How to investigate mild to moderate bleeding disorders and bleeding disorder of unknown cause.

A bleeding tendency is one of the most common complaints observed by hematologists. It is challenging to differentiate a clinically insignificant bleeding from a bleeding phenotype that requires hemostatic evaluation and medical intervention. A thorough review of personal and familial history, objective assessment of bleeding severity using a bleeding assessment tool, and a focused physical examination are critical to correctly identifying suspected patients with mild to moderate bleeding disorders (MBDs). A basic laboratory work-up should be performed in all patients referred for a bleeding tendency. If a hemostatic abnormality is found such as evidence of von Willebrand disease, a platelet function disorder, or a coagulation factor deficiency, more extensive testing should be performed to further characterize the bleeding disorder. Conversely, if all results are normal the patient is considered to have bleeding disorder of unknown cause (BDUC). For patients with BDUC, further evaluation may include non-routine testing to look for rare bleeding disorders not detected by routine hemostasis tests, such as thrombomodulin-associated coagulopathy, tissue factor pathway inhibitor-related bleeding disorder, hyperfibrinolytic-bleeding disorders or impaired tissue factor production. In this review, we summarize the stepwise diagnostic procedure in MBDs and provide some insights into the biological features of BDUC.

Standardization of definition and management for bleeding disorder of unknown cause: communication from the SSC of the ISTH.

In many patients referred with significant bleeding phenotype, laboratory testing fails to define any hemostatic abnormalities. Clinical practice with respect to diagnosis and management of this patient cohort poses significant clinical challenges. We recommend that bleeding history in these patients should be objectively assessed using the International Society on Thrombosis and Haemostasis (ISTH) bleeding assessment tool. Patients with increased bleeding assessment tool scores should progress to hemostasis laboratory testing. To diagnose bleeding disorder of unknown cause (BDUC), normal complete blood count, prothrombin time, activated partial thromboplastin time, thrombin time, von Willebrand factor antigen, von Willebrand factor function, coagulation factors VIII, IX, and XI, and platelet light transmission aggregometry should be the minimum laboratory assessment. In some laboratories, additional specialized hemostasis testing may be performed to identify other rare causes of bleeding. We recommend that patients with a significant bleeding phenotype but normal laboratory investigations should be registered with a diagnosis of BDUC in preference to other terminology. Global hemostatic tests and markers of fibrinolysis demonstrate variable abnormalities, and their clinical significance remains uncertain. Targeted genomic sequencing examining candidate hemostatic genes has a low diagnostic yield. Underlying BDUC should be considered in patients with heavy menstrual bleeding since delays in diagnosis often extend to many years and negatively impact quality of life. Treatment options for BDUC patients include tranexamic acid, desmopressin, and platelet transfusions.

Activated protein C and free protein S in patients with mild to moderate bleeding disorders.

BACKGROUND: Underlying mechanisms for bleeding and impaired thrombin generation (TG) and plasma clot formation (PCF) in patients with mild to moderate bleeding disorders (MBDs) are still to be elucidated, especially in bleeding disorder of unknown cause (BDUC). The role of the natural anticoagulants activated protein C (APC) and free protein S (PS) has not yet been investigated in this patient population. AIMS: To analyze antigen levels of APC and PS in patients with MBDs and BDUC and investigate associations to clinical bleeding phenotype and severity as well as and hemostatic capacity. METHODS: Antigen levels of APC and free PS were measured in 262 patients from the Vienna Bleeding Biobank (VIBB), a single-center cohort study, by ELISA and compared to 61 healthy controls (HC). RESULTS: Antigen levels of APC were higher in MBD patients than in HC when adjusted for age, sex and BMI (median (IQR) 33.1 (20.6-52.6) and 28.6 (16.4-47.2) ng/mL). This was most pronounced in patients with BDUC (35.3 (21.7-54.3) ng/mL). No differences in PS antigen levels between patients and HC were seen overall, or according to specific diagnoses. Further, no association between APC or PS and bleeding severity or global tests of hemostasis or TG were identified, while paradoxically APC weakly correlated with shorter lag time and time to peak of PCF in BDUC. CONCLUSION: Our data demonstrate increased antigen levels of APC in BDUC, which might contribute to the bleeding tendency in some patients and could be a future therapeutic target in BDUC.

