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In many patients referred with significant bleeding phenotype, laboratory testing fails to define any hemostatic abnormalities. Clinical practice with respect to diagnosis and management of this patient cohort poses significant clinical challenges. We recommend that bleeding history in these patients should be objectively assessed using the International Society on Thrombosis and Haemostasis (ISTH) bleeding assessment tool. Patients with increased bleeding assessment tool scores should progress to hemostasis laboratory testing. To diagnose bleeding disorder of unknown cause (BDUC), normal complete blood count, prothrombin time, activated partial thromboplastin time, thrombin time, von Willebrand factor antigen, von Willebrand factor function, coagulation factors VIII, IX, and XI, and platelet light transmission aggregometry should be the minimum laboratory assessment. In some laboratories, additional specialized hemostasis testing may be performed to identify other rare causes of bleeding. We recommend that patients with a significant bleeding phenotype but normal laboratory investigations should be registered with a diagnosis of BDUC in preference to other terminology. Global hemostatic tests and markers of fibrinolysis demonstrate variable abnormalities, and their clinical significance remains uncertain. Targeted genomic sequencing examining candidate hemostatic genes has a low diagnostic yield. Underlying BDUC should be considered in patients with heavy menstrual bleeding since delays in diagnosis often extend to many years and negatively impact quality of life. Treatment options for BDUC patients include tranexamic acid, desmopressin, and platelet transfusions., Competing Interests: Declaration of competing interests No funding was sought for design, data interpretation, or manuscript writing. Individual conflict of interest declaration is listed below. R.I.B.’s institution received grants for research clinical research from Bayer, Takeda, Pfizer, Daiichi Sankyo, CSL Behring, Roche, Amgen, Celgene, Rigel Pharmaceuticals, Ionis Pharmaceuticals, AbbVie, Sanofi, MorphoSys AG, Acerta Pharma, Jansen-Cileg, Bristol-Myers Squibb, Boehringer Ingelheim, and Astra Zeneca. The institution is in receipt of equipment and reagents for research from Werfen and Technoclone. He received honoraria for speaking at educational meetings from Bayer, Bristol Myers Squibb, Cardinal Health, and Astra Zeneca. He provided expert testimony for Sandoz. He has participated on a Data Safety Monitoring Board or Advisory Boards for Roche, Janssen-Celeg, CSL Behring, Astra Zeneca, and George Institute. A.M. has received consultancy fees from Pfizer and CSL Behring and speaker fees from Astra Zeneca. W.T.: speakers fees from Bayer, Pfizer, AstraZeneca, Takeda, Novo Nordisk, CSL Behring, Alexion, Portola, Sanofi, and Sobi. Advisory boards for Pfizer, AstraZeneca, Sanofi, Ablynx, Takeda, Novo Nordisk, Grifols, LFB Biopharmaceuticals, and Daiichi Sankyo. Support to attend educational meetings from CSL Behring and Bayer. M.L. has served on advisory boards for CSL Behring, as a consultant for Sobi, CSL Behring, Takeda, and Band Therapeutics, received speaker fees from Sobi and Takeda, and has research funding from Takeda. R.S.: consultancy fees from Band/Guardian Therapeutics, Star/Vega Therapeutics, Roche, BioMarin Pharmaceuticals, Genentech, LFB, Bayer, Novo Nordisk. Octapharma AG, and Takeda. Her institution received investigator-initiated research grants from Takeda, Octapharma, LFB, and Genentech. P.J. received consultancy fees from Band/Guardian Therapeutics, Star/Vega Therapeutics, Roche, and BioMarin Pharmaceuticals. M.N. received research grants or lecture fees from Roche Diagnostics, Siemens Healthineers, Werfen, Stago, Technoclone, Pentapharm, Buhlmann laboratories, Sysmex, Euroimmun, Bayer Healthcare, and Viatris. J.G. received honoraria, advisory boards, and scientific support from CSL Behring, Takeda, Novartis, SOBI, and Amgen. G.L. received research funding from BioMarin and teaching honoraria from Sobi, Sanofi, Abbvie, Novo Nordisk, Alexion, Novartis, and Takeda. J.O.D. has served on the speaker’s bureau for Baxter, Bayer, Novo Nordisk, Sobi, Boehringer Ingelheim, Leo Pharma, Takeda, and Octapharma; served on the advisory boards of Baxter, Sobi, Bayer, Octapharma CSL Behring, Daiichi Sankyo, Boehringer Ingelheim, Takeda, and Pfizer; and received research grant funding awards from 3M, Baxter, Bayer, Pfizer, Shire, Takeda, and Novo Nordisk. Other authors declared no conflict of interest., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Rare genetic diseases affect millions, and identifying causal DNA variants is essential for patient care. Therefore, it is imperative to estimate the effect of each independent variant and improve their pathogenicity classification. Our study of 140 214 unrelated UK Biobank (UKB) participants found that each of them carries a median of 7 variants previously reported as pathogenic or likely pathogenic. We focused on 967 diagnostic-grade gene (DGG) variants for rare bleeding, thrombotic, and platelet disorders (BTPDs) observed in 12 367 UKB participants. By association analysis, for a subset of these variants, we estimated effect sizes for platelet count and volume, and odds ratios for bleeding and thrombosis. Variants causal of some autosomal recessive platelet disorders revealed phenotypic consequences in carriers. Loss-of-function variants in MPL, which cause chronic amegakaryocytic thrombocytopenia if biallelic, were unexpectedly associated with increased platelet counts in carriers. We also demonstrated that common variants identified by genome-wide association studies (GWAS) for platelet count or thrombosis risk may influence the penetrance of rare variants in BTPD DGGs on their associated hemostasis disorders. Network-propagation analysis applied to an interactome of 18 410 nodes and 571 917 edges showed that GWAS variants with large effect sizes are enriched in DGGs and their first-order interactors. Finally, we illustrate the modifying effect of polygenic scores for platelet count and thrombosis risk on disease severity in participants carrying rare variants in TUBB1 or PROC and PROS1, respectively. Our findings demonstrate the power of association analyses using large population datasets in improving pathogenicity classifications of rare variants., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In patients with severe congenital factor X deficiency, spontaneous intracranial hemorrhage (ICH) is particularly frequent in early childhood. We describe a case of fetal death at 26 weeks due to massive ICH. Gene panel analysis of postmortem samples revealed homozygosity for a pathologic F10 gene variant (c.1210T>C, p.Cys404Arg), which impedes correct folding of the catalytic serine protease domain and, therefore, causes a significant reduction in FX levels. The parents, not consanguineous but of the same ethnic community, were found to be heterozygous for this variant and did not have any personal or family history of abnormal bleeding. To the best of our knowledge, this is the first reported case of severe FX deficiency resulting in ICH diagnosed through postmortem genetic analysis. It illustrates the importance of exploring the etiology of fetal or neonatal ICH, which may impact future pregnancies, and the treatment of a potential coagulopathy in the child., Competing Interests: Declaration of competing interests The authors declare no conflict of interest., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

