurate assessment of anticoagulation intensity is essential, to optimize anticoagulant dosing and facilitate thrombus resolution; minimize the risk of recurrent thrombosis or bleeding; inform assessment of whether recurrent thrombosis is related to breakthrough thrombosis while on therapeutic anticoagulation, subtherapeutic anticoagulation, non-adherence, or spurious results; and guide the management of bleeding. Knowledge of anticoagulant intensity also informs assessment and comparison of anticoagulation regimens in clinical studies. Considerations regarding anticoagulation dosing and/or monitoring of thrombotic APS patients underpin appropriate management in special situations, notably APS-related severe renal impairment, which can occur in APS or APS/systemic lupus erythematosus-related nephropathy or catastrophic APS; and APS-related thrombocytopenia. Anticoagulant dosing and monitoring in thrombotic APS patients also require consideration in anticoagulant-refractory APS and during pregnancy. In this review, we summarize the tests generally used in monitoring anticoagulant therapy, use of the main anticoagulants considered for thrombotic APS, lupus anticoagulant effects on anticoagulation monitoring tests, and strategies for appropriate anticoagulant monitoring in thrombotic APS., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of InternationalSociety on Thrombosis and Haemostasis.)

Background: One objective of the Belgian Rare Diseases plan is to improve patients' management using phenotypic tests and, more specifically, the access to those tests by identifying the biochemical analyses used for rare diseases, developing new financing conditions and establishing reference laboratories., Methods: A feasibility study was performed from May 2015 until August 2016 in order to select the financeable biochemical analyses, and, among them, those that should be performed by reference laboratories. This selection was based on an inventory of analyses used for rare diseases and a survey addressed to the Belgian laboratories of clinical pathology (investigating the annual analytical costs, volumes, turnaround times and the tests unavailable in Belgium and outsourced abroad). A proposal of financeable analyses, financing modalities, reference laboratories' scope and budget estimation was developed and submitted to the Belgian healthcare authorities. After its approval in December 2016, the implementation phase took place from January 2017 until December 2019., Results: In 2019, new reimbursement conditions have been published for 46 analyses and eighteen reference laboratories have been recognized. Collaborations have also been developed with 5 foreign laboratories in order to organize the outsourcing and financing of 9 analyses unavailable in Belgium., Conclusions: In the context of clinical pathology and rare diseases, this initiative enabled to identify unreimbursed analyses and to meet the most crucial financial needs. It also contributed to improve patients' management by establishing Belgian reference laboratories and foreign referral laboratories for highly-specific analyses and a permanent surveillance, quality and financing framework for those tests.

This guidance focuses on methodological aspects of lupus anticoagulant (LA) testing, as well as interpretation of results for clinicians. The main changes in how to test for LA compared with the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee 2009 guidelines, in the preanalytical phase are more detailed recommendations on how to handle testing in anticoagulated patients, and the timing of testing. Also, routine coagulation tests are advised to obtain more information on the coagulation background of the patient, and when necessary, anti-Xa activity measurement for heparins or specific assays for direct oral anticoagulants should be performed. The three-step procedure with two test systems (diluted Russell's viper venom time and activated partial thromboplastin time [aPTT]) is essentially not changed. Silica remains the preferable activator in the aPTT assays, but ellagic acid is not excluded. We advise simultaneous performance of the mixing and confirmatory step, in each sample with a prolonged screening test. The confirmatory step can also be performed on a mixture of patient plasma and normal pooled plasma. Cutoff values should be established in-house on at least 120 normals, with transference of the manufacturer's cutoffs as an alternative. Reporting of results has not been changed, although more attention is focused on what clinicians should know. Patient selection for LA testing has been expanded., (© 2020 International Society on Thrombosis and Haemostasis.)