Bleeding Disorder of Unknown Cause: A Diagnosis of Exclusion.

Patients with an unexplained mild to moderate bleeding tendency are diagnosed with bleeding disorder of unknown cause (BDUC), a classification reached after ruling out other mild to moderate bleeding disorders (MBD) including von Willebrand disease (VWD), platelet function defects (PFDs), coagulation factor deficiencies (CFDs), and non-hemostatic causes for bleeding. This review outlines our diagnostic approach to BDUC, a diagnosis of exclusion, drawing on current guidelines and insights from the Vienna Bleeding Biobank (VIBB). According to guidelines, we diagnose VWD based on VWF antigen and/or activity levels ≤50 IU/dL, with repeated VWF testing if VWF levels are <80 IU/dL. This has been introduced in our clinical routine after our findings of diagnostically relevant fluctuations of VWF levels in a high proportion of MBD patients. PFDs are identified through repeated abnormalities in light transmission aggregometry (LTA), flow cytometric mepacrine fluorescence, and glycoprotein expression analysis. Nevertheless, we experience diagnostic challenges with regard to reproducibility and unspecific alterations of LTA. For factor (F) VIII and FIX deficiency, a cutoff of 50% is utilized to ensure detection of mild hemophilia A or B. We apply established cutoffs for other rare CFD being aware that these do not clearly reflect the causal role of the bleeding tendency. Investigations into very rare bleeding disorders due to hyperfibrinolysis or increase in natural anticoagulants are limited to cases with a notable family history or distinct bleeding phenotypes considering cost-effectiveness. While the pathogenesis of BDUC remains unknown, further explorations of this intriguing area may reveal new mechanisms and therapeutic targets.

The necessity of repeat testing for von Willebrand disease in adult patients with mild to moderate bleeding disorders.

BACKGROUND: In patients with mild-to-moderate bleeding disorders (MBD), von Willebrand disease (VWD) is diagnosed at von Willebrand factor (VWF) levels ≤50 IU/dL. Although VWF levels are unstable, repeated testing for VWD diagnosis is not necessarily advised in recent guidelines. OBJECTIVES: To analyze the relevance of repeated VWF testing to diagnose VWD in patients with MBD. METHODS: Data of 277 patients with MBD from the Vienna Bleeding Biobank with at least 2 separate assessments of VWF antigen (VWF:Ag) and activity (VWF:Act) were analyzed. RESULTS: In repeated VWF measurements, 36 patients (13.0%) had "changing" VWF levels (≤/>50 IU/dL), 27 (9.7%) had persistent levels ≤50 IU/dL ("pathologic"), and 214 (77.3%) had levels >50 IU/dL ("normal"). Of the 36 changing patients, 22 (61%) were diagnosed with VWD at baseline, whereas the others only met VWD diagnostic criteria at repeated measurements. Using logistic regression, we estimated a probability of change of 26.4% (95% CI, 12.5-47.4) at baseline VWF levels of 30 IU/dL, 50.8% (95% CI, 35.6-65.8) at 50 IU/dL, 18.8% (95% CI, 12.3-27.6) at 60 IU/dL, and 1.2% (95% CI, 0.3-4.9) at 80 IU/dL. Baseline VWF was a strong predictor for changing status (Χ2 = 49.9; P < .001), while age, sex, Vicenza score, and blood type O had limited added value (Χ2 = 5.1; P = .278). Baseline VWF:Ag or VWF:Act cutoffs of 80 IU/dL had negative predictive values of 98.1% and 99.1% for changing status, respectively. CONCLUSION: Our data emphasize an overlap between patients with VWD and MBD with bleeding disorder of unknown cause and underline the need for repeated VWF testing, especially in patients with VWF levels <80 IU/dL.

