Coagulation Factor Deficiencies Research Articles

Impact of Blood Group O and Von Willebrand Factor on Bleeding Severity in Patients with Von Willebrand Disease and Bleeding Disorder of Unknown Cause

Background: In patients with bleeding, von Willebrand disease (VWD) is diagnosed at von Willebrand factor (VWF) levels of ≤50 IU/dL ( James et al. Blood Adv. 1:280-300. 2021). Nevertheless, the majority of patients with a mild to moderate bleeding disorder (MBD) is diagnosed with bleeding disorder of unknown cause (BDUC), upon exclusion of VWD, platelet function defects (PFD), and coagulation factor deficiencies (CFD). Bleeding severity in patients with VWD does not correlate well with VWF levels in type 1 VWD, suggesting other modifiers of the bleeding tendency, when patients with severe VWD are excluded. Blood group O (BG O) is associated with lower VWF levels and has been found to be overrepresented in VWD. In BDUC patients, BGO is also overrepresented and recognized as an independent risk factor for bleeding severity and recurrent bleeding ( Mehic et al. Blood Adv.20: 5157-5164. 2020; Mehic et al. JTH. 7: 1757-1768. 2023). Aim: This study aimed to comprehensively investigate the clinical phenotype of patients with VWD or BDUC from the Vienna Bleeding Biobank (VIBB) and evaluate the direct effect of ABO BG on bleeding severity beyond its effect on VWF levels. Methods: Patients with VWD or BDUC from the VIBB, a single-center cohort study (EC 603/2009), were categorized by lowest VWF, as measured by VWF:antigen (VWF:Ag) or VWF ristocetin co-factor activity (VWF:RCo), into four groups: <30 IU/dL (very low VWF), 30-50 IU/dL (low VWF), 51-80 IU/dL, and >80 IU/dL. Patients diagnosed with PFD (n=221), CFD (n=28), or other diagnoses (n=4) were excluded. To study the association of BG O with bleeding severity irrespective of VWF levels, we fitted linear regression models adjusting for age, sex, and lowest VWF. We allowed for non-linearity by modelling age and sex with restricted cubic splines. Results: A total of 637 patients, 18 (2.8%) with very low VWF, 55 (8.6%) with low VWF, 237 (37.2%) with VWF levels ranging from 51 to 80 IU/dL and 327 (51.3%) with VWF levels >80 IU/dl, were included in this analysis, as summarized in Table 1. BG O was more prevalent in patients with very low VWF levels (61%), low VWF levels (71%), and VWF levels 51-80 IU/dL (66%) than in patients with VWF levels >80 IU/dL (30%). Patients with low VWF were youngest, followed by very low VWF and those with VWF 51-80 IU/dL. Age was highest in the patient group with VWF levels >80 IU/dL at clinical presentation and might influence VWF levels and bleeding scores. With regard to bleeding severity, patients with very low VWF levels had highest median bleeding scores or number of bleeding manifestations, while the bleeding severity was similar amongst the other three categories (Table 1). VWF levels did not correlate with bleeding severity scores (Vicenza score: Spearman rho=-0.03, p=0.53; ISTH BAT: rho=-0.02, p=0.75; number of bleeding manifestations: rho=-0.02, p=0.66). In linear regression, holding age, sex, and lowest VWF levels constant, predicted mean bleeding scores and number of bleeding manifestations were higher with BG O (Vicenza score mean difference [MD], 0.64; 95% confidence interval [95%CI], 0.12 to 1.25; p=0.015; ISTH BAT MD 0.52; 95%CI, -0.25 to 1.28; p=0.18; number of bleeding manifestations MD, 0.24; 95%CI, -0.06 to 0.54; p=0.12) (Figure 1). Conclusion: Our findings demonstrate an overlap between VWD and BDUC with a similar clinical phenotype and bleeding phenotype, but increased severity in patients with VWF levels <30 IU/dL. Patients with BG O had increased bleeding scores compared to those with BG non-O. BG O appears to be an independent factor in modifying bleeding severity in both patients with VWD or BDUC across all VWF ranges.

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

Advancing Hemophilia Prophylaxis: Personalized Low-Dose CFC and Emicizumab in Resource-Limited Settings

Introduction: Hemophilia, a congenital bleeding disorder resulting from clotting factor deficiency, significantly impacts the lives of persons due to recurrent bleeding. Prophylaxis, involving clotting factor replacement, has been recognized as a vital approach to reduce complications and to improve the quality of life in people with hemophilia. However, access to this remains limited, in low-income countries like India. This study examines the effectiveness of personalized low-dose clotting factor concentrates (CFC) prophylaxis and Emicizumab in children with hemophilia, aiming to achieve zero annual bleed rates (ABR), thereby enhancing their well-being and reducing healthcare burden. Patients and Methods: This retrospective observational study was conducted at the Hemophilia Treatment Centre (HTC) in Aluva, Kerala, India, spanning from May 31, 2022, to June 1, 2023. Study included children under 18 years with hemophilia A/B, with or without inhibitors, who had commenced prophylaxis with personalized low-dose CFC since 2015 and completed one year of prophylaxis. The total sample size was 31. Data on ABR, Annual Joint Bleed Rate (AJBR), Hemophilia Joint Health Status (HJHS) score, and Functional Independence Score Hemophilia (FISH) were extracted from the HTC registry.Standard half-life CFCs were used for prophylaxis, with a starting frequency of twice a week for Factor VIII and once a week for Factor IX. Patients were regularly followed up in the Hemophilia Clinic, and the frequency/doses of prophylaxis were increased in case of breakthrough bleeds. Additionally, children were advised to organize peak activities to coincide with the days of CFC injections. Personalization of doses was not based on pharmacokinetic software but was determined through close monitoring and individual adjustments.Health education on identifying and managing bleeding episodes followed ISTH guidelines. Prophylaxis outcomes were assessed with changes in ABR and AJBR during the last year of prophylaxis and compared to the bleed rates in the one year prior to prophylaxis. Changes in HJHS and FISH were documented for older children. All analysis were done using SPSS version24. For quantitative variables results were presented as mean with standard deviation and for qualitative variables, results were presented in percentages. Comparison achieved with standard paired t-test, and p value < 0.05 considered as significant. Results: Out of 36 children receiving prophylaxis, 31 met the inclusion criteria. Among them,10 (32%) received Emicizumab prophylaxis, while 21 (68%) were managed with personalized low-dose CFC prophylaxis. Hemophilia A constituted 24(77%) of cases, mostly classified as severe disease-29 (94%). Table :1 summarises the patient characteristics, prophylaxis dose, cost and outcome measures of the study population.The personalized low-dose CFC prophylaxis had significant measurable improvements, reducing the ABR from 14.90 to 2.90( p 0.000) and the AJBR from 4.95 to 1.43( p 0.004). Additionally, HJHS improved from 3.30 to 0.95( p 0.102), and FISH increased from 29.90 to 32.00( p 0.020).Children on Emicizumab achieved exceptional results, with all reporting zero ABR ( p 0.027), and AJBR ( p 0.131) even with lower than recommended doses(1.02mg/kg/week). In contrast, personalized low-dose CFC prophylaxis (Factor VIII: 36.35/kg/week, Factor IX:32/kg/week) showed varied outcomes, with only 4(19%) achieving zero ABR. HJHS and FISH were not evaluated in Emicizumab group as 7(70%) were belonging to the age group of less than 5 years. Conclusion: This study highlights the effectiveness of personalized low-dose CFC prophylaxis and Emicizumab in reducing bleeding rates and improving musculoskeletal health and functional independence in children with hemophilia. Emicizumab prophylaxis demonstrated remarkable outcomes, achieving zero bleeds even with lower doses. However, personalized low-dose CFC prophylaxis struggled to achieve zero bleed rates for most of them. The findings underscore the need for alternative strategies and increased accessibility to Emicizumab and other newer drugs to achieve the goal of zero bleed rates for children with hemophilia in resource-limited settings. The study's results provide hope for achieving zero bleed rates, ultimately enhancing the well-being of children with hemophilia in resource-limited regions.

