Evaluation of thromboelastometry, thrombin generation and plasma clot lysis time in patients with bleeding of unknown cause: A prospective cohort study.

INTRODUCTION: Diagnostic evaluation of patients with a bleeding tendency remains challenging, as no disorder is identified in approximately 50% of patients. An impaired interplay of several haemostatic factors might explain bleeding phenotype in these patients. OBJECTIVE: To investigate whether global haemostasis assays are able to identify haemostatic abnormalities in patients with a bleeding tendency unexplained by current diagnostic laboratory tests. MATERIALS AND METHODS: Patients of ≥12 years with a bleeding tendency were included from a tertiary outpatient clinic. Bleeding phenotype was assessed with the ISTH-BAT. Patients were classified as having bleeding of unknown cause (BUC) or a mild bleeding disorder (MBD) based on abnormalities assessed by routine haemostatic tests. Global haemostasis tests (rotational thromboelastometry (ROTEM), thrombin generation test (TG) and plasma clot lysis time (CLT)) were measured in all patients. The results were compared with 76 controls. RESULTS: One hundred and eighty-one patients were included, and 60% (109/181) was classified as having BUC. BUC patients demonstrated a significantly prolonged lag time in TG (median 7.7 minutes, IQR 6.7-8.7) and a significantly prolonged CLT (median 60.5 minutes, IQR 54.7-66.1) compared to controls. No differences in ROTEM variables were found. Patients with MBD showed an impaired thrombin generation with a significantly decreased ETP (median 1024 nmol/L*min, IQR 776-1355) and peak height (median 95 nmol/L, IQR 76-138), compared to BUC patients and controls. CONCLUSION: No major differences were found in ROTEM and TG variables in BUC patients compared to controls. BUC patients did have a significantly prolonged clot lysis time. The underlying mechanism for this finding is unknown.

FIBTEM clot firmness parameters correlate well with the fibrinogen concentration measured by the Clauss assay in patients and healthy subjects.

The Clauss assay is the assay most often used for measuring plasma fibrinogen levels. However, the FIBTEM-assay, determined using thromboelastometry (ROTEM) can also be used to estimate fibrinogen levels. A major advantage of the FIBTEM is that it can provide information about fibrinogen levels within minutes, while the Clauss assay needs 30-60 min before the result is available. The aim of this study was to investigate the correlation between fibrinogen levels measured by the Clauss assay and results from the FIBTEM-assay. We included 111 patients ≥18 years for whom both ROTEM analyses and a fibrinogen measurement using the Clauss assay were available. In addition, ROTEM and Clauss measurements from 75 healthy subjects were included. Spearman correlation was used to determine the association between the results of both assays. The patients included were mostly patients with major trauma or undergoing large surgery (e.g. cardiac surgery or liver transplantation). Strong correlations were found between FIBTEM clot firmness parameters and fibrinogen levels measured by the Clauss assay in patients (Spearman's correlation coefficients (rs) above 0.80 (p < .001) for all subgroups) and healthy subjects (rs = 0.66, p < .001). The correlation between early FIBTEM parameters (clot firmness at 5 or 10 min) and the maximum clot firmness was almost perfect (rs above 0.96). Also, the correlation between the α-angle and FIBTEM parameters was strong (rs above 0.7). In conclusion, strong correlations were found between early FIBTEM parameters and fibrinogen levels.

Severe postpartum haemorrhage as first presenting symptom of an inherited bleeding disorder.

INTRODUCTION: Postpartum haemorrhage (PPH) is the major cause of maternal death worldwide. Haemostatic abnormalities are independently associated with a significantly increased risk for severe PPH. In this study, the value of haemostatic evaluation in women with severe PPH was explored. AIM: To investigate the occurrence of previously unknown inherited bleeding disorders in women with severe PPH. METHODS: Women with severe PPH (blood loss of ≥2000 mL) between 2011 and 2017, referred to the haematology outpatient clinic for haemostatic evaluation, were retrospectively included. A bleeding disorder was diagnosed based on (inter)national guidelines, or when having a clear bleeding phenotype, not fulfilling any diagnostic criteria or laboratory abnormalities, this being classified as Bleeding of Unknown Cause (BUC). Logistic regression was used to model the association between diagnosis and obstetrical causes and risk factors for PPH. RESULTS: In total, 85 women with PPH were included. In 23% (n = 16), a mild bleeding disorder was diagnosed, including low von Willebrand factor (Low VWF 8/16), platelet function disorders (PFD 5/16), BUC (2/16) and von Willebrand disease type 1 (1/16). No significant associations were found between obstetrical causes or risk factors for PPH and the presence of a bleeding disorder. CONCLUSION: In 23% of women with severe PPH, a mild bleeding disorder was diagnosed, independent of obstetrical causes or risk factors for PPH. This implies that severe PPH can be the first clinical symptom of an inherited bleeding disorder. Therefore, to optimize clinical management, haemostatic evaluation after severe PPH is recommended.

Proteomic landscapes of inherited platelet disorders with different etiologies.