This study investigates the feasibility of centrifugal spinning for producing fibrous membranes containing pullulan, chitosan, and danshen extract. The danshen extract is composed of 20 wt% salvianolic acid B (SA). Citric acid was added to the mixture as a crosslinking agent to promote its use in the aqueous medium. The influence of the danshen concentration (25 wt% and 33 wt%) on fiber morphology, thermal behavior, and the biochemical effect was analyzed. Developed fiber-based membranes consist of long, continuous, and uniform fibers with a sparse scattering of beads. Fiber diameter analysis shows values ranging from 384 ± 123 nm to 644 ± 141 nm depending on the concentration of danshen. The nanofibers show adequate aqueous stability after crosslinking. Thermal analysis results prove that SA is loaded into nanofibers without compromising their structural integrity. Cell-based results indicate that the developed nanofiber membranes promote cell growth and are not detrimental to fibroblast cells. Anticancer studies reveal a promising inhibition to the proliferation of HCT116 colon cancer cells. The developed systems show potential as innovative systems to be used as a bioactive chemotherapeutic drug that could be placed on the removed tumor site to prevent development of colon cancer microdeposits., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests or personal/financial contributed conflicts that could influence the work imparted in this manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Congenital factor VII (FVII) deficiency is an inherited bleeding disorder, with heterogenous bleeding symptoms. Women with FVII deficiency face hemostatic challenges during menstruation, ovulation, and childbirth. This systematic review evaluated prevalence and management of bleeding symptoms associated with gynecological and obstetric issues in women with FVII deficiency., Methods: Databases (BIOSIS Previews, Current Contents Search, Embase, and MEDLINE) were searched for studies reporting FVII deficiency and gynecological or obstetric issues in women. Articles were screened using Joanna Briggs Institute checklists and relevant data extracted., Results: One hundred fourteen women were identified from 62 publications. Forty-six women had severe deficiency (FVII:C < 5% or <5 IU/dl). Heavy menstrual bleeding (HMB) was the most common bleeding symptom (n = 94; 82%); hospitalization and urgent medical/surgical interventions for acute HMB episodes were required in 16 women (14%). Seven women reported ovarian bleeding (6%); other bleeding symptoms varied. Patient management was inconsistent and included hemostatic and hormonal treatments. Only four women (7%) reporting vaginal bleeding during pregnancy. Postpartum hemorrhage (PPH) occurred following 12/45 deliveries (27%; 5 [42%] requiring blood transfusion) and was not necessarily prevented by prophylaxis (8 women)., Conclusion: Women with congenital FVII deficiency have an increased risk of HMB, ovarian bleeding, and PPH, impacting quality of life. Recognition of a bleeding disorder as the cause is often delayed. Management of bleeding complications is heterogeneous due to lack of treatment guidelines. Harmonizing severity classification of FVII deficiency may help standardize treatment strategies and development of specific guidelines for these women., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