Randomized, Active-Control Phase 3 Study of Four-Factor Prothrombin Complex Concentrate Versus Frozen Plasma in Bleeding Adult Cardiac Surgery Patients

Background and Significance: Patients following cardiac surgery often develop coagulopathic bleeding and associated poor outcomes. The development of coagulopathy is multifactorial, including anticoagulation, hemorrhage, hemodilution and consumptive losses after tissue injury and during cardiopulmonary bypass (CPB). Reduced thrombin generation due to coagulation factor deficiency is an important contributor to post-CPB bleeding. Prothrombin complex concentrate (PCC; off-label) and frozen plasma (FP) are administered for coagulation factor replacement during surgery. The LEX-211 (FARES-II) study will determine if four-factor PCC (4F-PCC, Octaplex, Octapharma) is clinically non-inferior to FP regarding hemostatic effectiveness in cardiac surgery patients requiring coagulation factor replacement. Study Design and Methods: LEX-211 (FARES-II; NCT05523297) is a multicenter, randomized, active-control, prospective, Phase 3 trial that is being conducted at 13 hospitals in Canada and the United States. The study is being conducted in accordance with the Declaration of Helsinki. The study includes patients ≥18 years old undergoing cardiac surgery with CPB who require coagulation factor replacement due to bleeding and known (e.g., as indicated by international normalized ratio) or suspected coagulation factor deficiency. Exclusion criteria include heart transplant, insertion/removal of ventricular assist devices, high probability of death within 24 hours, severe right heart failure, heparin contraindications, thromboembolic events within the prior 3 months, and IgA deficiency. Patients will be randomized to 4F-PCC or FP when the blood bank receives the first order for coagulation factor replacement (Figure 1). For 4F-PCC dosing, patients weighing ≤60 kg will receive 1,500 international units (IU), and those >60 kg will receive 2000 IU. For FP, patients weighing ≤60 kg will receive 3 U and patients weighing >60 kg will receive 4 U. Patients are treated according to their assigned group until a maximum of 2 doses of 4F-PCC/FP have been administered during the treatment period (24 hours after initiation). If additional treatment is required, patients in both groups receive FP. The primary endpoint is the hemostatic response to 4F-PCC vs. FP, rated ‘effective’ if no further hemostatic intervention (systemic hemostatic agents, i.e., platelets, cryoprecipitate, other coagulation factor products, or a second dose of study drug, or surgical re-opening for bleeding) is required within 60 minutes to 24 hours after initiation of the first dose. Secondary and safety endpoints, with their timings, are described in Table 1. An unblinded interim analysis (100 evaluable patients/group) will test sample size assumptions and enable re-estimation if necessary. Depending upon the interim results, and accounting for dropouts (20% anticipated), the total sample size will range between 513-1,250 patients. The non-inferiority of the primary endpoint of ‘haemostatic response’ will be tested for 4F-PCC vs. FP using a Farrington-Manning score test with a non-inferiority margin of 0.10 at a one-sided significance level alpha of 2.5%. If non-inferiority is demonstrated, the superiority of 4F-PCC with regard to the primary endpoint will be investigated. LEX-211 (FARES-II) is in progress, with the first study site initiated in Q4 2022. Currently, >150 patients have been included in the study. Completion is expected in Q4 2024. The results of this study will inform clinical practice for bleeding cardiac surgery patients requiring coagulation factor replacement, potentially reducing allogeneic blood product usage and improving patient outcomes.

Health Care Resource Use, Comorbidities, and Costs of Hemophilia A Patients in France: A Nationwide Claims Database Analysis

Background and objectives Hemophilia A is an inherited bleeding disorder characterized by a deficiency in blood clotting factor VIII (FVIII). Its severity varies based on the degree of FVIII deficiency, with the most severe cases experiencing frequent spontaneous hemorrhages (FVIII levels < 1 IU/dL). Treatment options include on-demand administration following a hemorrhage or prophylaxis to prevent bleeding. The most frequent complication is the production of inhibitory antibodies against the coagulation factors, leading to more aggressive and expensive treatments. The aim of this study was to describe Hemophilia A patients according to severity in France in 2021, including sociodemographic characteristics and comorbidities, current treatments, healthcare resource use and related costs. Methods This study used the French health-insurance claims database (SNDS) containing pseudonymized individual data for over 66 million people. Hemophilia A patients are fully covered in France and might be identified by the ICD-10 code D66. Patients alive on January 1st, 2021, were selected after exclusion of those presenting with Willebrand disease and other rare bleeding disorders. Comorbidities were identified through validated algorithms. An age and gender matched control group without Hemophilia A was randomly selected in the general population: for each HA patient a subject of the same age, gender and region of residence was randomly selected among the overall population of French citizens (excluding HA patients). Sub-group analyses were performed according to the treatment pattern (on demand / in prophylaxis) and the presence of inhibitors (defined with the treatment: emicizumab before October 2019, high dose of factor VIII or bypassing agents). Direct costs were estimated in a societal perspective. Results 5,507 patients with Hemophilia A were identified. Mean age was 36 years and 97.3% were males. Compared to controls, hemophilia patients had significantly (p<0.05) more frequently an HIV infection, hepatitis B or C, and a hepatic disease (Figure 1). There were no significant differences for chronic cardiopathy, coronary artery disease, chronic heart failure and arrhythmia. Respectively 4,056 (73.7%), 36 (0.7%), 1,243 (22.6%) and 172 (3.1%) patients were treated on demand without and with inhibitors and in prophylaxis without and with inhibitors. Compared to controls, patients with hemophilia A had more frequent consultations with general practitioners and hospital specialists, visits to emergency unit, with nurses and physiotherapists (Table 1). They also were more frequently treated with analgesics and corticosteroids but less frequently with non-steroid anti-inflammatory drugs. The proportion of hemophilia A patients hospitalized was higher than controls, overall and also for bleeding episodes and for orthopedic surgery. The mean annual direct medical costs (drugs, consultations, hospitalizations, ...) varied strongly according to treatment modalities and presence of inhibitors: €11,471 for patients treated on demand without inhibitors (versus €1,806 for their matched controls)€76,332 for patients treated on demand with inhibitors (versus €2,288 for their matched controls)€247,937 for patients treated in prophylaxis without inhibitors (versus €1,159 for their matched controls)€349,616 for patients treated in prophylaxis with inhibitors (versus €1,342 for their matched controls). The majority of the costs was related to antihemophilic drugs: 64%, 60%, 94% and 93% respectively in the 4 treatment groups. Conclusion: These results highlight the burden of hemophilia A in terms of comorbidities, healthcare consumptions and cost. The cost of Hemophilia A varied greatly with disease severity and presence of inhibitors and was mostly due to the antihemophilic drugs.

