Scientific Standardization Committee Research Articles

Antiphospholipid Syndrome and Lupus Anticoagulant: Where do we stand today ?

Antiphospholipid Syndrome and Lupus Anticoagulant: Where do we stand today ?

Report of Scientific Standardization Committee 2024

Report of Scientific Standardization Committee 2024

Consensus report on flow cytometry for platelet function testing in thrombocytopenic patients: communication from the SSC of the ISTH

BackgroundPlatelet count alone does not reliably predict bleeding risk, suggesting platelet function is important to monitor in patients with thrombocytopenia. There is still an unmet need for improved platelet function diagnostics in patients with low platelet count in many clinical situations. Flow cytometry is a promising tool allowing reliable platelet function study in this setting. ObjectivesThe goal of this joint project between the International Society on Thrombosis and Haemostasis (ISTH) Scientific Standardization Committee (SSC) Subcommittees on Platelet Physiology and Platelet Immunology is to provide expert consensus guidance on the use of flow cytometry for the evaluation of platelet function, particularly activation, in patients with low platelet counts. MethodsA literature review was performed to identify relevant questions and areas of interest. An electronic expression of interest form was thereafter announced on the ISTH webpage, followed by a survey encompassing 37 issues regarding preanalytical, analytical, postanalytical, and performance aspects. Areas of disagreement or uncertainty were identified and formed the basis for 2 focus group discussions. ResultsConsensus recommendations relative to patient sample collection, preanalytical variables, sample type, platelet-count cutoff, any potential specific modification of the standard flow cytometry protocol, and results expression and reporting are proposed based on the current practices of experts in the field as well as on literature review. ConclusionThe proposed consensus recommendations would allow standardization of protocols in upcoming clinical studies. The clinical utility of platelet function testing using flow cytometry to predict bleeding risk still needs rigorous multicenter outcome studies in patients with thrombocytopenia.

Report of Scientific Standardization Committee 2023

Report of Scientific Standardization Committee 2023

Report of Scientific Standardization Committee 2022

Report of Scientific Standardization Committee 2022

Report of Scientific Standardization Committee 2021

Report of Scientific Standardization Committee 2021

Towards standardization of thrombin generation assays: Inventory of thrombin generation methods based on results of an International Society of Thrombosis and Haemostasis Scientific Standardization Committee survey

BackgroundThrombin generation (TG) is a better determinant of the overall function of the hemostatic system than the routinely used clotting time‐based assays. Nowadays, TG is widely used in hemostasis research teams, for both clinical and basic research. However, there is significant variability between laboratories regarding preanalytics, reagents, TG protocol, analysis interpretation, and normalization. ObjectivesTo document the extent of variation in the methodology of TG, we aim to collect all the methods that are being used to measure TG in a survey. MethodsWe organized a questionnaire through the Standardization committee for Lupus Anticoagulant/Antiphospholipid Antibodies of the International Society of Thrombosis and Haemostasis Scientific Standardization committee. The questionnaire consisted of 51 questions regarding the different aspects of TG: type of users, methods, sample type, analysis, and interpretation of results, normalization, and quality control. ResultsOf the 240 surveys that were started, 108 were completed (45%). However, not all questions were in scope for all laboratories. One‐half of the laboratories were research laboratories and half diagnostic laboratories. The most used TG assay was the calibrated automated thrombinography‐based assay (56%). There was a divergence regarding several aspects of the TG assay: type of needle for blood collection, blood tubes, centrifugation, sample storage and thawing, reagents, sample dilution, calibration, reference ranges, data normalization, and so on. ConclusionsThere is an important variation in the methods used for measuring TG. A standardized protocol and data normalization should lead to a better reproducibility and for comparing data from different laboratories.

Advance in the Management of Sepsis-Induced Coagulopathy and Disseminated Intravascular Coagulation.

Coagulopathy commonly occurs in sepsis as a critical host response to infection that can progress to disseminated intravascular coagulation (DIC) with an increased mortality. Recent studies have further defined factors responsible for the thromboinflammatory response and intravascular thrombosis, including neutrophil extracellular traps, extracellular vesicles, damage-associated molecular patterns, and endothelial glycocalyx shedding. Diagnosing DIC facilitates sepsis management, and is associated with improved outcomes. Although the International Society on Thrombosis and Haemostasis (ISTH) has proposed criteria for diagnosing overt DIC, these criteria are not suitable for early detection. Accordingly, the ISTH DIC Scientific Standardization Committee has proposed a new category termed “sepsis-induced coagulopathy (SIC)” to facilitate earlier diagnosis of DIC and potentially more rapid interventions in these critically ill patients. Therapy of SIC includes both treatment of the underlying infection and correcting the coagulopathy, with most therapeutic approaches focusing on anticoagulant therapy. Recently, a phase III trial of recombinant thrombomodulin was performed in coagulopathic patients. Although the 28-day mortality was improved by 2.6% (absolute difference), it did not reach statistical significance. However, in patients who met entry criteria for SIC at baseline, the mortality difference was approximately 5% without increased risk of bleeding. In this review, we discuss current advances in managing SIC and DIC.