The last decade has seen large increases in the number of patients registered with heritable platelet disorders in national databases of bleeding disorders. Although individually rare, collectively they are a relatively common cause of heritable bleeding. This revolution has come about through the application of high-throughput sequencing strategies and efforts to standardize diagnostic testing. There is renewed interest in established parameters such as platelet volume and utilising simple tools such as blood smears. The diagnostic yield from peripheral blood smears can be improved with new microscopy techniques that could potentially assist in determining which patients need to be referred to tertiary centres for specialist testing. A better understanding of the other clinical features that can accompany abnormalities of platelet number or function, can lead to better clinical management and prevention of serious complications. There are challenges for clinicians who need to be aware of these developments, understand the limitations of new diagnostic techniques and keep abreast of strategies for incorporation into clinical practice. This review discusses some of these approaches, the limitations that clinicians need to be aware of and techniques that may enter clinical use in the future., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The implementation of high-throughput sequencing (HTS) technologies in research and diagnostic laboratories has linked many new genes to rare bleeding, thrombotic, and platelet disorders (BTPD), and revealed multiple genetic variants linked to those disorders, many of them being of uncertain pathogenicity when considering the accepted evidence (variant consequence, frequency in control datasets, number of reported patients, prediction models, and functional assays). The sequencing effort has also resulted in resources for gathering disease-causing variants associated with specific genes, but for BTPD, such well-curated databases exist only for a few genes. On the other hand, submissions by individuals or diagnostic laboratories to the variant database ClinVar are hampered by the lack of a submission process tailored to capture the specific features of hemostatic diseases. As we move toward the implementation of HTS in the diagnosis of BTPD, the Scientific and Standardization Committee for Genetics in Thrombosis and Haemostasis has developed and tested a REDCap-based interface, aimed at the community, to submit curated genetic variants for diagnostic-grade BTPD genes. Here, we describe the use of the interface and the initial submission of 821 variants from 30 different centers covering 14 countries. This open-access variant resource will be shared with the community to improve variant classification and regular bulk data transfer to ClinVar., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