Health-related quality of life is impaired in bleeding disorders of unknown cause: results from the Vienna Bleeding Biobank.

Background: Bleeding disorder of unknown cause (BDUC) is a diagnosis of exclusion after extensive investigation of coagulation and platelet function and is commonly seen among patients with mild-to-moderate bleeding disorders. Despite increasing awareness among treating physicians, little is known about the health-related quality of life (HrQoL) in BDUC. Objectives: To investigate HrQoL in patients with BDUC in comparison to the general population and patients diagnosed with other established bleeding disorders. Methods: Patients with mild-to-moderate bleeding disorders from the Vienna Bleeding Biobank, a prospective cohort study, were contacted via mail and phone to complete the 36-Item Health Survey Questionnaire form. Results: In total, 333/657 (50.7%) patients completed the 36-Item Health Survey Questionnaire. Patients with BDUC (n = 207, 62%) had significantly impaired HrQoL both in physical (47.8 vs 49.2) and mental health parameters (42.9 vs 51.0) compared to the general population (n = 2914, 56% females), which remained after adjustment for sex and age in multivariable linear regression. The impairment in HrQoL, compared to patients with von Willebrand disease, platelet function defects, or mild clotting factor deficiencies, did not prevail after adjustment for age and sex. In patients with BDUC, age and the presence of at least 1 comorbidity were associated with impaired physical health but not sex or bleeding severity. Of all analyzed bleeding symptoms, only joint bleeding was associated with impaired physical health and gastrointestinal bleeding with mental health in BDUC. Conclusion: The impairments in HrQoL in patients with BDUC emphasize the burden of BDUC on mental and physical well-being, encouraging early recognition and better counseling of patients with BDUC.

Hemostatic biomarkers associated with postpartum hemorrhage: a systematic review and meta-analysis.

Postpartum hemorrhage (PPH) is a leading cause of maternal morbi-mortality. Although obstetric risk factors are well described, the impact of predelivery hematologic and hemostatic biomarkers remains incompletely understood. In this systematic review, we aimed to summarize the available literature on the association between predelivery hemostatic biomarkers and PPH/severe PPH. Searching MEDLINE, EMBASE, and CENTRAL databases from inception to October 2022, we included observational studies on unselected pregnant women without bleeding disorder reporting on PPH and on predelivery hemostatic biomarkers. Two review authors independently performed title, abstract and full-text screening, upon which quantitative syntheses of studies reporting on the same hemostatic biomarker were conducted, calculating the mean difference (MD) between women with PPH/severe PPH and controls. A search on 18 October 2022 yielded 81 articles fitting our inclusion criteria. The heterogeneity between studies was considerable. With regard to PPH, the estimated average MD in the investigated biomarkers (platelets, fibrinogen, hemoglobin, Ddimer, activated partial thromboplastin time, and prothrombin time) were not statistically significant. Women who developed severe PPH had lower predelivery platelets than controls (MD = -26.0 109/L; 95% confidence interval, -35.8 to -16.1), whereas differences in predelivery fibrinogen concentration (MD = -0.31 g/L; 95% confidence interval, -0.75 to 0.13) and levels of factor XIII or hemoglobin were not statistically significant in women with and without severe PPH. Predelivery platelet counts were, on average, lower in women with severe PPH compared with controls, suggesting the potential usefulness of this biomarker for predicting severe PPH. This trial was registered at the International Prospective Register of Systematic Reviews as CRD42022368075.

Bleeding phenotype in nonsevere hemophilia by International Society on Thrombosis and Haemostasis bleeding assessment tool, bleeding frequency, and the joint status.