Health Care Resource Use, Comorbidities, and Costs of Hemophilia B Patients in France: A Nationwide Claims Database Analysis

Background and objectives Hemophilia B is an inherited bleeding disorder characterized by a deficiency in blood clotting factor IX (FIX). Its severity varies based on the degree of FIX deficiency, with the most severe cases experiencing frequent spontaneous hemorrhages (FIX levels < 1 IU/dL). Treatment options include on-demand administration following a hemorrhage or prophylaxis to prevent bleeding. A complication is the production of inhibitory antibodies against the coagulation factors in some patients, leading to more aggressive and expensive treatments. The aim of this study was to describe Hemophilia B patients according to severity in France in 2021, including sociodemographic characteristics and comorbidities, current treatments, healthcare resource use and related costs. Methods This study used the French health-insurance claims database (SNDS) containing pseudonymized individual data for over 66 million people. Hemophilia B patients are fully covered in France and might be identified by the ICD-10 code D67. Patients alive on January 1st, 2021, were selected after exclusion of those presenting with Willebrand disease and other rare bleeding disorders. Comorbidities were identified through validated algorithms. An age and gender matched control group without Hemophilia B was randomly selected in the general population: for each HB patient a subject of the same age,gender and region of residence was randomly selected among the overall population of French citizens (excluding HB patients). Sub-group analyses were performed according to the treatment pattern (on demand / in prophylaxis) and the presence of inhibitors (defined with the treatment: high dose of factor IX or bypassing agents). Direct costs were estimated in a societal perspective. Results 1,311 patients with Hemophilia B were identified. Mean age was 36 years and 83.5% were males. Compared to controls, hemophilia patients had significantly (p<0.05) more frequently an HIV infection, hepatitis B or C, and a chronic cardiopathy (Figure 1). There were no significant differences for coronary artery diseases, chronic heart failure, arrhythmia, and hepatic diseases. Respectively 996 (76.0%) and 303 (23.1%) patients were treated on demand and in prophylaxis both without inhibitors. Less than 10 patients were treated either on demand with inhibitors or in prophylaxis with inhibitors. Compared to controls, patients with hemophilia B had more frequent consultations with general practitioners and hospital specialists, visits to emergency unit, with nurses and physiotherapists (Table 1). They also were more frequently treated with analgesics and corticosteroids but less frequently with non-steroid anti-inflammatory drugs. The proportion of hemophilia B patients hospitalized was higher than controls, overall and also for bleeding and for orthopedic surgery. The mean annual direct medical costs (drugs, consultations, hospitalizations, ...) varied strongly according to treatment modalities: €6,506 for patients treated on demand without inhibitors (versus €1,795 for their matched controls)€162,635 for patients treated in prophylaxis without inhibitors (versus €1,071 for their matched controls). The majority of the costs was related to antihemophilic drugs: 33% and 92% respectively for these 2 treatment groups. Conclusion: These results highlight the burden of hemophilia B in terms of comorbidities, healthcare consumptions and cost. The cost of Hemophilia B varied greatly with disease severity and was mostly due to the antihemophilic drugs

Targeted genome engineering based on CRISPR/Cas9 system to enhance FVIII expression in vitro

BackgroundHemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease. MethodsInitially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression. ResultsWe identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro. ConclusionsKnock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases.

Increased Circulating Levels of YKL-40 Protein in Children and Adolescents with Hemophilia: Correlation with the Degree of Hemophilic Arthropathy

Introduction: Hemophilia A and B are rare X-linked inherited bleeding disorders caused by complete or partial deficiency of coagulation factors VIII and IX, respectively. Recurrent joint bleeding (hemarthrosis) is the most frequent clinical manifestation of severe hemophilia. Unless appropriately managed, even subclinical hemarthrosis can lead to hemophilic arthropathy, that severely affects the quality of life of patients. Chitinase-3-like protein 1 (CHI3L1), also known as YKL-40, is a 40-kDa heparin-human cartilage glycoprotein without enzymatic activity, that is encoded by the CHI3L1 gene in humans and its name derives from the one letter code for the three terminal amino acids - tyrosine (Y), lysine (K) and leucine (L). YKL-40 is secreted in vitro and in vivo in the arthritic joint by various cell types, such as activated macrophages, chondrocytes, synoviocytes and neutrophils. Actually, it is a major protein that has been identified in the chondrocytes from arthritic knee joints.In fact, synovial as well as serum YKL-40 levels are found increased in joint diseases like rheumatoid arthritis and osteoarthritis, indicating its potential role as a biomarker of inflammation and tissue remodeling or degradation, whereas it is absent in normal joints. In our knowledge, there are not any reports linking YKL-40 expression with hemophilic arthropathy, thus we aimed to investigate if this protein could be implicated in hemophilic arthropathy severity, by measuring its circulating levels and correlate them with hemophilic arthropathy features and laboratory parameters in children and adolescents with hemophilia A and B. Patients and Methods: Eighty children and adolescents (median age:14 years, range 10-18 years) with severe (n= 60), moderate (n= 8) and mild (n= 12) hemophilia A or B (n= 7), on prophylaxis or on demand treatment (n= 12) were included in the study, while 35 apparently healthy male individuals matched for age served as controls. No signs of hemophilic arthropathy (NHAP), i.e. Hemophilia Joint Health Score 2.1 ≤ 2 and Hemophilia Early Detection Arthropathy with Ultrasound Score ≤ 1, were detected in 34/80 patients with median age of 13.5 years. In 37/80 patients with median age of 14.25 years arthropathy (HAP) was confirmed, while 18/80 patients with median age of 15.75 years presented with acute bleeding (AB)-hemarthrosis or muscle bleed- at the time of the laboratory evaluation. In patients and controls YKL-40 concentrations were determined by means of an immunoenzymatic technique. Results: We found that: a) Circulating YKL-40 levels in patients with hemophilia were significantly higher compared to controls, 21.9±2.3 ng/mL (95% Confidence Intervals of the mean (CI): 19.6;24.2 ng/mL) vs 17.0±2.5 ng/mL (CI: 14.5;19.4 ng/mL), respectively, p=0.003. b) Patients with arthropathy (HAP) tend to express higher circulating YKL-40 levels (25.5±2.0 ng/mL) than patients without arthropathy (NHAP)(20.1±1.8 ng/mL) and patients with acute bleeding (AB) (19.7±2.4 ng/mL), respectively, p-trend=0.06. Conclusions: To the best of our knowledge, this is the first study to evaluate the potential clinical significance of circulating YKL-40 levels in patients with hemophilia and demonstrated that it could reflect hemophilic arthropathy activity in clinical or even in subclinical level. As YKL-40 in synovial fluid is derived from cells in the inflamed/damaged synovium, such as chondrocytes, we postulate that it may be involved in the pathophysiology of the hemophilic arthropathy processes and reflect local disease activity. Further studies are essential on the role of YKL-40 in the evolution of hemophilic arthropathy, in order to fully elucidate its clinical significance and prognostic utility.