T-type calcium channels regulate neuronal excitability and are important contributors of pain processing. CaV3.2 channels are the major isoform expressed in nonpeptidergic and peptidergic nociceptive neurons and are emerging as promising targets for pain treatment. Numerous studies have shown that CaV3.2 expression and/or activity are significantly increased in spinal dorsal horn and in dorsal root ganglia neurons in different inflammatory and neuropathic pain models. Pharmacological campaigns to inhibit the functional expression of CaV3.2 for treatment of pain have focused on the development of direct channel blockers, but none have produced lead candidates. Targeting the proteins that regulate the trafficking or transcription, and the ones that modify the channels via post-translational modifications are alternative means to regulate expression and function of CaV3.2 channels and hence to develop new drugs to control pain. Here we synthesize data supporting a role for CaV3.2 in numerous pain modalities and then discuss emerging opportunities for the indirect targeting of CaV3.2 channels., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Context: There are no validated Spanish tools to assess symptom burden in pediatric cancer. The Pediatric Memorial Symptom Assessment Scale (Pediatric-MSAS) is an English valid multidimensional and comprehensive instrument., Objectives: To validate Pediatric-MSAS-Spanish (MSAS-Child, MSAS-Teen, and MSAS-Caregiver versions) in patients with cancer treated in two public hospitals in Buenos Aires, Argentina., Methods: Cross-sectional study, classical psychometric theory. We recruited a convenience sample of 148 caregivers of children ≥ two years, 51 young children (seven to 12 years), and 48 adolescents (≥13 years). We assessed feasibility, comprehensibility, internal consistency, and convergent and known-groups validity., Results: Pediatric-MSAS-Spanish was feasible, acceptable, and comprehensible. Reliability of MSAS-total and subscale scores was satisfactory (Cronbach alpha: 0.90, 0.89, 0.71, respectively, for caregiver, teen, and child MSAS-total score). MSAS-total caregiver, teen, and child scores met a priori criteria for convergent validity correlating with Pediatric Quality of Life Inventory total scores (Spearman correlation (r s ) = -0.59, -0.66, and -0.32, respectively) and visual -analogue well-being scores (r s  = -0.63, -0.46, and -0.4, respectively). Caregiver-teen correlation was strong for total (r s  = 0.78) and physical (r s  = 0.85) scores, and moderate for global distress index (r s  = 0.64) and psychological (r s  = 0.45) scores. MSAS-total caregiver-child correlation was moderate (r s  = 0.30) and Kappa analysis showed poor agreement. All MSAS-Caregiver scores and MSAS-Teen total and physical scores differentiated inpatients/outpatients and patients on/off-treatment, while MSAS-Teen psychological and global distress index subscales or MSAS-Child scores did not., Conclusion: Pediatric-MSAS-Spanish is feasible and reliable for assessing symptom burden in children with cancer. Validity of MSAS-Caregiver and MSAS-Teen was largely supported. Further work on MSAS-Child is warranted., (Copyright © 2020 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease., (© 2020 by The American Society of Hematology.)

Molecular diagnostics of inherited platelet disorders (IPD) has been revolutionized by the implementation of high-throughput sequencing (HTS) approaches. A conclusive diagnosis using HTS tests can be obtained quickly and cost-effectively in many, but not all patients. The expanding use of HTS tests has raised concerns regarding complex variant interpretation and the ethical implications of detecting unsolicited findings such as variants in IPD genes RUNX1, ETV6, and ANKRD26, which are associated with increased leukemic risk. This guidance document has been developed and written by a multidisciplinary team of researchers and clinicians, with expertise in hematology, clinical and molecular genetics, and bioethics, alongside a RUNX1 patient advocacy representative. We recommend that for clinical diagnostics, HTS for IPD should use a multigene panel of curated diagnostic-grade genes. Critically, we advise that an HTS test for clinical diagnostics should only be ordered by a clinical expert that is: (a) fully aware of the complexity of genotype-phenotype correlations for IPD; (b) able to discuss these complexities with a patient and family members before the test is initiated; and (c) able to interpret and appropriately communicate the results of a HTS diagnostic report, including the implication of variants of uncertain clinical significance. Each patient should know what an HTS test could mean for his or her clinical management before initiating a test. We hereby propose an exemplified informed consent document that includes information on these ethical concerns and can be used by the community for implementation of HTS of IPD in a clinical diagnostic setting. This paper does not include recommendations for HTS of IPD in a research setting., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Impaired thrombin generation (TG) in patients with acquired coagulopathy, is due to low coagulation factors and thrombocytopenia. The latter is typically treated with platelet transfusions and the former with plasma and occasionally with prothrombin complex concentrates (PCCs). We hypothesized that manipulating the concentrations of coagulation factors might result in restoration of platelet-dependent TG over and above that of simple replacement therapy., Objective: To investigate the influence of PCCs on impaired TG secondary to thrombocytopenia., Methods: TG was evaluated by thrombin generation assay using a thrombocytopenia model in which normal plasma samples with varying platelet counts (20-300 × 10 9 /L) were spiked with PCCs (25%-150% increase in plasma PCC levels)., Results: PCCs and platelets significantly increased TG in a dose-dependent manner in vitro. Two-way repeated measures of analysis of variance showed variance in peak height, area under the curve, time to peak, and velocity. This variance explained, respectively, by levels of PCC was 47, 59, 25 and 53%; by platelet count was 45, 28, 44, and 14%; by the combination was 80, 67, 70, and 62% variance; and a combination with additional interaction was 91, 84, 76, and 68%. TG at a platelet count 40 × 10 9 /L with an approximate 25% increase in PCC concentration was similar to TG at 150 × 10 9 /L. Similarly, patient samples spiked ex vivo with PCCs also showed highly significant improvements in TG., Conclusions: Impaired TG of thrombocytopenia is improved by PCCs, supporting the need for additional studies in complex coagulopathies characterized by mild to moderate thrombocytopenia and abnormal coagulation., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals, Inc on behalf of International Society on Thrombosis and Haemostasis.)

