von Willebrand factor alloantibodies in type 3 von Willebrand disease The development of neutralizing antibodies is a rare complication of von Willebrand disease treatment. In major surgical procedures for severe forms of the disease, the recognition of ineffective therapy and alternative treatment protocols are lifesaving. We report the case of a 6-year-old girl with type 3 von Willebrand disease in whom inhibitors were sought due to ineffective haemostasis together with lower than expected von Willebrand factor (VWF) recoveries after a surgical procedure. Replacement therapy first with recombinant factor VIIa and then with high doses of recombinant factor VIII in continuous infusion successfully stopped the bleeding. A high level of anti-VWF antibodies was determined by the immunological method. A frameshift mutation associated with premature termination codon (c.2435delC, p.Pro812ArgfsTer31) was determined in our patient. Although the reports on association of this mutation with inhibitor risk are inconsistent, it represents an evidence-based diagnostic and management practice in recognition of high-risk VWF genotype. Correspondence to Barbara Faganel Kotnik, MD, PhD, Department of Haematology and Oncology, University Medical Centre Ljubljana, University Children's Hospital, Bohoričeva 20, 1000 Ljubljana, Slovenia. Tel: +386 1 522 9215; fax: +386 1 522 4038; e-mail: barbara.faganel@kclj.si Received 16 May, 2019 Revised 22 August, 2019 Accepted 26 September, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Evaluation of biomarkers for monitoring thrombogenic potential of FXaI16L A zymogen-like activated factor X variant (FXaI16L) is being developed for treating acute bleeding conditions. Activated factor V is an essential cofactor to FXaI16L for activating prothrombin to thrombin. Thrombi/emboli formation was observed microscopically in FXaI16L toxicity studies in animals. The objective of this research was to evaluate candidate biomarkers for FXaI16L-induced thrombi/emboli formation to inform safety monitoring and dose-escalation decisions in FXaI16L clinical trials. Effects of intravenous FXaI16L administration on platelets, fibrinogen, activated partial thromboplastin time (aPTT), prothrombin time (PT), D-dimer, tissue factor pathway inhibitor, thrombin : antithrombin complex, antithrombin, and factor V, and protein C (PC) activities were evaluated in mice, rats, and monkeys. Mice had endogenous factor V activity 10× that of monkeys and were overly sensitive to FXaI16L-induced thrombi/emboli formation. In monkeys, decreases in fibrinogen and prolongation in aPTT and PT emerged as potential biomarkers for impending FXaI16L-induced thrombi/emboli formation, based on association of changes with microscopically observable thrombi/emboli (0–97 thrombi/emboli per monkey). PC decreases, measured by a clot-based assay, were also observed. A similar reduction in PC activity, when measured by clot-based assay, was observed in a phase 1 clinical trial. However, an in-vitro experiment with human plasma spiked with increasing concentrations of FXaI16L indicated dose-dependent FXaI16L-induced interference with clot-based assays and no depletion of PC or S by FXaI16L in non-clot-based assays. Nonclinical biomarker studies identified fibrinogen, aPTT and PT as potential biomarkers for monitoring the clinical safety of FXaI16L. Results of clot-based assays with FXaI16L treatment should be interpreted with caution. Correspondence to Michael W. Bolt, PhD, DABT, Drug Safety Research and Development, Pfizer Inc, 1 Portland St, Cambridge, MA 02139, USA Tel: +1 617 674 6408; e-mail: Michael.Bolt@Pfizer.com Received 3 May, 2019 Revised 27 August, 2019 Accepted 26 September, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Congenital afibrinogenemia in a patient with vascular abnormalities and a novel variant: clinical-molecular description and literature review The objective is to report a patient with congenital afibrinogenemia and vascular abnormalities and also review the clinical and molecular issues. The female proband, diagnosed with congenital afibrinogenemia, was admitted at a hospital due to a hemorrhagic shock. Angiotomography revealed ectasias from ascending branch to the abdominal aorta, with multiple calcifications and atheroma. Clinical exome identified a homozygous novel pathogenic variant in FGG gene. In our review the main symptom, at diagnosis, was umbilical cord bleeding and the degree of clinical involvement varied from asymptomatic to severe. The FGA gene was the most affected and possible hot spots were observed. Variants considered as loss of function were the most frequent. The association of vascular abnormalities in a patient with congenital afibrinogenemia alerts for a closer follow-up of vascular issues in these patients. Correspondence to Luiza A. Virmond, Clinical Genetics Department, Universidade Federal de São Paulo, Rua Botucatu, 394, Vila Clementino, São Paulo 04023-061, SP, Brazil E-mail: virmondluiza@gmail.com Received 2 August, 2019 Accepted 15 October, 2019 Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.bloodcoagulation.com). Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (www.bloodcoagulation.com). Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