Report of Scientific Standardization Committee 2019

Report of Scientific Standardization Committee 2019

Report of Scientific Standardization Committee 2018

Report of Scientific Standardization Committee 2018

Laboratory Diagnosis of the Lupus Anticoagulant

OBJECTIVE: This review describes consensus guidelines and laboratory methods for diagnosing a lupus anticoagulant (LAC). BACKGROUND: A prolonged APTT due to the presence of a LAC is a frequent finding in the clinical laboratory in patients referred for preadmission testing. Accurate detection of the LAC can often be very challenging for the laboratory. While no single assay is specific for diagnosing a LAC, there is a combination of assays that can be used to improve laboratory diagnosis. METHOD: This review describes several assays used to detect a LAC; reviews consensus guidelines that influence the choice of assays by laboratories; and associated preanalytical variables that may lead to a missed diagnosis. CONCLUSION: A thorough understanding of the principles involved in LAC testing and preanalytical variables may lead to a more accurate diagnosis of the LAC. ABBREVIATIONS:aB2GPI - anti-B2 glycoprotein I, aCL - anti-cardiolipin, APLAs - antiphospholipid antibodies, BCSH - British Committee for Standards in Haematology, CLSI - Clinical and Laboratory Standards Institute, DOAC - direct oral anticoagulant, dRVVT - dilute Russell9s viper venom time, ISTH-SSC - International Society for Thrombosis and Haemostasis – Scientific Standardization Committee, LAC - lupus anticoagulant, VKA - vitamin K antagonist, SCT - silica clotting time.

第11 回日本血栓止血学会学術標準化委員会（Scientific Standardization Committee: SSC）2017 シンポジウムプログラム

第11 回日本血栓止血学会学術標準化委員会（Scientific Standardization Committee: SSC）2017 シンポジウムプログラム

第10 回日本血栓止血学会学術標準化委員会（SSC）2016 シンポジウムプログラム

第10 回日本血栓止血学会学術標準化委員会（SSC）2016 シンポジウムプログラム

第9回日本血栓止血学会学術標準化委員会（SSC）2015シンポジウム

第9回日本血栓止血学会学術標準化委員会（SSC）2015シンポジウム

第8回日本血栓止血学会学術標準化委員会（SSC）2014 シンポジウム

第8回日本血栓止血学会学術標準化委員会（SSC）2014 シンポジウム

Measurement of Lupus Anticoagulants: An Update on Quality in Laboratory Testing

Lupus anticoagulants (LAs) are antiphospholipid antibodies that interfere with in vitro phospholipid-dependent clotting tests, but are associated in vivo with significant clinical manifestations such as recurrent pregnancy loss and venous and arterial thrombosis. Although their detection is important for the diagnosis of thrombotic disorders such as the antiphospholipid syndrome, laboratory identification has historically been fraught with many issues. These have included variability in the sensitivity of assays and reagents; high false-negative and false-positive detection rates; a lack of consensus for the use of mixing tests; and, to some extent, lack of compliance with guidelines published by the Lupus Anticoagulant/Antiphospholipid Antibody Scientific Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH). Since the most recently updated guidelines in 2009, external quality assurance (EQA) programs have conducted surveys to provide a "snapshot" of laboratory practices related to the investigation of LA and to identify problems and monitor improvements in testing for LA. This article will review the impact of the most recently updated ISTH guidelines for LA testing and discuss the findings of recent EQA surveys.

第7回日本血栓止血学会学術標準化委員会（SSC）2013シンポジウム報告

第7回日本血栓止血学会学術標準化委員会（SSC）2013シンポジウム報告

Single vs. swarm detection of microparticles and exosomes by flow cytometry

Microparticles and exosomes are cell-derived vesicles and potential biomarkers for disease. Recently, the Scientific Standardization Committee collaborative workshop of the ISTH initiated standardization of vesicle detection by flow cytometry with polystyrene beads. Because polystyrene beads have different optical properties from biological vesicles, and because the mechanisms causing the detection signal are incompletely understood, there are contradictions between expected and observed results. To develop a model with which to relate the detection signal of a flow cytometer to the diameter of vesicles and clarify observed discrepancies. We combined measurements of polystyrene and silica beads with an estimated refractive index of vesicles and performed Mie calculations of light scattering. We established the relationship between measured light scattering and the diameter of vesicles. The Megamix gating strategy proposed by the Scientific Standardization Committee selects single vesicles and cells with diameters between 800 and 2400 nm when applied on the forward-scattering detector of regular flow cytometers. Nevertheless, we demonstrated that, irrespective of the applied gating, multiple vesicles smaller than 220 nm or multiple 89-nm silica beads were counted as a single event signal at sufficiently high concentrations. Vesicle detection by flow cytometry is attributed to large single vesicles and swarm detection of smaller vesicles; that is, multiple vesicles are simultaneously illuminated by the laser beam and counted as a single event signal. Swarm detection allows the detection of smaller vesicles than previously thought possible, and explains the finding that flow cytometry underestimates the concentration of vesicles.