Background: Although the phenotype of severe hemophilia has been well studied, there are still knowledge gaps in nonsevere hemophilia. Objectives: The objective of this study was to characterize the clinical bleeding phenotype in nonsevere hemophilia and its association with different factor VIII/IX assessments. Methods: This was a cross-sectional, multicenter study to investigate the bleeding phenotype in adults with nonsevere hemophilia by the number of bleeding and joint bleeding in the past 5 years, a joint score, and the International Society on Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT). Factor levels were analyzed by 1-stage (lowest in history and at study inclusion) and chromogenic assay (at study inclusion). Patients were enrolled between March 2015 and May 2019. Results: Of the 111 patients (86 with mild and 25 with moderate hemophilia), 57 patients (54.8%) reported any bleeding and 24 (23.1%) any joint bleeding in the past 5 years. A joint score ≥1 was found in 44 patients (41.9%), an ISTH-BAT ≥4 in 100 patients (90.1%), and an ISTH-BAT joint item ≥1 in 50 patients (45.0%). Within the ISTH-BAT, muscle and joint bleeds showed the largest difference between mild and moderate hemophilia. The lowest factor VIII/IX level in patients' history was best associated with bleeding outcomes. Factor was inversely associated with joint bleeds (incidence rate ratio 0.88; 95% CI, 0.79-0.98), joint score, and ISTH-BAT (odds ratios from proportional odds ordinal logistic regression 0.92; 95% CI, 0.87-0.97; and 0.89; 95% CI, 0.86-0.93, respectively). Conclusion: The occurrence of joint bleeding differentiated persons with mild and moderate hemophilia. The ISTH-BAT and lowest factor in patients' history provided valuable information of the bleeding phenotype in nonsevere hemophilia.

Risk factors for future bleeding in patients with mild bleeding disorders: longitudinal data from the Vienna Bleeding Biobank.

BACKGROUND: The future bleeding risk, especially after hemostatic challenges, and thus the requirement for hemostatic treatment in patients with mild-to-moderate bleeding disorders (MBDs) is largely unknown. OBJECTIVES: This study aimed to prospectively examine the recurrence of bleeding symptoms and clinical risk factors for future bleeding in patients with MBD, including patients with bleeding disorder of unknown cause (BDUC). METHODS: Bleeding symptoms of patients with MBD included in the Vienna Bleeding Biobank were re-evaluated at in-person follow-up visits or by mail. RESULTS: In total, 392 patients, including 62.8% with BDUC, were investigated for the recurrence of bleeding events. During the follow-up time of median (IQR) 4.3 years (2.6-6.7), 72% of patients had at least 1 bleeding event. Most persistent bleeding manifestations were hematomas (n = 146/245, 59.6%) and bleeding from small wounds (n = 69/141, 48.9%), followed by epistaxis (n = 42/132, 31.8%), oral mucosal bleeding (n = 26/87, 29.9%), and joint bleeding (n = 7/14, 50.0%). Patients with previous postinterventional bleeding had a significantly increased risk for bleeding events after surgery (n = 33/114, 29.0 %) or tooth extraction (n = 16/39, 41.0%). A high bleeding score (OR [95% CI], 1.14 [1.05 to 2.94], per 1 unit) and follow-up time (OR [95% CI], 1.23 [1.12 to 1.36], per 1 year) were independently associated with any bleeding event. For bleeding after hemostatic challenges, blood group O (OR, 3.17 [1.57 to 6.40]), previous postsurgical bleeding (OR, 2.40 [1.06 to 5.46]), and an established diagnosis (OR, 2.07 [1.04 to 4.10]) were independent risk factors. CONCLUSION: Patients with MBD have a high risk for recurrent bleeding. This encourages prophylactic hemostatic treatment in patients with MBD, particularly when they face hemostatic challenges.

Platelet function and soluble P-selectin in patients with primary immune thrombocytopenia.

