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.

Argatroban to achieve therapeutic anticoagulation in two patients with acute thrombosis and heparin resistance We present two cases where argatroban was successfully used in patients with acute thrombosis requiring anticoagulant treatment where heparin resistance with unfractionated heparin had been encountered. The first case was a woman with abdominal arterial thrombosis, of unknown cause, treated with therapeutic low molecular weight heparin that developed pulmonary embolism despite therapeutic anticoagulation (and had evidence of heparin resistance on anti-Xa monitoring). The second patient had provoked abdominal arterial thrombosis from sepsis and could not attain therapeutic anticoagulation with intravenous unfractionated heparin. In both cases therapeutic anticoagulation was achieved with the use of argatroban, as a temporizing measure to treat the acute thrombotic event. Conventionally, argatroban has been described for use in heparin-induced thrombocytopenia. The use of argatroban is briefly discussed, especially in the context of heparin resistance where anticoagulation can be challenging. Further research using argatroban in heparin resistant patients could be justified. Correspondence to Will Thomas, Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK Tel: +44 01223 257 039; fax: +44 01223 216 891; e-mail: w.thomas1@nhs.net Received 4 June, 2019 Revised 3 September, 2019 Accepted 5 September, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Rare bleeding disorders and advances in gene therapy Rare bleeding disorders usually begin in childhood and manifest as varying degrees of bleeding, which can be life-threatening in severe cases. With the development of gene editing technology, it is expected that hereditary coagulation factor disorders will someday be fundamentally cured by gene therapy. On account of their rarity, comprehension of these diseases is essential for the application of new treatment strategies. We have compiled the features of some newly discovered mutations of prothrombin, factor VII, and factor X in recent years. In addition, this review introduces the advances and obstacles in gene therapy. Correspondence to Zhigang Yang, Department of Hematology and Rheumatology, Affiliated Zhanjiang Central People's Hospital of Guangdong Medical University, Zhanjiang, Guangdong, China. Tel: +86 759-3152128; e-mail: yangzg@gdmu.edu.cn Received 24 June, 2019 Revised 8 August, 2019 Accepted 21 August, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Plasma phenotypes of protein S Lys196Glu and protein C Lys193del variants prevalent among young Japanese women Protein S Tokushima (p.Lys196Glu) and two protein C gene variants (p.Arg189Trp, p.Lys193del) are hereditary thrombophilia in Japanese and Chinese populations, respectively; however, their diagnosis by plasma analyses is difficult because of the type II deficiency phenotype. Three gene variant genotypes were examined in young Japanese women (n = 231). Plasma total protein S activity and total protein S antigen levels were measured using a total protein S assay system, protein C and protein S activities by clot-based methods, and protein C and free protein S antigen levels by latex agglutination methods. protein S Tokushima (p.Lys196Glu) and protein C p.Lys193del variants were prevalent among participants with allele frequencies of 1.08 and 0.86%, respectively, whereas any carrier of protein C p.Arg189Trp variant was not identified. The plasma phenotype of the type II deficiency of protein S Tokushima heterozygotes was demonstrated by decreased total protein S activity with a normal total protein S antigen level; however, the protein C activities of protein C p.Lys193del heterozygotes were within reference intervals, whereas their protein C antigen levels were elevated. We compared the diagnostic accuracy of the total protein S activity/total protein S antigen ratio for identifying protein S Tokushima heterozygotes with that of the clot-based protein S activity/free protein S antigen ratio and found that sensitivity and specificity of 100% each was only achieved by the former. Protein S Tokushima and protein C p.Lys193del are prevalent among young Japanese women, and a plasma analysis using the total protein S assay system is more accurate than the clot-based protein S activity/free protein S antigen ratio for diagnosing protein S Tokushima carriers. Correspondence to Hiroko Tsuda, MD, PhD, Graduate School of Health and Nutrition Sciences, Nakamura Gakuen University, 5-7-1, Befu, Johnan-ku, Fukuoka 814-0198, Japan E-mail: tsuda@nakamura-u.ac.jp Received 26 April, 2019 Revised 5 August, 2019 Accepted 21 August, 2019 This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Severe acquired platelet dysfunction because of primary myelofibrosis with full functional and morphological recovery after allogeneic hematopoietic cell transplantation Primary myelofibrosis (PMF) is a clonal hematopoietic stem cell disorder characterized by fibrosis of the marrow cavity, marked megakaryocyte atypia and progressive cytopenias. Although thrombosis predominates, bleeding is the primary manifestation in up to 20% of patients and may be life-threatening. In this report, we document restoration of megakaryocyte and platelet structure and function in PMF after allogeneic hematopoietic cell transplantation (HCT). A 59-year-old man presented with recurrent episodes of postoperative bleeding preceding a diagnosis of primary myelofibrosis (PMF). Platelet aggregation and secretion studies showed abnormal responses to all agonists tested (epinephrine, ADP, arachidonic acid, U46619, collagen, ristocetin) despite the presence of thrombocytosis. After an allogeneic HCT, platelet morphology and function studies were all normal. The pathophysiology of platelet dysfunction in myeloid neoplasia is not well understood but, as highlighted in our report, restoration of platelet function by HCT supports a clonal process involving an early hematopoietic progenitor cell. Correspondence to Yevgeniy A. Linnik, MD, Department of Pathology & Laboratory Medicine, Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, One Medical Center Drive, Lebanon, NH 03756, USA Tel: +1 603 650 8523; fax: +1 603 650 7214; e-mail: Yevgeniy.Linnik@vumc.org Received 25 March, 2019 Accepted 14 August, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.