Circulating Versus Progenitor Endothelial Cells Levels Are Abnormal in Patients with Different Types of Von Willebrand Disease and Correlate with Markers of Angiogenesis

Background:Von Willebrand disease (VWD) is the most common inherited bleeding disorder that is characterized by deficiency or abnormality of von Willebrand factor (VWF). VWF, synthesized by endothelium and magakaryocytes, circulates in plasma and is present in sub-endothelium and platelets: therefore it is an ideal marker for endothelial formation/damage and megakaryocytopoiesis. Circulating (CEC) and progenitor (EPC) endothelial cells have been also proposed as markers of bone marrow-derived angiogenesis.Aims of the study: to evaluate the association of CEC/EPC with cellular and circulating VWF, we have measured the number of CEC/EPC together with VWF and cytokines involved in angiogenesis in a cohort of 84 patients with different VWD types.Methods: Eighty-four VWD patients were diagnosed according to the recommendations of the Scientific Standardization Committee Sub-committee on VWF of the ISTH. The VWD cohort was composed by the following VWD types (number): 1 (27); 2A (10); 2B (23); 2M (17), 3 (7). Twenty healthy individuals were used as controls. CEC (CD146+, CD31+,CD45−) and EPC (CD34+, CD133+, CD45−) were evaluated by cytometry. Circulating levels of VEGF, E-selectin and EPO were determined by ELISA. Both CEC/EPC and cytokines were tested in blind. i.e. without knowing in advance the type of VWD diagnosis.Results: Median (range)[* = p <0.05] of CEC/EPC measured in controls and in different VWD types are shown below together with those of VEGF, E-selectin and EPO (Table).Patients (n)CECs (cells ×106/l)EPCs (cells ×106/l)VEGF (pg/ml)E-Selectin (ng/ml)EPO (mIU/ml)Controls (20)13.4 (4.3–33.0)0.24 (0.04–0.60)224 (92–392)28 (14.6–34.2)7.1 (3,0–16,6)All VWD (84)20.0 (2.0–133.2)*0.09 (0.00–1.14)*334 (88–1286)*34.1 (13.0–125.5)*10.5 (1.7–53.0)*VWD1 (27)19.2 (4.2–133.2)0.08 (0.00–1.14)*296 (121–1103)*28.7 (13.0–74.3)11.9 (1.7–26.6)*VWD2A/2M (27)16.5 (2.0–58.9)*0.08 (0.00–0.53)*371 (88–1286)*41.9 (19.6–117.3)*10.2 (6.3–44.7)*VWD2B (23)30.1 (9.0–108.8)*0.13 (0.00–0.96)*330 (142–675)*33.8 (13.8–103.9)*12.9 (2.9–53.0)*VWD3 (7)41.3 (5.2–78.9)*0.13 (0.00–0.37)475 (131–676)33.8 (22.6–125.5)*10.5 (7.2–19.0)The global VWD population had higher CEC (p=.002) and lower EPC (p=.001) than controls. These data were associated with high VEGF (p=.003), E-selectin (p=.001) and EPO (p=.001) levels. As far as VWD types, VWD1 patients showed slightly high CEC (p=.06), normal E-selectin, markedly low EPC (p=.002) and high VEGF (p=.02). VWD2A/2M, grouped together, showed higher CEC (p=.05) and low EPC (p=.009) together with high VEGF (p=.005), EPO (p=.004) and markedly higher E-selectin (p<.0001). Conversely, VWD2B, characterized by enhanced interactions with platelet glycoproteins Ib (GpIb), and VWD3, by virtually complete deficiency of VWF, showed high CEC (p=.0002 and .01) and relatively reduced EPC (p=.03 and .05) with elevated VEGF (p=.01 and .13), E-selectin (p=.002 and .02) and EPO (p=.005 and 0.1).Discussion and conclusions: Based on these results, we can conclude that CEC are increased in VWD patients, especially in VWD2B and 3: high CEC are associated with increased levels of cytokines involved in angiogenesis (up-regulation). Conversely, EPC are always decreased in VWD patients, especially in VWD1 and VWD2A/2M, suggesting down-regulation of bone marrow-derived angiogenesis. These preliminary data might be important to better understand the role of VWF in the pathogenesis of different VWD types as well as in bone marrow-derived angiogenesis