Prevalence of Rare Bleeding Disorders in the Central California Cohort: A Retrospective Single Institution Analysis

Abstract Rare bleeding disorders (RBD) constitute 3- 5% of all inherited clotting factor deficiencies and include inherited deficiencies of fibrinogen (FI), factors II, V, VII, X, XI and XIII, and combined factor V and VIII, and affect both sexes in an autosomal recessive manner. Rare platelet disorders can also present with bleeding diathesis which are recessively inherited. Significant clinical heterogeneity is present in these disorders with varying bleeding symptomatology causing diagnostic and therapeutic challenges. The objective of this study was to analyze the prevalence, characteristics, and treatment of rare bleeding disorders at the Valley Children's Hemophilia Treatment Center(HTC) in Central California.We conducted a retrospective analysis of all individuals who were referred for abnormal bleeding to our institution from 2018- 2023. We analyzed the clinical features, laboratory variables, and treatment of patients identified with RBD. Rare platelet disorders were included in this study. We excluded factor VIII, IX deficiencies and Von Willebrand disease (VWD) from this analysis. Results We identified a total of 157 patients (age range 18 months-46 years old) with RBD, 46% were of female gender (Table 1). Majority of the patients (n=92, 58%) were of Hispanic ethnicity. Major RBD in the cohort was Factor 7 (F7) deficiency (n= 96, 60.4%). F11 deficiency constituted 10.1%, followed by fibrinogen deficiency (6.3%), and F13 deficiency (5.7%). F2 and Plasminogen activator inhibitor-1 (PAI-1) deficiency was identified in one individual each. Glanzmann thrombasthenia was the most common platelet disorder noted in our population (n=7, 4.4%), followed by giant platelet disorder (n=6, 3.8%). MYH9- related disorder was identified in 5 (3.1%) individuals with macrothrombocytopenia and bleeding. Wiskott-Aldrich Syndrome was detected in 3 (1.9%) cases of RBD. Combined bleeding disorders (CBDs) were observed in 15 individuals (9.5%) (Table 2). These disorders were distinguished by genotyping, factor activity, and chromogenic activity assays. F7 and Type 1 VWD combined disorder was observed in 46.7% (n=7) of these individuals. Genetic testing was done in 38% (n=60) of the affected individuals. Genetic analysis was done through American Thrombosis & Hemostasis Network (ATHN 10) in 19% (n=30). F7 gene mutation was the most commonly identified mutation and variants c.1238G>A (p.Arg413Gln), c.1172G>A (p.Arg353Gln), and c.1091G>A (p.Arg364Gln) were observed in those with F7 deficiency. All individuals with F7 deficiency with variants of uncertain significance were presumed to be positive. Treatment was specific to the factor deficiency. Most individuals received additional anti-fibrinolytic therapy during acute bleeding episodes. Recombinant F7 was used in platelet disorders for bleeding. Major bleeding events and death in the cohort was 1.2% (n= 2), and 2 individuals with F13 deficiency developed inhibitors to F13 concentrate. These individuals have been managed with recombinant F7 and anti-fibrinolytic therapy. Conclusion Ethnically diverse distribution of rare bleeding disorders was evident in our Central California cohort. Similar to previous reports, F7 deficiency was of the highest prevalence. A high prevalence of CBDs was also observed in this cohort. Genetic testing enabled diagnosis in some complex cases and needs to be used more widely to facilitate timely and accurate identification, and treatment of CBDs, and RBD in general. Further, these results help to identify novel mutations in different ethnicities, and provide additional information in understanding the clinical impact of new mutations.

Bleeding Disorders in Women- a Unique Challenge Requiring a Unique Approach

Introduction: Bleeding disorders in women, particularly in developing countries, have been largely neglected due to various factors such as social stigma, limited access to comprehensive laboratory facilities, and the misconception of physiological bleeding during menstruation. Heavy menstrual bleeding (HMB) is a common symptom in women, and a significant percentage of cases may be associated with inherited bleeding disorders. However, the underreporting and underdiagnosis of these conditions remain prevalent, partly due to the stigma surrounding menorrhagia, leading to delayed medical care seeking. This study aims to explore the clinical and laboratory profiles of females with bleeding disorders who presented to our institute. Methods: A consecutive series of female patients referred to our advanced coagulation laboratory for complete workup with bleeding manifestations or abnormal screening coagulogram over a 59-month period (September 2018 to July 2023) were assessed. Patients with liver disease, chronic inflammation, or on anticoagulation therapy were excluded. Data on age at presentation and diagnosis, ISTH BAT score, annual bleeding rate, family history, hospitalizations, surgical interventions, and follow-up were retrospectively retrieved from our database. Heavy menstrual bleeding was subjectively defined based on specific criteria. Various laboratory parameters were analyzed for all patients. Data were presented using median/range and percentage. Missing data were not imputed, and MS Excel 2016 was used for analysis. Results: Over the study duration, 1019 patients were evaluated at our institute, with 488 females (47.9%) and 531 males (52.1%). Among the 235 patients (23%) diagnosed with bleeding disorders, 87 (37%) were women. The median age at diagnosis was 29 years (range: 9 months-84 years), while the median age of first symptom was 15 years (range: 7 days-81 years). The median time interval between presentation and diagnosis was 8 years (range: 6 months-30 years). The most common presenting symptom was HMB (34.4%), followed by ecchymotic patches (17.2%) and epistaxis (16%). Significant ISTH BAT scores (>6) were found in 17 patients (19.5%). A family history of bleeding was observed in 4.5% of patients, with 3 of them born to consanguineous parents. Incidentally detected abnormal screening coagulogram was present in 12.6% of patients. The most common bleeding disorder diagnosed was VWD (35.6%), followed by Factor VII deficiency (13.8%), Vitamin K dependent coagulation factor deficiency (9.2%), and Acquired Haemophilia A (9.2%). Intracranial bleeding (4.5%) was primarily associated with specific deficiencies. No carriers were detected in the study. Conclusion: Addressing bleeding disorders in women requires specific attention. The ISTH BAT score proved to be a user-friendly yet powerful tool, with 19.5% of patients showing significant scores that prompted comprehensive diagnostic workup. This study highlights the long latent period between the age of first symptom and the age of diagnosis, emphasizing the need to raise awareness among patients and primary physicians, especially gynaecologists. Menorrhagia starting from menarche was the most common bleeding symptom, emphasizing the importance of prompt and accurate diagnosis to prevent life-threatening bleeds. The consideration of acquired hemophilia A is crucial in cases of unexplained postpartum hemorrhage. Developing countries face additional challenges due to limited specialized laboratories, leading to outsourcing and increased testing costs. To improve diagnosis and care, early identification of female bleeders is essential, facilitated by outreach programs and advanced coagulation testing centers.