BACKGROUND: The bleeding phenotype in immune thrombocytopenia (ITP) is heterogeneous, but usually mild and only partly dependent on the severity of thrombocytopenia. Platelet reactivity has previously been suggested to underly the mild phenotype. METHODS: Platelet function was assessed as basal and agonist-induced surface expression of P-selectin and activation of GPIIb/IIIa via flow cytometry, and soluble (s)P-selectin levels were assessed in plasma of 77 patients with primary ITP, 19 hemato-oncologic thrombocytopenic controls (TC) and 20 healthy controls (HC). The association of platelet function with laboratory and clinical parameters such as bleeding manifestations at inclusion and previous thrombosis was analyzed. RESULTS: ITP patients showed tendency towards increased surface P-selectin and elevated levels of activated GPIIb/IIIa. Platelet activation after stimulation with all agonists including TRAP-6, ADP, arachidonic acid and CRP was decreased compared to HC. Compared to TC, only GPIIb/IIIa activation but not surface P-selectin was higher in ITP. Levels of soluble (s)P-selectin were significantly higher in ITP patients compared to TC, but similar to HC. Higher sP-selectin levels were associated with blood group O and current therapy, with highest levels in TPO-RA treated patients. Platelet reactivity was not associated with platelet count or size, platelet antibodies, treatment regime, or blood group. No correlation between platelet activation with the bleeding phenotype or previous thrombotic events could be observed. CONCLUSION: ITP patients did not have hyper-reactive platelets compared to HC, but partly higher reactivity compared to TC. Further studies are needed to understand the underlying mechanism behind the bleeding and pro-thrombotic phenotype in ITP. 250/250.

Investigating patients for bleeding disorders when most of the "usual" ones have been ruled out.

A State of the Art lecture titled "Investigating Patients for Bleeding Disorders When Most of the Usual Ones Have Been Ruled Out" was presented at the International Society on Thrombosis and Haemostasis Congress in 2023. Mild to moderate bleeding disorders (MBDs) in patients in whom no diagnosis of an established disorder, such as platelet function defect, von Willebrand disease, or a coagulation factor deficiency, can be identified are classified as bleeding disorders of unknown cause (BDUCs). Prospective data from the Vienna Bleeding Biobank and other studies have revealed a high proportion of BDUCs of up to 70% among patients with MBD who have a similar bleeding phenotype as other MBDs. As BDUC is a diagnosis of exclusion, the accuracy of the diagnostic workup is essential. For example, repeated testing for von Willebrand disease should be considered if von Willebrand factor values are <80 IU/dL. Current evidence does not support the clinical use of global assays such as thromboelastography, platelet function analyzer, or thrombin generation potential. Rare and novel bleeding disorders due to genetic variants in fibrinolytic factors or natural anticoagulants are rare and should only be analyzed in patients with specific phenotypes and a clear family history. In BDUC, blood group O was identified as a risk factor for increased bleeding severity and bleeding risk after hemostatic challenges. Future studies should improve the phenotypical characterization and ideally identify novel risk factors in BDUC, as a multifactorial pathogenesis is suspected. Finally, we summarize relevant new data on this topic presented during the 2023 International Society on Thrombosis and Haemostasis Congress.

Lupus anticoagulant test persistence over time and its associations with future thrombotic events.

Data on lupus anticoagulant (LA) test stability in patients persistently positive for LA are limited, and its implications on clinical outcomes are lacking. We investigated the rate and predictors of a negative LA test and whether experiencing a negative test affected a patient's risk of future thrombotic events or death in a prospective observational study of persistently LA+ patients. We followed 164 patients (84% women) for a median of 9.2 years and a total of 1438 follow-up visits. During the observation period, 50 thrombotic events (23 arterial and 27 venous events) occurred, and 24 patients died. Forty-six of the patients had at least 1 negative LA test during the observation period, corresponding to a 10-year cumulative incidence of a negative LA test of 28% (95% confidence interval, 20-35). The majority of patients with available follow-up after a negative LA test (n = 41) had at least 1 subsequent positive test for LA (n = 28/41, 68%). Vitamin K antagonist (VKA) treatment at baseline was associated with a negative LA test during follow-up. Using a multistate time-to-event model with multivariable adjustment, a negative LA test had no impact on a patient's prospective risk of thrombosis or mortality. We conclude that a negative LA test during observation cannot be used clinically to stratify a patient's risk for future events.