BACKGROUND: Inherited platelet disorders (IPDs) are a heterogeneous group of rare diseases that are caused by the defects in early megakaryopoiesis, proplatelet formation, and/or mature platelet function. Although genomic sequencing is increasingly used to identify genetic variants underlying IPD, this technique does not disclose resulting molecular changes that impact platelet function. Proteins are the functional units that shape platelet function; however, insights into how variants that cause IPDs impact platelet proteomes are limited. OBJECTIVES: The objective of this study was to profile the platelet proteomics signatures of IPDs. METHODS: We performed unbiased label-free quantitative mass spectrometry (MS)-based proteome profiling on platelets of 34 patients with IPDs with variants in 13 ISTH TIER1 genes that affect different stages of platelet development. RESULTS: In line with the phenotypical heterogeneity between IPDs, proteomes were diverse between IPDs. We observed extensive proteomic alterations in patients with a GFI1B variant and for genetic variants in genes encoding proteins that impact cytoskeletal processes (MYH9, TUBB1, and WAS). Using the diversity between IPDs, we clustered protein dynamics, revealing disrupted protein-protein complexes. This analysis furthermore grouped proteins with similar cellular function and location, classifying mitochondrial protein constituents and identifying both known and putative novel alpha granule associated proteins. CONCLUSIONS: With this study, we demonstrate a MS-based proteomics perspective to IPDs. By integrating the effects of IPDs that impact different aspects of platelet function, we dissected the biological contexts of protein alterations to gain further insights into the biology of platelet (dys)function.

Automated Fiber Diameter and Porosity Measurements of Plasma Clots in Scanning Electron Microscopy Images.

Scanning Electron Microscopy (SEM) is a powerful, high-resolution imaging technique widely used to analyze the structure of fibrin networks. Currently, structural features, such as fiber diameter, length, density, and porosity, are mostly analyzed manually, which is tedious and may introduce user bias. A reliable, automated structural image analysis method would mitigate these drawbacks. We evaluated the performance of DiameterJ (an ImageJ plug-in) for analyzing fibrin fiber diameter by comparing automated DiameterJ outputs with manual diameter measurements in four SEM data sets with different imaging parameters. We also investigated correlations between biophysical fibrin clot properties and diameter, and between clot permeability and DiameterJ-determined clot porosity. Several of the 24 DiameterJ algorithms returned diameter values that highly correlated with and closely matched the values of the manual measurements. However, optimal performance was dependent on the pixel size of the images-best results were obtained for images with a pixel size of 8-10 nm (13-16 pixels/fiber). Larger or smaller pixels resulted in an over- or underestimation of diameter values, respectively. The correlation between clot permeability and DiameterJ-determined clot porosity was modest, likely because it is difficult to establish the correct image depth of field in this analysis. In conclusion, several DiameterJ algorithms (M6, M5, T3) perform well for diameter determination from SEM images, given the appropriate imaging conditions (13-16 pixels/fiber). Determining fibrin clot porosity via DiameterJ is challenging.

Outcome of Surgical Interventions and Deliveries in Patients with Bleeding of Unknown Cause: An Observational Study.

BACKGROUND: The most optimal management for patients with bleeding of unknown cause (BUC) is unknown, as limited data are available. OBJECTIVE: Evaluate management and outcome of surgical procedures and deliveries in patients with BUC. MATERIALS AND METHODS: All patients ≥12 years of age, referred to a tertiary center for a bleeding tendency, were included. Bleeding phenotype was assessed and hemostatic laboratory work-up was performed. Patients were diagnosed with BUC or an established bleeding disorder (BD). Data on bleeding and treatment during surgical procedures and delivery following diagnosis were collected. RESULTS: Of 380 included patients, 228 (60%) were diagnosed with BUC and 152 (40%) with an established BD. In 14/72 (19%) surgical procedures major bleeding occurred and 14/41 (34%) deliveries were complicated by major postpartum hemorrhage (PPH). More specifically, 29/53 (55%) of the BUC patients who underwent surgery received prophylactic treatment to support hemostasis. Despite these precautions, 4/29 (14%) experienced major bleeding. Of BUC patients not treated prophylactically, bleeding occurred in 6/24 (25%). Of pregnant women with BUC, 2/26 (8%) received prophylactic treatment during delivery, one women with and 11 (46%) women without treatment developed major PPH. CONCLUSION: Bleeding complications are frequent in BUC patients, irrespective of pre- or perioperative hemostatic treatment. We recommend a low-threshold approach toward administration of hemostatic treatment in BUC patients, especially during delivery.

Thrombophilia: Women-Specific Reference Ranges Can Prevent Misdiagnosis in Women.

BACKGROUND: Thrombophilia is a state where abnormalities of the hemostatic system predispose a patient to thrombosis. Some coagulation factors are generally lower in women than in men. Therefore, the use of routine reference ranges (RRRs) based on male or mixed-sex groups may be misleading in the diagnosis of thrombophilia in women. We hypothesize that this affects the analysis of thrombophilia after pregnancy complications. Therefore, the aim of our study was to investigate the effect of women-specific reference ranges (WRRs) on the interpretation of hemostatic variables in postpartum women. METHODS: Coagulant and anticoagulant variables were measured 3 months postpartum in 61 healthy women with an uncomplicated pregnancy and in 197 women who experienced preeclampsia (PE). In 55 of the healthy women, these variables were also measured at least 6 months after an uncomplicated pregnancy and used to calculate WRR. RESULTS: In total, 48% of healthy women had normal results when using routine reference ranges compared with 89% when using WRRs (P < 0.05). In the women with PE, there were normal results in 26% of women when using routine reference ranges compared with 66% when using WRRs (P < 0.05). CONCLUSION: When using WRRs, fewer abnormalities were seen in healthy women as well as in women with a history of PE, which may prevent misdiagnosis of thrombophilia.