Proactive Systematic Hemophilia Carrier Screening: A Step Towards Gender Equity in Hemophilia Care

Introduction Despite multiple awareness-raising initiatives, and in contrast with male persons with hemophilia (PwH), a considerable number of carriers and female PwH still go undiagnosed. We here present the findings of a proactive and extensive hemophilia carrier screening project. Methods From May 2021 until April 2023, potential and obligate carriers of hemophilia A (HA) and B (HB) were identified by updating family trees of all PwH regularly followed at the Cliniques universitaires Saint-Luc. Data from previously screened female individuals (bleeding history, coagulation factor assays and testing for the index patient's pathological genetic variant) were collected retrospectively through electronic medical records (EMRs). Female individuals who had not been previously screened or with missing data were provided with a total of 125 invitation letters by related index hemophilia patients. The study protocol received approval of the local ethics committee. Results All 287 male PwH (226 HA, 61 HB) from 228 families (180 HA, 48 HB) were included in the analysis. Seven female PwH (3 HA, 4 HB) were considered as index cases in their families. In total, based on the family trees, there were 900 females, among which 454 were obligate and/or genetically proven carriers of hemophilia and 118 were found to be non-carriers (Figure 1). Mean age at diagnosis was 30.7 years (range 1-82) in families with HA and 30.1 years (range 0.5-93) in families with HB, without significant difference between these two groups (p=0.809) or between families with severe, moderate or mild hemophilia (p=0.535). Genetic testing for pathological variants has been conducted in 133 obligate, 237 potential and 4 sporadic carriers, and remains to be performed in 190 obligate and 328 potential carriers. Eight females were obligate non-carriers. The group of mothers, sisters and daughters of PwH comprised 543 females, of which at least 178 (35.7% of those with known date of birth) were of reproductive age (18-49 years). Seventy-three percent of females in the latter group have had genetic testing. The proportion of female individuals genetically tested did not significantly differ between families of different severities of HA (p=0.132). In families with moderate HB, female individuals were more likely to be tested (genetic status known in 59.4%) than in other HB severity groups (p=0.01), yet this may be attributed to the presence of one large family comprising 6 male persons with moderate HB and 22 female individuals, of whom a large proportion (n=18, 82%) has had genetic testing. Among carriers with known coagulation factor levels (261/454), 42 (23.0%) and 23 (29.5%) carriers of HA and HB, respectively, had a factor level below 40 IU/dL, and 65 (35.5 %) and 32 (41.0%) had a factor level below 50 IU/dL. The proportion of carriers with a factor level below 40 IU/dL did not significantly differ between families with severe, moderate and mild hemophilia (22.6%, 34.7% and 22.7% respectively, p=0.213), neither did mean coagulation factor levels in carriers with a factor deficiency in these groups (26.2, 30.4 and 27.1 IU/dL in families with severe, moderate and mild HA and HB, p=0.344). Carriers with a coagulation factor deficiency of less than 40 IU/dL were screened earlier than other female individuals (mean age at diagnosis 25.8 and 31.8 years, respectively, p=0.034). Conclusions This study represents the first reported attempt to systematically identify all (potential) hemophilia carriers within families of all PwH followed at a single hemophilia treatment center. To date, this initiative has allowed 44% of the entire cohort to be screened, and carrier status to be genetically confirmed in 73% of mothers, sisters and daughters of PwH who are of childbearing age, based on the date of birth documented in their EMR. Although original and promising, these data reflect the major challenges and difficulties in reaching out and providing appropriate screening to all women genetically linked to one or more PwH. The carrier screening efforts that have been initiated in our center, and which should ideally be replicated across the hemophilia community globally, appear as a critical step towards providing equal access to haemophilia diagnosis and care to all potentially affected individuals, regardless of their gender.

Differentiation of Acquired Hemophilia a from Lupus Anticoagulant Using Global Coagulation Assays and Magnesium-Dependent Activated Partial Thromboplastin Time Assay

CONCLUSION Rapid and reliable differentiation between bleeding and thrombotic tendency in cases with prolonged activated partial thromboplastin time (APTT) remains challenging in clinical practice, in particular, distinguishing cases positive for coagulation factor inhibitor or lupus anticoagulant (LA). To differentiate acquired hemophilia A (AHA) from LA using three global coagulation assays: rotational thromboelastometry (ROTEM), thrombin generation assay (TGA), and clot waveform analysis (CWA); also to validate the effectiveness of magnesium-dependent APTT (APTT-Mg) assay. This method uses APTT-Mg and normal APTT using Ca to screen for LA, and APTT-Mg/APTT ratio is smaller in LA. We included patients with prolonged APTT without congenital coagulation factor deficiency who were referred to our department between November 2019 and September 2021. APTT prolongation was defined as >35 s (normal range: 25-35 s) measured routinely with Coagpia APTT-N (Sekisui Medical). The AHA group had a confirmed diagnosis and underwent treatment of AHA. The LA group was divided into LA1 (possible LA) and LA2 (definite LA). LA1 was defined by any of the following conditions: (1) index of circulation anticoagulant ≥15 in the cross-mixing test performed with Coagpia APTT-N; (2) positive dilute Russell's viper venom time (DRVVT) phospholipid neutralization method using Gladipore LA (Medical & Biological Laboratories); (3) positive APTT phospholipid neutralization test using Hemosil SCT (Instrumentation Laboratory); (4) positive for anti-CL antibody; (5) positive for anti-CLβ2GPI antibody; or (6) positive for phosphatidylserine-dependent anti-PT antibody. LA2 was defined by index of circulation anticoagulant >12.4 in the cross-mixing test performed by Thrombocheck APTT-SLA (Sysmex Corporation), or item (2) or (3) above, in accordance with criteria of LA in the International Society of Thrombosis and Haemostasis. LA1 and LA2 did not overlap, because cases that did not meet the conditions of LA2 were treated as LA1, and global coagulation assays were performed in all LA cases. In the global coagulation assays, 12 healthy volunteer samples were tested and regarded as the normal goroup. For the APTT-Mg assay, equal volumes of 25 mmol MgCl 2 and 25 mmol CaCl 2 were used instead of the normally used CaCl 2, and Thrombocheck APTT-SLA was used to measure APTT-Mg, which was then divided by the normal APTT concentration measured with the same APTT reagent. Ten patients in the AHA, 12 in the LA1, and 32 in the LA2 groups were eligible. ROTEM showed that clotting time in the non-activated rotational thromboelastometry assay (NATEM) mode was >3600 s (minimum 1363 s) for AHA, 501 s (IQR: 430-632 s) for LA1, 533 s (IQR: 443-641 s) for LA2, and 500 s (IQR: 429-645 s) for the normal group. TGA showed that peak thrombin concentration was 16 nM (IQR: 13-29 nM) in the AHA group, 242 nM (IQR: 175-273 nM) in the LA1 group, 174 nM (IQR: 129-259 nM) in the LA2 group, and 190 nM (IQR: 125-288 nM) in the normal group. In the CWA, Ad|min1| was 2.1 (IQR: 1.7-3.2) in the AHA group, 8.7 (IQR: 8.0-9.5) in the LA1 group, 6.7 (IQR: 5.8-8.4) in the LA2 group, and 10.3 (IQR: 9.8-10.6) in the normal group. APTT-Mg/APTT was 1.23 (IQR: 1.18-1.29), 1.26 (IQR: 1.23-1.38), and 1.05 (IQR: 0.95-1.20) in the AHA, LA1, and LA2 groups, respectively. ROTEM, TGA, and CWA effectively differentiated between the LA1/2 and AHA groups. Clotting time in non-activated rotational thromboelastometry assay mode (ROTEM) and peak height (TGA) were better at distinguishing high-titer LA cases from LA-positive AHA cases. APTT-Mg/APTT could be suitable for distinguishing AHA from high-titer LA; however, it may not be useful for differentiating AHA from low-titer or suspected LA.

