Natural Anticoagulant Protein Levels in Patients With Beta-Thalassemia Major: A Case-Control Study.

Background: ß-thalassemia is a group of inherited blood disorders that affect the production of ß-globin chains, leading to the reduction or absence of these chains. One of the complications observed in patients with ß-thalassemia major (ß-TM) is thrombosis, especially in those who receive frequent blood transfusions. This may be due to a decrease in the levels of the natural anticoagulants: protein C (PC), total protein S (PS), and antithrombin (AT). Methods: In this case-control study, patients with ß-TM, who had received at least 20 packed cell transfusions during their lifetime, were included. Patients with other underlying diseases like bleeding or thrombotic disorders were excluded. Totally, 118 patients with ß-TM and 120 healthy individuals were included. Results: The mean level of PC and AT was significantly lower in patients with ß-TM (48.2 ± 65.4 and 57.42 ± 13.6, respectively) compared to the control group (97.1 ± 21.46 and 81.79 ± 14.3, respectively), with P value of 0.001 and 0.01, respectively. Although the difference was not statistically significant (P = 0.1), a similar trend was observed for total PS (61.12 ± 21.12 for patients versus 72.2 ± 35.2 for the control group). Of note, the decrease in PC, AT, and total PS levels compared to the control group was 50.36%, 27.5%, and 15.34%, respectively. Conclusions: It seems that ß-TM patients who receive prolonged blood transfusions frequently are at an increased risk of decreased in natural anticoagulants levels and therefore potentially are at risk of thrombosis.

Falsely prolonged prothrombin time test in a patient with erythrocytosis: a case report.

The prothrombin time (PT) test is commonly used to monitor deficiencies in coagulation factors. A prolonged PT may indicate a deficiency of factors II, V, VII, X, and fibrinogen, or the presence of an inhibitor. However, further tests are required to differentiate between a true factor deficiency and the presence of an inhibitor. It is important to note that falsely prolonged PT can lead to misdiagnosis and inappropriate clinical intervention that can have life-threatening consequences. A 19-year-old woman with elevated hematocrit levels and prolonged PT was diagnosed with secondary erythrocytosis due to cyanotic congenital heart disease with ventricular septal defect (VSD). However, further investigation revealed that the prolonged PT result was false. Excess citrate in the blood sample, caused by polycythemia, led to this misleading outcome, resulting in unnecessary and potentially harmful treatment. This incident emphasizes the importance of laboratory personnel and clinicians being aware of the test's limitations. Not only should specialists in thrombosis and hemostasis possess this knowledge, but it is also pertinent for general laboratory staff, as well as laboratory directors and specialists. The significance of accurate laboratory testing for the proper diagnosis and treatment of patients is highlighted in this case.

Interleukin 10, but not tumor necrosis factor-alpha, gene variations are associated with factor VII inhibitor development.

OBJECTIVE: Development of alloantibodies against coagulation factor VII (FVII) is the main therapeutic challenge in severe congenital FVII deficiency. About 7% of patients with severe congenital FVII deficiency develop an inhibitor against FVII. In this research, the relationship between interleukin (IL)-10 and tumor necrosis factor-alpha (TNF)-α gene variants and inhibitor development was evaluated for a group of Iranian patients with severe congenital factor VII deficiency. METHODS: Patients with FVII deficiency were divided into 2 groups: 6 cases and 15 controls. Genotyping was performed using the amplification-refractory mutation system polymerase chain reaction. RESULTS: We found that IL-10 rs1800896 A>G gene variant is associated with the risk of FVII inhibitor development (OR = 0.077, 95% CI = 0.016-0.380, P = .001), whereas the TNFα-rs1800629G>A variant has no relation with inhibitor development in severe FVII deficiency. CONCLUSION: The results show that the IL-10 rs1800896 A>G variant increases the risk of developing an inhibitor in patients with severe congenital FVII deficiency.

Novel Insights into Heterozygous Factor XIII Deficiency.