Background:  Anticardiolipin (aCL) and anti-β2 glycoprotein I (aβ2GPI) immunoglobulin A (IgA) antiphospholipid antibodies (aPL) have shown to associate with thrombosis and pregnancy morbidity. However, inclusion of IgA aPL in the classification criteria of the antiphospholipid syndrome (APS) has been debated. We investigated the value of aCL and aβ2GPI IgA aPL in the detection of thrombosis and pregnancy morbidity in addition to the current aPL panel for APS., Methods:  We included 1,068 patients from eight European medical centers: 259 thrombotic APS patients, 122 obstetric APS patients, 204 non-APS thrombosis patients, 33 non-APS obstetric patients, 60 APS patients with unspecified clinical manifestations, 196 patients with autoimmune diseases, and 194 controls. aCL and aβ2GPI IgG/M/A were detected with four commercial assays and lupus anticoagulant was determined by the local center., Results:  Positivity for IgA aPL was found in 17 to 26% of the patients with clinical manifestations of APS and in 6 to 13% of the control population. Both aCL and aβ2GPI IgA were significantly associated with thrombosis and pregnancy morbidity. Isolated IgA positivity was rare in patients with clinical manifestations of APS (0.3-5%) and not associated with thrombosis and/or pregnancy morbidity. Addition of IgA to the current criterion panel did not increase odds ratios for thrombosis nor pregnancy morbidity., Conclusion:  aCL and aβ2GPI IgA are associated with clinical manifestations of APS. However, isolated IgA positivity was rare and not associated with thrombosis or pregnancy morbidity. These data do not support testing for aCL and aβ2GPI IgA subsequent to conventional aPL assays in identifying patients with thrombosis or pregnancy morbidity., Competing Interests: G. W. Moore reports consultancy fees from Technoclone, outside the submitted work. W. Chayoua, D. Yin, and B. de Laat are employees of Synapse Research Institute, part of Diagnostica Stago SAS. The other authors state that they have no relevant conflict of interest., (Thieme. All rights reserved.)

Introduction: Light transmission aggregometry (LTA) is the gold standard for diagnosing bleeding disorders. Although LTA is laborious, requires large volumes of blood and is relatively insensitive to small changes in platelet function, there is still no competing alternative approach to replace LTA for the diagnosis of platelet bleeding disorders., Materials and Methods: This study investigates the correlation between flow cytometry-based whole blood platelet activation test (WB-PACT) and LTA and whether WB-PACT is of additional value for the identification of bleeding disorders. In total, 161 patients with suspected bleeding diathesis were tested., Results: A correlation of 0.41 between LTA and WB-PACT was found, and there was agreement between tests in 62% of cases (κ = 0.23). The WB-PACT is of additional value to LTA to detect platelet function disorders (PFD) as 10 patients with elevated bleeding score (BS) were detected with WB-PACT, 4 with LTA and 7 patients were positive with both tests. Interestingly, in contrast to LTA, WB-PACT has an additional option to detect VWF disfunctions., Conclusion: WB-PACT may have added value for the routine diagnostic work-up in patients who need to have platelet function tested., Competing Interests: Declaration of competing interest The authors report no declarations of interest. Some of the authors are employees of the research company Synapse BV (member of the Diagnostica Stago group)., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: High incidence of thrombosis in COVID-19 patients indicates a hypercoagulable state. Hence, exploring the involvement of antiphospholipid antibodies (aPL) in these patients is of interest., Objectives: To illustrate the incidence of criteria (lupus anticoagulant [LAC], anticardiolipin [aCL] immunoglobulin G [IgG]/IgM, antibeta2-glycoprotein I antibodies [aβ2GPI] IgG/IgM) and noncriteria (anti-phosphatidyl serine/prothrombin [aPS/PT], aCL, and aβ2GPI IgA) aPL in a consecutive cohort of critically ill SARS-CoV-2 patients, their association with thrombosis, antibody profile and titers of aPL., Patients/methods: Thirty-one consecutive confirmed COVID-19 patients admitted to the intensive care unit were included. aPL were measured at one time point, with part of the aPL-positive patients retested after 1 month., Results: Sixteen patients were single LAC-positive, two triple-positive, one double-positive, one single aCL, and three aCL IgG and LAC positive. Seven of nine thrombotic patients had at least one aPL. Sixteen of 22 patients without thrombosis were aPL positive, amongst them two triple positives. Nine of 10 retested LAC-positive patients were negative on a second occasion, as well as the double-positive patient. Seven patients were aPS/PT-positive associated to LAC. Three patients were aCL and aβ2GPI IgA-positive., Conclusion: Our observations support the frequent single LAC positivity during (acute phase) observed in COVID-19 infection; however, not clearly related to thrombotic complications. Triple aPL positivity and high aCL/aβ2GPI titers are rare. Repeat testing suggests aPL to be mostly transient. Further studies and international registration of aPL should improve understanding the role of aPL in thrombotic COVID-19 patients., (© 2020 International Society on Thrombosis and Haemostasis.)