Bleeding phenotype and hemostatic evaluation by thrombin generation in children with Noonan syndrome: A prospective study.

This study aimed to evaluate the bleeding phenotype and to conduct a comprehensive hemostatic evaluation in individuals with Noonan syndrome (NS), a dominantly inherited disorder caused by pathogenic variants in genes associated with the Ras/MAPK signaling pathway. Children with a genetically confirmed diagnosis of NS underwent clinical evaluation, routine laboratory tests, platelet function testing, and thrombin generation (TG) assessment. The study included 24 children. The most frequently reported bleeding symptoms were easy bruising and epistaxis, while bleeding complications were observed in 15% of surgical procedures. Various hemostatic abnormalities were identified, including platelet dysfunction, von Willebrand disease, and clotting factor deficiencies. Abnormal platelet function was observed in 50% of the patients, and significantly lower TG parameters were found compared to controls. However, no significant correlation was observed between bleeding symptoms and TG results. The study suggests that the bleeding diathesis in NS is multifactorial, involving both platelet dysfunction and deficiencies of plasma coagulation factors. The potential role of TG assay as an ancillary tool for predicting bleeding tendencies in individuals with NS undergoing surgery warrants further investigation.

Pathophysiology, diagnosis, and therapy for the management of acquired clotting factor deficiency

Pathophysiology, diagnosis, and therapy for the management of acquired clotting factor deficiency

Utility of the Platelet Function Analyzer (PFA-100) in Patients with Bleeding Disorder of Unknown Cause

Introduction: Bleeding disorder of unknown cause (BDUC) is a diagnosis of exclusion and patients share a similar phenotype with established mild bleeding disorders (MBDs) such as platelet function defects (PFD), mild von Willebrand disease (VWD), or mild coagulation factor deficiencies (CFD). The integration of global assays, such as the Platelet Function Analyzer (PFA)-100, as screening tools for MBDs lacks universal endorsement and is currently not recommended in clinical routine. While the PFA-100 has demonstrated noteworthy sensitivity in the detection of VWD, data regarding its clinical utility in PFD are conflicting and data on PFA-100 in BDUC patients are scarce. Aims: To evaluate the diagnostic utility of PFA-100 in BDUC compared to other MBDs, and to investigate the influence of various parameters on PFA-100 results. Methods: This study included 821 patients enrolled in the Vienna Bleeding Biobank (VIBB), a single-center cohort study on individuals with non-trivial bleeding tendencies who were referred to our tertiary center until December 2022 (EC 604/2009). Patients under anticoagulation or antiplatelet treatment were excluded. PFA-100® assessments (Dade Behring Inc., Newark, Delaware, USA) were conducted on citric acid anticoagulated whole blood samples obtained at study inclusion using two different cartridges: Dade® PFA-100 Collagen/Epinephrine (EPI) and Dade® PFA-100 Collagen/adenosindiphosphat (ADP; Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Results: PFA-100 data of 535 BDUC patients (65%) were compared to those of 79 patients (10%) with VWD, 180 (22%) with PFD, and 27 (3%) with CFD. Clinical characteristics are shown in Table 1. In patients with BDUC, the median (interquartile range) closure time (CT) measured by the PFA-100-EPI assay was 102 seconds (88-117), and 135 seconds (112-163; Table 1) with the PFA-100-ADP assay. The CTs were shorter in BDUC compared to VWD (PFA-100-EPI: p<.001, PFA-100-ADP: <p.001) and PFD (PFA-100-EPI: p<.001, PFA-100-ADP: <p.001), while no significant difference was observed compared to CFD (PFA-100-EPI: p=0.2; PFA-100-ADP: p=0.7). Based on in-house reference ranges, CT was normal in both PFA-100 assays in 180 (34%) of all BDUC patients, while 341 patients (64%) had at least one abnormal CT and 11 patients (2%) had abnormal CTs in both PFA-100 assays. A similar distribution was observed in patients with CFD (no abnormal CT: 33%, 1 abnormal CT: 63%, 2 abnormal CTs: 4%). Statistical analysis revealed a significant difference in the number of pathological CTs between BDUC and VWD (no abnormal CT: 9%, 1 abnormal CT: 58%, 2 abnormal CTs: 33%; p<.001), as well as between BDUC and PFD (no abnormal CT: 24%, 1 abnormal CT: 69%, 2 abnormal CTs: 7%; p=0.003). In respect to bleeding severity, in BDUC patients no correlation was found between CTs in the PFA-100-EPI and PFA-100-ADP assays with the Vicenza bleeding score (PFA-100-EPI Spearman rho (r)=0.06, p=0.06; PFA-100-ADP r=0.02, p=0.6), ISTH-BAT (PFA-100-EPI r= 0.06, p=0.3; PFA-100-ADP r= 0.10, p=0.06), or the number of bleeding manifestations (PFA-100-EPI r=0.01, p=0.81; PFA-100-ADP r=-01, p=0.8). The number of pathological PFA-100 was not associated with specific bleeding symptoms assessed by the Vicenza score in BDUC (data not shown). Using a Tobit regression model, we analyzed the association of clinical and laboratory parameters with prolonged CTs in both PFA-100 assays in BDUC, as shown in Table 2. In multivariable linear regression, reduced hematocrit, lower VWF:Ag/VWF:RCo levels, reduced platelet count and higher fibrinogen levels were associated with longer CTs. Increasing age was additionally associated with a longer CT in PFA-100-ADP. Conclusion: In a minority of BDUC patients CT was prolonged in both PFA-100 (EPI and ADP) assays. PFA-100 was not associated with the bleeding severity or specific symptoms in BDUC. In BDUC patients, lower hematocrit, lower platelet count, lower VWF levels and interestingly higher fibrinogen levels were associated with a prolonged CT. Our descriptive results do not suggest the integration of PFA-100 as screening tool for BDUC patients.