The prevalence and clinical significance of heterozygous factor XIII (FXIII) deficiency has long been debated, with controversial reports emerging since 1988. In the absence of large epidemiologic studies, but based on a few studies, a prevalence of 1 per 1,000 to 5,000 is estimated. In southeastern Iran, a hotspot area for the disorder, a study of more than 3,500 individuals found an incidence of 3.5%. Between 1988 and 2023, a total of 308 individuals were found with heterozygous FXIII deficiency, of which molecular, laboratory, and clinical presentations were available for 207 individuals. A total of 49 variants were found in the F13A gene, most of which were missense (61.2%), followed by nonsense (12.2%) and small deletions (12.2%), most occurring in the catalytic domain (52.1%) of the FXIII-A protein and most frequently in exon 4 (17%) of the F13A gene. This pattern is relatively similar to homozygous (severe) FXIII deficiency. In general, heterozygous FXIII deficiency is an asymptomatic condition without spontaneous bleeding tendency, but it can lead to hemorrhagic complications in hemostatic challenges such as trauma, surgery, childbirth, and pregnancy. Postoperative bleeding, postpartum hemorrhage, and miscarriage are the most common clinical manifestations, while impaired wound healing has been rarely reported. Although some of these clinical manifestations can also be observed in the general population, they are more common in heterozygous FXIII deficiency. While studies of heterozygous FXIII deficiency conducted over the past 35 years have shed light on some of the ambiguities of this condition, further studies on a large number of heterozygotes are needed to answer the major questions related to heterozygous FXIII deficiency.

The History of Factor XIII Deficiency.

Despite the early discovery of factor XIII (FXIII) in 1944, the diagnosis of FXIII deficiency was not made until 1960, after all the other coagulation factor deficiencies, most likely due to the normality of routine coagulation testing in FXIII deficiency. Although the first case was detected by the clot solubility test and this test has long since been used to detect FXIII deficiency, the test is no longer recommended by experts. Over the past 60 years, knowledge about FXIII deficiency has expanded considerably, between 1992, when the first variant was identified, and 2022, 197 mutations have been reported. Almost all missense mutations have a similar effect on FXIII, leading to instability and faster degradation of mutant FXIII protein. Therapeutic options have evolved from historical fresh frozen plasma (FFP), old plasma, whole blood, and cryoprecipitate, to plasma-derived and recombinant FXIII concentrates, respectively available since 1993 and 2012. These concentrate products were respectively approved by the Food and Drug Administration in 2011 and 2013. This historical review covers various aspects of FXIII related disorders, including the discovery of the FXIII, associated disorders, molecular basis, diagnosis, and treatment of FXIII deficiency.

International Council for Standardization in Haematology Guidance for New Lot Verification of Coagulation Reagents, Calibrators, and Controls.

The clinical laboratory uses commercial products with limited shelf life or certain expiry dates requiring frequent lot changes. Prior to implementation for clinical use, laboratories should determine the performance of the new reagent lot to ensure that there is no significant shift in reagent performance or reporting of patient data. This guideline has been written on behalf of the International Council for Standardization in Haematology (ICSH) to provide the framework and provisional guidance for clinical laboratories for evaluating and verifying the performance of new lot reagents used for coagulation testing. These ICSH Working Party consensus recommendations are based on good laboratory practice, regulatory recommendations, evidence emerged from scientific publications, and expert opinion and are meant to supplement regional standards, regulations, or requirements.

COVID-19 vaccine-induced immune thrombotic thrombocytopenia: pathophysiology and diagnosis.

Coronavirus disease-19 (COVID-19) vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious clinical condition with high mortality rate in apparently healthy individuals without noticeable risk factors. VITT typically arises due to the administration of vaccines that possess recombinant adenoviral vectors, including ChAdOx1 nCov-19 (AstraZeneca) and Ad26 COV2.S (Johnson & Johnson/Janssen). Thrombosis frequently occurs at atypical sites, such as the cerebral or splanchnic circulations, in this particular pathological state. Similar to heparin-induced thrombotic thrombocytopenia (HITT), it seems that the cause of VITT is the misdirection of anti-platelet factor 4 antibodies (anti-PF4 Abs), an ancient antimicrobial mechanism. Anti-PF4 Abs in patients with VITT activates the coagulation system, leading to thrombosis. This process occurs through the stimulation of platelets (Plts) and neutrophils and subsequently release of neutrophil extracellular traps (NETs). Due to the potentially fatal consequences of VITT, early diagnosis is mandatory. In addition to thrombocytopenia, thrombosis, and the presence of anti-PF4 Abs, the day of symptoms onset and the elevation of D-dimer are also required for definitive diagnosis of VITT. The absence of one or more criteria can result in the exclusion of definitive VITT and lead to the diagnosis of probable, possible, or unlikely VITT.

Fibrinogen Aα gene genotyping in patients with inherited afibrinogenemia deficiency; a novel mutation in Iranian afibrinogenemia patients.