An obscure case of using apixaban anti-Xa levels in a morbidly obese patient who was nil per os with enterocutaneous fistula Apixaban anti-Xa levels have been introduced to monitor apixaban activity. Presented is a fundamental use of anti-Xa monitoring in questionable absorption in enterocutaneous fistula. A 57-year-old morbidly obese male (150 kg, BMI 42.5) presented to the emergency department with deep venous thromboses and pulmonary embolisms. He also had high-output enterocutaneous fistula managed with an abdominal collection device, total parental nutrition therapy, and nil per os status. He was able to take some oral (PO) medications; however, he reported finding whole capsules in his collection device. He refused enoxaparin injections for venous thromboembolism treatment. The decision was made to load with apixaban therapy with anti-Xa monitoring. After two 10-mg doses, peak apixaban anti-Xa level was 146 ng/ml which fell within on-therapy levels in the AMPLIFY study, prothrombin time 18 s, partial thromboplastin time 35.5 s, international normalized ration 1.5. Monitoring was an important factor in this patient with questionable absorption and further complicated by his morbid obesity which has not been adequately studied in clinical trials. Correspondence to Jennifer L. Cole, PharmD, BCPS, BCCCP, Clinical Pharmacy Specialist, Veterans Healthcare System of the Ozarks, Department of Pharmacy, 1100 North College Avenue, Fayetteville, AR 72703, USA Tel: +1 479 444 4020; e-mail: Jennifer.cole@va.gov Received 24 June, 2019 Revised 15 August, 2019 Accepted 6 October, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Mixing-based inhibitor screening in haemophilia A: challenges in interpretation Inhibitor development in haemophilia A patients is a dreaded complication of factor VIII (FVIII) replacement therapy. With increasing use of FVIII replacement therapy, there is an imperative need for cost-effective and standardized screening. To evaluate the efficacy of mixing-based inhibitor screening (MBIS) in the detection of FVIII inhibitors and to assess the best cut-off values for MBIS. Forty inhibitor positive and 40 inhibitor negative haemophilia A patients, diagnosed by standard criteria, with detailed clinical, haematological and on-demand treatment records were included. MBIS was evaluated in all 80 cases and a classical Bethesda assay and Nijmegen modification of Bethesda assay (NBA) were used as gold standards for inhibitor diagnosis. Classical Bethesda assay missed eight cases, most with low titres, which were confirmed by NBA. A systematic analysis of cut-offs for MBIS using an receiver operating characteristic curve fixed the cut-off at more than 5 s. MBIS detected 36 out of 40 inhibitor positive haemophilia A patients with a sensitivity, specificity, PPV and NPV of 90.0, 95, 94.7, 90.5%, respectively, whereas at the conventional cut-off of more than 10 s, MBIS detected only 25 of 40 cases with a low sensitivity of 62.5%. The likelihood ratio of a positive test was 11. The false-negative haemophilia A patients had low titres from 1.6 to 4.2 BU/ml. MBIS at a cut-off of 5 s can be considered as an effective screening test in low-resource situations. In clinical situations and in cases with clinical evidence of inhibitors we recommend that a direct NBA should be done. Correspondence to Namrata P. Awasthi, Department of Pathology, Dr Ram Manohar Lohia Institute of Medical Sciences, Vibhuti Khand, Gomti Nagar, Lucknow 226010, India E-mail: namratapunit@yahoo.co.in Received 3 October, 2018 Revised 29 August, 2019 Accepted 26 September, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Congenital factor XI deficiency, complete genotype and phenotype of two Iranian families Congenital factor XI (FXI) deficiency is a mild trauma-related bleeding disorder with estimated worldwide prevalence of one per 1 million. The disorder is less frequent in Iran and a few studies have been performed on Iranian patients. In the current study, we assessed molecular, laboratory and clinical features of two Iranian patients with congenital FXI deficiency and their families. Clinical features and demographic data of the patients were assessed by the physician and a staff member trained specifically to deal with patients with bleeding disorders. FXI activity and antigen assays were performed for seven members of the two families and genotyping was performed by direct sequencing of all F11 gene exons and intron-exon boundaries as well as the untranslated regions. Five members of the two families were affected by FXI deficiency. Both patients experienced prolonged epistaxis, whereas other family members were asymptomatic. Two gene defects were observed in the patients and their families. Two disease-causing mutations were c.943G>A (p.Glu315Lys) missense and the four-nucleotide deletion (g.27849-27852del) in exon 15. The gene deletion was observed in homozygote state in the patient with severe FXI deficiency (FXI activity <1%) and heterozygote state in the parent, whereas the c.943G>A mutation was detected in heterozygote state and was accompanied by epistaxis in the patient. FXI deficiency is a mild bleeding disorder that is caused by heterogeneous molecular defects. Correspondence to Majid Safa, Cellular and Molecular Research Center, Department of Hematology and Blood Transfusion, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran E-mail: safa.m@iums.ac.ir Received 9 June, 2019 Revised 27 August, 2019 Accepted 26 September, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