Random X-Chromosome Inactivation in Adult Hemophilia B Carriers Does Not Attenuate Bleeding Risk

Introduction: The X-linked bleeding disorder Hemophilia B (HB) results from deficiency in coagulation Factor IX (FIX). In contrast to Hemophilia A, genotype-phenotype correlations in HB males are complex, as some F9 variants in males are associated with low plasma FIX activity (FIX:C) but milder than expected bleeding phenotypes. At least 75% of FIX is extravascular and not adequately measured from plasma. Hemostasis is likely influenced by this extravascular FIX which is bound reversibly to type IV collagen in the subendothelial basement membrane. We predict that genotype-phenotype correlations in female carriers of F9 mutations may have additional complexity as the mutant FIX can competitively interfere with wild type (WT) FIX for extravascular binding sites. F9 expression in female HB carriers is influenced by X-chromosome inactivation (XCI), a process that silences one X in XX females. XCI results in cells that randomly inactivate either the X carrying the mutant or wild type F9, although skewed XCI (>80:20) is found in ~25% of the general population. XCI skewing in HB carriers can lead to preferential expression of the X carrying the F9 mutation, which in turn influences plasma and extravascular levels of FIX and bleeding tendency. How frequently XCI is skewed in HB carriers, and the role of XCI in modulating FIX levels and bleeding phenotypes, remains poorly understood. We sought to determine whether XCI skewing may further differentiate bleeding risk in HB carriers. Methods: Subjects were enrolled in a prospective, observational cross-sectional study with informed consent. Both the Condensed MCMDM-1 bleeding assessment tool (BAT) and Pictorial Blood-loss Assessment Chart (PBAC) were completed at the time of the visit. F9 mutation analysis confirmed carrier status. FIX:C and FIX antigen ELISA assays were performed on all subjects. XCI skewing was assessed using established assays at the Androgen Receptor (AR) and Fragile X Mental Retardation (FMR1) loci. Results: A total of 15 subjects (11 adults) were confirmed F9 mutation carriers; six missense mutations and one frameshift mutation were identified. Abnormal bleeding is frequent in adult HB carriers, with 5/11 (45%) having BAT scores >4. Heavy menstrual bleeding (HMB) is particularly frequent, with all adult HB carriers reporting PBAC > 100, and 8/11 with BAT HMB subcategory score >1. FIX assessment by activity (mean 56% ± 28) and antigen (mean 0.55 IU/mL ± 0.2) assays are highly correlated (r 2= 0.83, p < 0.0001). Notably, only two adult HB carriers with significant bleeding have FIX levels < 50%, while two HB carriers with FIX levels < 50% have BAT scores < 4, indicating that neither FIX:C nor FIX antigen levels adequately predict bleeding risk in all HB carriers. Indeed, for all 11 adult HB carriers, no significant differences in either FIX:C or FIX antigen were observed between those with BAT scores ≥ 4 and < 4. We asked if XCI explained FIX levels or bleeding phenotypes. Nearly half (5/11) of the adult carriers have moderately skewed XCI (> 70:30); only one is extremely XCI skewed (> 80:20), and of the individuals with skewed XCI, 3/5 have BAT scores ≥ 4. Although random XCI is proposed to attenuate HB carrier bleeding phenotype, 2/6 HB carriers with random XCI also have BAT scores ≥ 4. XCI weakly correlated with FIX antigen (n = 15; r 2= 0.27, p < 0.05) but not FIX:C. When assessing this relationship for only the individuals with F9 c.1025C>T (n = 6), there was significant correlation with FIX:C (R 2= 0.73, p < 0.05) but not with FIX antigen or bleeding phenotype. The lack of correlation between XCI and FIX antigen in individuals with an identical F9 mutation argues for additional influences on intravascular, and likely extravascular, FIX levels and will require further investigation. Conclusions: Bleeding tendency in HB carriers is not rare and does not correlate with plasma FIX:C or FIX antigen. Random XCI is not fully protective against abnormal bleeding. Future studies are needed to better understand bleeding phenotypes in HB carriers that consider XCI, F9 mutation type and influences on circulating levels of WT and mutant FIX.

Longitudinal Patient Analysis Suggests a Positive Correlation of IL-10/IL-6 and FVIII Inhibitor Amount

Hemophilia A (HA) is the most common X-linked bleeding disorder caused by a deficiency in coagulation factor VIII (FVIII). Inhibitor development in up to 30% of HA patients is the most challenging complication in HA treatment and has been associated with several patient-related risk factors such as gene mutation and residual plasma factor VIII concentrations. Treatment related risk factors are under debate. However, HCV infections were recently shown to correlate with low level inhibitor abundance, potentially as a result of chemokine and cytokine regulation. Therefore, the aim of this study was to evaluate a potential connection between proinflammatory cytokine levels and FVIII-specific B cell responses in HA patients without infections, but with other comorbidities that might modulate cytokine levels. Thus, several clinical parameters and concentrations of plasmatic proinflammatory cytokines (IL-1ß, IL-6, IL-8, IL-10, IL-12p70, TNF, IFNa, IFNg, TNFa, MCP-,1 IL-17A, IL-18, IL-23 and IL-33) were quantified from different patient samples. At first, the influence of proinflammatory cytokines on inhibitor development was assessed in a HA patient, who had an intron 22 inversion and was also diagnosed with type 1 diabetes. Prophylactic treatment began at the age of 9 months with 30IU/kg Octanate, and after four days of exposure, the formation of inhibitors was initially detected. Longitudinal cytokine analysis in plasma samples of that patient revealed differential regulation patterns of cytokines. IL-1b, IFNg, TNFa, IL-6, IL-10 and IL-12p70 tended to increase upon inhibitor occurrence, whereas other cytokines remained unchanged (MCP-1, IL-8, IL-17A, IL-18 and IL-23) or decreased (IFNa2 and IL-33). Next, a correlation analysis was performed, assuming that proinflammatory cytokines might stimulate B cells to produce antibodies. This analysis involved all cytokines that were found to be elevated upon inhibitor formation. The comparison of clinical and laboratory data from that patient revealed a strong association of inhibitor levels and IL-10 expression (Pearson r = 0,9391; R squared = 0,8818) as well as with IL-6 (Pearson r = 0,9290; R squared = 0,8630). Furthermore, a modest correlation was observed for IL-1b (Pearson r = 0,5277; R squared = 0,2785), IFNg (Pearson r = 0,3413; R squared = 0,1165), IL-12p70 (Pearson r = 0,3547; R squared = 0,1258), and TNFa (Pearson r = 0,3070; R squared = 0,09422). Other proinflammatory cytokines with a rather stable profile upon inhibitor initiation like MCP-1 (Pearson r = -0,06810; R squared = 0,004638) and IL-18 (Pearson r = 0,2245; R squared = 0,05042) seemed not to be directly correlated with inhibitor occurrence. Finally, we aim to investigate the potential impact of IL-10 and IL-6 on inhibitor production and/or FVIII immune tolerization across various patient groups and conditions. Therefore, we will compare patients with detectable inhibitor titers, those who have successfully eradicated inhibitors, and individuals without inhibitor titers (with normal recovery). Preliminary data suggest a positive correlation between both cytokines and active inhibitor presence. However, to reach a definitive conclusion, the sample size will be/should be increased.