BACKGROUND: Congenital fibrinogen deficiencies (CFD) are a group of rare bleeding disorders (RBD). Afibrinogenemia as a subclass of these disorders would occurs as a result of mutations in fibrinogen gene. Here in, the sequences of Aα chain of fibrinogen (FGA) in patients with inherited afibrinogenemia disorder in south-eastern of Iran were analysed. METHODS: The FGA gene exons were amplified using PCR method and the DNA sequences were analysed to study the mutations in Aα chain of Fibrinogen. RESULTS: Results showed that there was no large deletion in FGA gene. Although a frame shift mutation: c.196_197insT p.Ser66PhefsX10 in a patient and a novel mutation of IVS2-1G>A in two other patients were detected which were different from those detected in European population. CONCLUSION: Different mutations are responsible of afibrinogenemia deficiency which requires more relevant studies for confirmation. The type and distribution of mutations in fibrinogen gene in Iranian patients is significantly different with reported mutations in European patients.

Congenital Bleeding Disorders and COVID-19-A Systematic Literature Review.

Hypercoagulability is a prominent feature of coronavirus disease 2019 (COVID-19) and can lead to fatal consequences. Although the impact of COVID-19 on several disorders is well-established, its effect on congenital bleeding disorders (CBDs) is not well-documented. To address this ambiguity, a systematic review was conducted on the available studies to determine the impact of COVID-19 and vaccination aimed to prevent COVID-19 on patients with CBDs. We performed a systematic literature review using relevant keywords and followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocol. We conducted our search on the PubMed, Scopus, and Web of Science databases until July 2023. Out of 31 included studies, 12 case series covering 770 patients with CBD and COVID-19 were further analyzed. The majority of the patients had hemophilia A (n = 352, ∼46%) or hemophilia B (n = 74, ∼10%), while the remaining patients had von Willebrand disease (n = 43, 5.6%) or rare bleeding disorders (n = 27, 3.5%). A total of 25 deaths (3.2%) and 22 intensive care unit admissions (2.8%) were recorded. Bleeding complications were reported in the majority of the 12 case series (n = 7, 58.3%) and in most of the case reports (n = 8, ∼57%), while thrombotic complications were only reported in two studies (16.6%). The mortality rate ranged from 0% in five studies (41.6%) to 5.7% and the rate of hospitalization ranged from 0 to 40%. Bleeding complications were reported in a range of 0 to 81%, while the thrombotic complication rate in one study was 6.9%. The mortality rate varied from 0 to 5.7%, and the hospitalization rate ranged from 0 to 40%. Bleeding complications were reported in a range of 0 to 81%, while the rate of thrombotic complications in one study was 6.9%. Vaccination was reported in five case series, which included 821 patients with CBDs with the majority having hemophilia A (n = 479; 67.2%) and hemophilia B (n = 85; ∼12%). The most frequently reported side effects were myalgia (6.5%), flu-like symptoms (4.8%), fever (4.7%), and headache (4%). COVID-19 in patients with CBDs appears to provoke thrombotic complications and bleeding events more frequently, as well as a higher rate of hospitalization, which may be partially due to the increased risk of bleeding events. Although it seems that patients with CBD have lower mortality rates, further studies are necessary to fully understand this, especially considering comorbidities and low number of available studies.

Laboratory Diagnosis of Activated Protein C Resistance and Factor V Leiden.

The factor V Leiden (FVL) polymorphism is known as the most common inherited risk factor for venous thrombosis. In turn, FVL is the leading cause of an activated protein C resistance (APCR) phenotype, in which the addition of exogenous activated protein C to plasma does not result in the expected anticoagulant effect. In the routine laboratory approach to the formal diagnosis of FVL, an initial positive screening plasma-based method for APCR is often performed, and only if needed, this is followed by a confirmatory DNA-based assay for FVL. Multiple methods with accepted sensitivity and specificity for determining an APCR/FVL phenotype are commonly categorized into two separate groups: (1) screening plasma-based assays, including qualitative functional clot-based assays, for APCR, and (2) confirmatory DNA-based molecular assays, entailing several tests and platforms, including polymerase chain reaction-based and non-PCR-based techniques, for FVL. This review will describe the methodological aspects of each laboratory test and prepare suggestions on the indication of APCR and FVL testing and method selection.

Status of major hemostatic components in the setting of COVID-19: the effect on endothelium, platelets, coagulation factors, fibrinolytic system, and complement.

The coagulation, fibrinolytic, anticoagulation, and complement systems are in delicate balance with the vessel wall endothelium ensuring appropriate hemostasis. Coagulopathy in coronavirus disease 2019 (COVID-19) is not a simple disorder of one hemostatic component but a complicated process affecting most of the hemostasis system. COVID-19 disturbs the balance between the procoagulant systems and the regulatory mechanisms. Here, we investigate the effect of COVID-19 on key hemostatic components, including platelets, endothelial cells, coagulation factors, fibrinolytic system, anticoagulant protein system, and complement system, to improve our understanding of the pathophysiological processes underlying COVID-19 coagulopathy based on evidence.