Thrombomodulin in patients with mild to moderate bleeding tendency.

INTRODUCTION: A massive increase of soluble thrombomodulin (sTM) due to variants in the thrombomodulin gene (THBD) has recently been identified as a novel bleeding disorder. AIM: To investigate sTM levels and underlying genetic variants as a cause for haemostatic impairment and bleeding in a large number of patients with a mild to moderate bleeding disorder (MBD), including patients with bleeding of unknown cause (BUC). PATIENTS AND METHODS: In 507 MBD patients, sTM levels, thrombin generation and plasma clot formation were measured and compared to 90 age- and sex-matched healthy controls. In patients, genetic analysis of the THBD gene was performed. RESULTS: No difference in sTM levels between patients and controls was found overall (median ([IQR] 5.0 [3.8-6.3] vs. 5.1 [3.7-6.4] ng/ml, p = .762), and according to specific diagnoses of MBD or BUC, and high sTM levels (≥95th percentile of healthy controls) were not overrepresented in patients. Soluble TM levels had no impact on bleeding severity or global tests of haemostasis, including thrombin generation or plasma clot formation. In the THBD gene, no known pathogenic or novel disease-causing variants affecting sTM plasma levels were identified in our patient cohort. CONCLUSION: TM-associated coagulopathy appears to be rare, as it was not identified in our large cohort of patients with MBD. Soluble TM did not arise as a risk factor for bleeding or altered haemostasis in these patients.

Natural anticoagulants: A missing link in mild to moderate bleeding tendencies.

INTRODUCTION: There is a growing interest in natural anticoagulants as a cause of mild to moderate bleeding disorders (MBDs), particularly in patients with bleeding of unknown cause (BUC), which is defined as having a mild to moderate bleeding phenotype without a definite diagnosis despite exhaustive and repeated laboratory investigations. Recently, abnormalities in two natural anticoagulant pathways, thrombomodulin (TM), and tissue factor pathway inhibitor (TFPI), were identified in single patients or families as the underlying cause for a bleeding tendency. AIM: The objective of this review is to discuss the current understanding of the role of natural anticoagulants in MBDs using available clinical and translational data. METHODS: A Cochrane Library and PubMed (MEDLINE) search focusing on selected natural anticoagulants and their role in MBDs was conducted. RESULTS: Data on the influence of natural anticoagulants including protein C, protein S, antithrombin, TM, and TFPI or factors with anticoagulant properties like fibrinogen gamma prime (γ') on MBDs are scarce. Observations from sepsis treatment and from translational research highlight their importance as regulators of the haemostatic balance, especially via the activated protein C-related pathway, and suggest a role in some MBDs. CONCLUSION: Similar to the distinct genetic variants of natural anticoagulants linked to thrombosis, we hypothesize that novel variants may be associated with a bleeding tendency and could be identified using next generation sequencing.

Fibrinolysis and bleeding of unknown cause.