A Single-Center Study of Patients with Isolated Acquired Clotting Factor Deficiency: Clinical Features, Treatments, and Prognosis

Introduction and aim: As a group of rare hemorrhagic disorders, isolatedacquired clotting factor deficiency (ACFD) is mainly caused by the existence of anti-factor antibodies or substances with absorbability of clotting factors. The most widely spread isolated ACFD is the acquired FVIII deficiency known as acquired hemophilia (AHA), and the remaining factors of deficiency are much less prevalent. Isolated ACFD is characterized by bleeding diathesis ranging from mild to life-threatening bleeding events. Their rarity and heterogeneity may lead to difficulty in diagnosis and management. We performed a retrospective study to analyze the characteristics of isolated ACFDs other than AHA, including acquired factor (F) II, FV, FIX, FX, FXI, FXII, FXIII, and VWF deficiencies, to enhance our understanding of these diseases. Methods: All patients with isolated ACFD other than AHA admitted to the Blood Disease Hospital, Chinese Academy of Medical Sciences, from July 1997 to December 2021, were retrospectively enrolled in the present study. The follow-up time started from when the patient was admitted to our center till March 2022. Clinical information, such as demographics, associated diseases, manifestations, laboratory tests, treatments, and prognosis, was collected. Results: A total of 54 patients with isolated ACFD admitted to our center were enrolled in our final analysis, including 28 males and 26 females. Acquired factor V deficiency (AFVD) was the most frequently diagnosed (20/54, 37.0%) isolated ACFD, followed by acquired factor X deficiency (AFXD) (n=16, 29.6%). The median age at diagnosis of all isolated ACFD patients was 55 (4-78) years. However, all four patients with acquired FII deficiency (AFIID) were young women (< 20 years). The median time from presenting bleeding diathesis or being found with an abnormal laboratory test to be diagnosed as isolated ACFD for all patients was 60 (2-2555) days. Underlying conditions were found in 35 (64.8%) patients with isolated ACFD, including malignancies (n=19, 35.2%), autoimmune diseases (n=12, 22.2%), and infections (n=4, 7.4%). Of the 19 patients associated with malignancies, 18 patients were secondary to hematologic malignancies. Plasma cell dyscrasias were the primary etiologies for patients with AFXD, with a proportion of 75.0% (12/16). A total of 44 (81.5%) patients with isolated ACFD experienced at least one bleeding episode. Subcutaneous hemorrhage was the most common bleeding phenotype affecting 61.4% (27/44) of patients. One acquired factor IX deficiency (AFIXD) patient and one AFXIID patient showed venous thromboembolism (VTE). Anti-factor neutralizing antibodies were detected in about half of the patients with isolated ACFD (24/54, 44.4%). Inhibitors were not found in two (10.0%) AFVD patients, one (6.3%) AFXD patient, two (50.0%) acquired factor XI deficiency (AFXID) patients, and five (100.0%) acquired factor XII deficiency (AFXIID) patients (Figure 1). Prothrombin complex concentrate (PCC) was administered to 20 (37.0%) isolated ACFD patients and fresh frozen plasma (FFP) was administered to 33 (61.1%) isolated ACFD patients. Thirty-seven (37/54, 68.5%) isolated ACFD patients underwent immunosuppressive therapy (IST). In addition, chemotherapy was administered to six (6/16, 37.5%) AFXD patients with light chain (AL) amyloidosis and four (4/5, 80%) AFXIID patients with hematologic malignancies to treat primary diseases. Overall, a majority of isolated ACFD patients achieved a high CR rate (≥ 50.0%) other than patients with AFXD, who achieved an extremely low CR rate of 18.2%. The efficiency of IST varied among different types of isolated ACFDs, ranging from 20.0% to 100.0%. The factor levels normalized in one (1/6, 16.7%) AFXD patient and all (4/4, 100.0%) AFXIID patients after receiving chemotherapy. The median time to achieve CR was shorter for AFVD patients compared with the AFXD patients (p < 0.05). Four (4/17, 23.5%) AFVD patients, four (4/11, 36.4%) AFXD patients, and one (1/2, 50.0%) acquired FXIII deficiency (AFXIIID) patient died. Four bleed-related deaths occurred in two AFVD patients, one AFXD patient, and one AFXIIID patient. Conclusions: Isolated ACFD is underestimated, delayed diagnosed, and lacks effective therapy. Therefore, clinicians should raise awareness for recognizing and managing isolated ACFD patients to avoid morbidity and mortality.

Epidemiological profile of hemophilia A in Karbala-Iraq.

Hemophilia is an X-linked congenital bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII) (in hemophilia A) or factor IX (FIX) (in hemophilia B) and is one of the most important hereditary conditions in Iraq. The current study tried to provide a glimpse into the epidemiological and clinical status, as well as complications and treatment used for patients with hemophilia A in Karbala, Iraq. This retrospective research was carried out by reviewing the medical records of 90 male patients diagnosed with hemophilia A registered at the Hereditary Blood Disease Center in Karbala Teaching Hospital for children in Karbala, Iraq. The data was collected from May 10, 2023, until June 15, 2023, and included age, severity, type of bleeding, therapeutic approach, chronic viral infections including hepatitis B, hepatitis C, and human immunodeficiency virus (HIV). The median age of the patients was 15 years (18.86±14.42). In this study, one-third of patients' presentations started in the first year of life. The most common type was severe hemophilia A (factor VIII activity < 1%). Consequently, more than half of the patients were treated with factor VIII concentrate. Four patients had hepatitis C, while HIV was confirmed in one patient. The epidemiological or clinical parameters of hemophilia A patients in Karbala seem similar to those in other cities in Iraq. By considering this preliminary data, further comprehensive studies for patients with hemophilia and associated complications in other provinces or cities in Iraq are highly suggested to provide a clear perspective about the prevalence and epidemiological burdens.

Evaluation of Cardiovascular Status in Hemophilia Patients in Dezful: Echocardiography, Electrocardiography, and Cardiac Risk Factors

Background: Hemophilia is a congenital bleeding disorder with X-linked inheritance that is associated with a deficiency of coagulation factor 8 (hemophilia A) or 9 (hemophilia B). The protective effect of hemophilia is challenging in the prevention of atherothrombotic heart diseases due to the lack of coagulation factors. Objectives: The present study aims to investigate congenital and acquired heart diseases and atherosclerosis risk factors in hemophilia patients. Methods: In the present cross-sectional descriptive study, electrocardiography (ECG) and echocardiography were performed and evaluated on 50 hemophilia patients in Dezful, southwest Iran. Fasting blood sugar, low-density lipoprotein (LDL), cholesterol, plasma high-density lipoprotein (HDL), systemic blood pressure, and body mass index (BMI) were also calculated. The data were analyzed in SPSS software version 21. The results showed 86% with hemophilia A, 12% with hemophilia B, and one patient (2%) with post-pregnancy hemophilia (mean age = 28.12 ± 14.12 years; age range = 6 - 76 years). Results: The most common congenital heart disease was mitral valve prolapse (MVP) with or without mitral regurgitation, consisting of 5 patients (10%); the most common acquired cardiac disorder was diastolic dysfunction grade I, consisting of 5 patients (10 %), and 2 patients (4%) also had systolic pulmonary artery hypertension (pulmonary artery systolic pressure more than 35 mmHg). Bicuspid aortic valve (BAV), atrial septal defect (ASD), and hypertrophic cardiomyopathy (HCM) were observed in one patient each. Ischemic heart disease was observed only in a 76-year-old patient. Examination of the electrocardiogram had no abnormal findings in the rest, except for two patients with HCM and ASD who had ventricular hypertrophy. The most common risk factor was obesity (16%), followed by hypertension (10%) and dyslipidemia (10%), and only 1 (2%) diabetic patient was identified. Patients with risk factors often had more than one risk factor, were over 30 years old, and had a severe form of hemophilia A. Conclusions: In the examination of echocardiography-ECG and risk factors in hemophilia patients, there was no significant abnormality report, while acquired heart diseases and risk factors proportional to age are evident from 30 years of age onwards.