Health-related quality of life in persons with haemophilia in Afghanistan.

INTRODUCTION: Haemophilia is the most common severe congenital bleeding disorder and can significantly influence patients' quality of life. The health-related quality of life (HRQoL) is a multi-dimensional concept that assess effect of different aspects of health status, including physical, mental, and social domains. Identification of the factors affecting the HRQoL of Persons with Haemophilia (PWH) can guide health care system to better management of patients. AIM: The aim of the present study is to evaluate HRQoL in PWH in Afghanistan. METHODS: This cross-sectional study was conducted on 100 PWH in Kabul City, Afghanistan. Data were collected using 36-Item-Short-Form Health Survey (SF-36) questionnaire and analysed using correlation coefficients and regression analysis. RESULTS: The mean scores for the SF-36 questionnaire 8 domains range from 33 ± 38.3 to 58.15 ± 20.5. The highest mean value belongs to physical function (PF) (58.15), whereas the lowest is related to restriction of activities due to emotional problems (RE) (33.00). A significant association (p < .005) was observed between all domains of SF-36 and patients' age except for PF (p = .055) and general health (GH) (p = .75). A significant association was also observed between all HRQoL domains and the severity of haemophilia (p < .001). The severity of haemophilia was the significant predictor for Physical Component Summary (PCS) and Mental Component Summary (MCS) (p < .001). CONCLUSION: Due to the reduced HRQoL in Afghan PWH, special attention by health care system should be paid to improve patients' quality of life.

Psychological complications among patients with congenital bleeding disorders.

Congenital bleeding disorders (CBDs), including inherited platelet function disorders and rare and common bleeding disorders, are a heterogeneous group of bleeding disorders with a wide range of clinical presentations, including psychological complications. Due to the chronic nature of CBDs, psychological complications are relatively common in these patients, which can affect treatment adherence, quality of life, and even the frequency of bleeding episodes. Chronic pain causes many psychological problems in CBDs, disrupting their social interactions, affecting all aspects of their lives, including their emotional functioning and behavior, and eventually leading to social exclusion. About one-third of patients with severe hemophilia A suffer from anxiety, 64% from depression, and 60% from other minor psychological complications such as withdrawal/depression, anxiety/depression, attention problems, and emotional problems. Anxiety, depression, and uncontrolled pain interfere with treatment adherence. For this reason, psychological interventions are needed in people with CBDs. Psychological interventions increase quality of life and treatment adherence. Therefore, early recognition of psychological complications in CBD patients may increase treatment adherence, leading to a reduction in bleeding episodes and thus an improvement in quality of life.

Factor VII Padua in Iran: clinical and laboratory findings of three unrelated patients.

The congenital factor VII (FVII) deficiency with an estimated incidence of one per 300 000 is the most common rare congenital bleeding disorder. The heterogeneous clinical pictures, including asymptomatic to life-threatening manifestations, are seen in patients with FVII deficiency. A variety of gene variants throughout the FVII ( F7 ) gene have been reported so far. In this setting, very rare FVII Padua polymorphism provokes an interesting condition in which results of prothrombin time and FVII activity are different based on the thromboplastin sources used in these tests. The current study aimed to report the phenotype and genotyping of patients with Padua variant. During the workup of the laboratory for FVII deficiency for diagnosis of FVII Padua, all patients with FVII deficiency who had prolonged prothrombin time, normal activated partial thromboplastin time, and variable FVII activity results using different sources of thromboplastin were included. Demographic data and clinical findings were recorded. For the molecular study, the F7 gene sequencing was performed using the Sanger sequencing technique. Five patients with FVII Padua and a history of mild-to-moderate bleeding, including easy bruising, epistaxis, gingivorrhagia, and bleeding after surgical challenges (including dental extraction and tonsillectomy), were detected during the study. DNA sequencing revealed a heterozygote CGG to CAG (Arg364Gln) variant in exon 9 at nucleotide position 1091, consistent with the genetic variant of FVII Padua. Timely diagnosis of FVII Padua is vital to avoid unnecessary exposure of patients to replacement therapy.

Molecular basis of rare congenital bleeding disorders.