The study of transcriptome sequencing in childhood immune thrombocytopenia No abstract available

Investigating the influence of LCT rs3754689 polymorphism on inhibitor development in Iranian and Afghan patients with severe hemophilia A Development of alloantibodies against factor VIII (FVIII) in patients with severe hemophilia A is the main complication of FVIII replacement therapy. There are many studies indicating several genetic factors associated with inhibitor development. A recent study showed that there is a correlation between the risk of inhibitor development and LCT rs3754689 polymorphism among Italian hemophilia A patients. The aim of this study was to speculate whether LCT rs3754689 polymorphism is correlated to inhibitor development in Afghan and Iranian patients. In addition, we assessed the association of F8 gene mutations and inhibitor development in Iranian patients. This case–control study was conducted on 33 severe hemophilia A patients with inhibitor and 119 samples without inhibitor. Genotyping was performed by Sanger sequencing, inverse and multiplex PCR. According to the obtained data, we found a significant correlation between LCT rs3754689 polymorphism and the risk of inhibitor development in Afghan patients (observed risk, 0.11; 95% confidence interval, 0.01–0.88; P = 0.012). Among Iranian patients, rs3754689 polymorphism showed no significant association with inhibitor development against FVIII (P > 0.05). However, we found a significant correlation between the risk of inhibitor formation and large deletions and nonsense mutations in F8 gene among Iranian patients (observed risk, 7.25; 95% confidence interval, 1.93–27.18; P = 0.003). Lack of association of rs3754689 polymorphism in Iranian population shows the various effects of genetic markers in different populations. More studies in different ethnicities or larger sample sizes are recommended. Correspondence to Sirous Zeinali, Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran; Dr Zeinali's Medical Genetics Laboratory, Kawsar Human Genetics Research Center, Tehran, Iran; Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran E-mail: zeinali@kawsar.ir Received 18 June, 2019 Revised 29 August, 2019 Accepted 26 September, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Thrombin generation test with the calibrated automated thrombogram and anticoagulant activity of Mentha crispa Evaluate the in-vitro effect of Mentha crispa extract on blood coagulation, compare the conventional coagulometric tests with thrombin generation test (TGT), and study the qualitative micromolecular composition of M. crispa. Extract of M. crispa was incubated with plasma and used in the coagulometric tests: prothrombin and activated partial thromboplastin times, fibrinogen, and TGT. A phytochemical prospection was performed to evaluate the chemical composition of this extract. The extract was efficient in prolonging prothrombin time and activated partial thromboplastin time, and reducing fibrinogen levels and TGT parameters, indicating that the extract of M. crispa inhibited the intrinsic and extrinsic pathways of blood coagulation. The results obtained in TGT are in agreement with the results of conventional coagulometric tests and the in-vitro anticoagulant activity of M. crispa suggests that its use by patients using oral anticoagulants deserves caution. Correspondence to Paula M. Leite, Department of Pharmaceutical Products, Faculty of Pharmacy, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, Minas Gerais, Brazil. Tel: +55 31 3409 6936; fax: +55 31 3409 6935; e-mail: paulamleite02@gmail.com; ORCID ID 0000-0002-8499-5791 Received 27 June, 2019 Revised 13 September, 2019 Accepted 26 September, 2019 Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.bloodcoagulation.com). Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Genetic analysis of a pedigree with hereditary coagulation factor XI deficiency To identify potential mutations of F11 gene in a family with hereditary coagulation factor XI (FXI) deficiency and explore the molecular pathogenesis. The FXI activity and FXI antigen were tested with clotting assay and ELISA, respectively. The FXI gene was amplified by PCR with direct sequencing. Three bioinformatics softwares were used to study the conservatism and harm of the mutation. The proband had a prolonged activated partial thromboplastin time (84.2 s), whose FXI activity and FXI antigen were 3.0 and 8.6%. Gene sequencing revealed that the propositus carried a heterozygous nonsense mutation c.738G>A in exon 7 resulting in a p.Trp228stop and deletions mutation c.1325delT in exon 12 resulting in a p.Leu424Cys. Two bioinformatics softwares all were indicated the mutation had affected the function of the protein. The c.738G>A heterozygous nonsense variation and the c.1325delT heterozygous deletion variation are associated with decreased FXI levels in this family, which is the first reported in the world. Correspondence to Lihong Yang, Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China. Tel: +86 57788069594; e-mail: YLH91@163.com Received 10 April, 2019 Revised 31 July, 2019 Accepted 21 August, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.