Patients with bleeding of unknown cause (BUC) present with a variety of mild to moderate bleeding symptoms, but no hemostatic abnormalities can be found. Hyperfibrinolysis is rarely evaluated as the underlying cause for bleeding in clinical practice, and well-established global assays for abnormal fibrinolysis are lacking. Few patients with definitive fibrinolytic disorders, including α2-antiplasmin deficiency, plasminogen activator inhibitor 1 deficiency, or Quebec platelet disorder, have been reported. This review aims to summarize data on established fibrinolytic disorders and to discuss assessments of fibrinolysis in prior bleeding cohorts. Furthermore, we review available global tests with the potential to measure fibrinolysis, such as turbidity fibrin clot assays and rotational thromboelastometry, and their relevance in the workup of patients with BUC. We conclude that, due to the lack of adequate global tests, hyperfibrinolysis might be an underdiagnosed cause for a bleeding disorder. The diagnosis of hyperfibrinolytic bleeding disorders would improve patient care as effective treatment with antifibrinolytic agents is available.

Elevated levels of tissue factor pathway inhibitor in patients with mild to moderate bleeding tendency.

High levels of tissue factor pathway inhibitor (TFPI), caused by a longer TFPIα half-life after binding to a factor V splice variant and variants in the F5 gene, were recently identified in 2 families with an as-yet-unexplained bleeding tendency. This study aimed to investigate free TFPIα in a well-characterized cohort of 620 patients with mild to moderate bleeding tendencies and its association to genetic alterations in the F5 gene. TFPIα levels were higher in patients with bleeding compared with healthy controls (median [interquartile range], 8.2 [5.5-11.7] vs 7.8 [4.3-11.1]; P = .026). A higher proportion of patients had free TFPIα levels more than or equal to the 95th percentile compared with healthy controls (odds ratio [OR] [95% confidence interval (CI)], 2.82 [0.98-8.13]). This was pronounced in the subgroup of patients in whom no bleeding disorder could be identified (bleeding of unknown cause [BUC; n = 420]; OR [95% CI], 3.03 [1.02-8.98]) and in platelet function defects (PFDs) (n = 121; OR [95% CI], 3.47 [1.09-11.08]). An increase in free TFPIα was associated with a mild delay in thrombin generation (prolonged lag time and time to peak), but not with alterations in routinely used global clotting tests. We could neither identify new or known genetic variations in the F5 gene that are associated with free TFPIα levels, nor an influence of the single-nucleotide variant rs10800453 on free TFPIα levels in our patient cohort. An imbalance of natural coagulation inhibitors such as TFPIα could be an underlying cause or contributor for unexplained bleeding, which is most probably multifactorial in a majority of patients.

Neutrophil subpopulations and their activation potential in patients with antiphospholipid syndrome and healthy individuals.

OBJECTIVES: Patients with APS are at increased risk of thromboembolism. Neutrophils have been shown to play a role in inducing thrombosis. We aimed to investigate differences in neutrophil subpopulations, their potential of activation and neutrophil extracellular trap (NET) formation comparing high and low-density neutrophils (HDNs/LDNs) as well as subpopulations in patients with APS and controls to gain deeper insight into their potential role in thrombotic manifestations in patients with APS. METHODS: HDNs and LDNs of 20 patients with APS and 20 healthy donors were isolated by density gradient centrifugation and stimulated. Neutrophil subpopulations, their activation and NET release were assessed by flow cytometry. RESULTS: LDNs of both groups showed higher baseline activation, lower response to stimulation (regulation of activation markers CD11b/CD66b), but higher NET formation compared with HDNs. In patients with APS, the absolute number of LDNs was higher compared with controls. HDNs of APS patients showed higher spontaneous activation [%CD11b high: median (interquartile range): 2.78% (0.58-10.24) vs 0.56% (0.19-1.37)] and response to stimulation with ionomycin compared with HDNs of healthy donors [%CD11b high: 98.20 (61.08-99.13) vs 35.50% (13.50-93.85)], whereas no difference was found in LDNs. NET formation was increased in patients' HDNs upon stimulation. CONCLUSION: HDNs and LDNs act differently, unstimulated and upon various stimulations in both healthy controls and APS patients. Differences in HDNs and LDNs between patients with APS and healthy controls indicate that neutrophils may enhance the risk of thrombosis in these patients and could thus be a target for prevention of thrombosis in APS.