Rare bleeding disorders (RBDs), including factor (F) I, FII, FV, FVII, combined FV and FVIII (CF5F8), FXI, FXIII and vitamin-K dependent coagulation factors (VKCF) deficiencies, are a heterogeneous group of hemorrhagic disorder with a variable bleeding tendency. RBDs are due to mutation in underlying coagulation factors genes, except for CF5F8 and VKCF deficiencies. FVII deficiency is the most common RBD with >330 variants in the F7 gene, while only 63 variants have been identified in the F2 gene. Most detected variants in the affected genes are missense (>50% of all RBDs), while large deletions are the rarest, having been reported in FVII, FX, FXI and FXIII deficiencies. Most were located in the catalytic and activated domains of FXI, FX, FXIII and prothrombin deficiencies. Understanding the proper molecular basis of RBDs not only can help achieve a timely and cost-effective diagnosis, but also can help to phenotype properties of the disorders.

Congenital Bleeding Disorders and COVID-19 - An Emphasis on the Role of Thrombosis as One of the Main Causes of Morbidity and Mortality in COVID-19.

A turbulent coagulation system is a prominent feature of Coronavirus Disease 2019 (COVID-19), with venous thromboembolism (VTE) a leading cause of death. Our hypothesis is that patients with inherited hypocoagulability, like congenital bleeding disorders (CBD), enjoy a protective effect against COVID-19-induced hypercoagulability and related fatal consequences. Our primary and follow-up observations revealed this effect, at least among patients with moderate to severe congenital bleeding disorders, particularly coagulation factor deficiencies. Theoretically, patients with inherited hypocoagulobility have only a potential protective effect against COVID-19-related hypercoagulability. Yet the lower rate of morbidity and mortality in patients with CBDs suggests that hypercoagulability and thrombotic events are the main cause of death in COVID-19. Therefore, appropriate and timely administration of anticoagulants could significantly decrease the rate of morbidity and mortality in COVID-19.

The most common disease-causing mutation of factor XIII deficiency is corrected by CRISPR/CAS9 gene editing system.

Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.

Risk and Management of Intracerebral Hemorrhage in Patients with Bleeding Disorders.

Intracerebral hemorrhage (ICH) is the most dreaded complication, and the main cause of death, in patients with congenital bleeding disorders. ICH can occur in all congenital bleeding disorders, ranging from mild, like some platelet function disorders, to severe disorders such as hemophilia A, which can cause catastrophic hemorrhage. While extremely rare in mild bleeding disorders, ICH is common in severe coagulation factor (F) XIII deficiency. ICH can be spontaneous or trauma-related. Spontaneous ICH occurs more often in adults, while trauma-related ICH is more prevalent in children. Risk factors that can affect the occurrence of ICH include the type of bleeding disorder and its severity, genotype and genetic polymorphisms, type of delivery, and sports and other activities. Patients with hemophilia A; afibrinogenemia; FXIII, FX, and FVII deficiencies; and type 3 von Willebrand disease are more susceptible than those with mild platelet function disorders, FV, FXI, combined FV-FVIII deficiencies, and type 1 von Willebrand disease. Generally, the more severe the disorder, the more likely the occurrence of ICH. Contact sports and activities can provoke ICH, while safe and noncontact sports present more benefit than danger. An important risk factor is stressful delivery, whether it is prolonged or by vacuum extraction. These should be avoided in patients with congenital bleeding disorders. Familiarity with all risk factors of ICH can help prevent occurrence of this diathesis and reduce related morbidity and mortality.

Noninvasive prenatal diagnosis of congenital factor XIII deficiency in Iran.

Congenital factor (F) XIII deficiency is a rare coagulation factor deficiency that is inherited in an autosomal recessive manner. FXIII deficiency presents various clinical manifestations, such as intracranial hemorrhage (ICH), which is the most common cause of morbidity and mortality. As ICH can occur in the neonatal period, prenatal diagnosis (PND) is an effective way to reduce neonatal ICH and its associated fatal consequences. In this study, we investigated a noninvasive prenatal diagnosis (NIPD) method, cell-free fetal DNA (cffDNA), for PND in FXIII deficiency. This study was conducted on seven pregnant women in the first trimester. After extraction of cffDNA from maternal plasma, PCR-restriction fragment length polymorphism (PCR-RFLP) was performed to find the underlying F13A gene mutations previously identified in the family members. PCR-RFLP was also performed on postnatal DNA samples. Sanger sequencing was performed to confirm the results. Four cases were heterozygous for F13A gene mutations, whereas three were unaffected. PCR- RFLP results for cffDNA and postnatal DNA samples were identical, and Sanger sequencing confirmed the results. cffDNA is a noninvasive and effective method for PND in congenital FXIII deficiency.