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD., (© 2019 by The American Society of Hematology.)

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans., (© 2019 by The American Society of Hematology.)

Background: Von Willebrand disease (VWD) results from quantitative or qualitative deficiency of von Willebrand factor (VWF) and is treated using VWF-containing concentrates. Several studies have compared the function of various VWF containing concentrates however this has not been performed using shear based assays., Objectives: To compare the platelet-capture potential of 10 commercially available, plasma-derived VWF concentrates under shear conditions., Methods: VWF containing concentrates were assessed for VWF:Ag, VWF:CB, VWF:RCo, factor VIII:C ADAMTS13 content, VWF multimeric profile and glycan content using lectin binding assays. Free-thiol content of each concentrate was investigated using MPB binding assays. An in vitro flow assay was used to determine the ability of each concentrate to mediate platelet capture to collagen., Results: VWF multimeric analysis revealed reduction of high molecular weight (HMW) forms in four of the concentrates (Alphante, Octanate and Haemoctin, and 8Y). The high MW multimer distribution of the remaining six concentrates (Optivate, Wilate, Fandhi, Wilfactin, Haemate P, and Voncento) was similar to the plasma control. Lectin analysis demonstrated that 8Y had increased amount of T-antigen. Although platelet capture after 5 minutes perfusion was similar for all concentrates; Alphante, Octanate, and Haemoctin, demonstrated the lowest levels of platelet capture after 60 seconds of perfusion. Free-thiol content and ADAMTS13 levels varied widely between the concentrates but was not correlated with function., Conclusion: Alphanate, Octanate, and Haemoctin, lacked HMW multimers and had the lowest initial platelet capture levels suggesting that the presence of VWF HMW multimers are required for initial platelet deposition.

Equine cyathostomin parasites are ubiquitous in grazing horses and have been shown to cause severe inflammatory disease in the large intestine of horses. Decades of intensive anthelmintic therapy have led to widespread anthelmintic resistance in cyathostomins across the world. In Cuba, no anthelmintic products are formulated and sold for equine usage and little is known about anthelmintic efficacy of ruminant and swine formulations used. A strongyle fecal egg count reduction test was used to assess the efficacy of a liquid formulation of ivermectin labelled for use in swine, ruminants and carnivores and a pelleted formulation of albendazole labelled for usage in ruminants. Nine farms in the province Camagüey were enrolled in the study comprising 149 horses in total. Albendazole efficacy was reduced on five farms and with the other four farms having no signs of reduced efficacy. Mean farm efficacies were ranging from 41.7% to 100% on the tested farms. Coprocultures found large strongyle larvae present on all farms, but all larvae identified post-treatment were cyathostomins. Ivermectin was found 100% efficacious on all studied farms. This study provided evidence of reduced albendazole efficacy in the study population. Further work is needed to evaluate whether these findings reflect true resistance or if they are due to pharmacokinetic or pharmacodynamic characteristics of the pelleted formulation tested here., (Published by Elsevier B.V.)

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity., (© 2016 by The American Society of Hematology.)

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD., (© 2016 by The American Society of Hematology.)

Background: Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals., Objective: To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome., Methods: Blood samples were taken from three patients with Hermansky-Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5, respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63 and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope., Results: SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence., Conclusions: A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak syndrome and could be used in the diagnosis of patients suspected of other platelet granule disorders., (© 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Background: Factor IX (FIX) is important in the coagulation cascade, being activated to FIXa on cleavage. Defects in the human F9 gene frequently lead to hemophilia B., Objective: To assess 1113 unique F9 mutations corresponding to 3721 patient entries in a new and up-to-date interactive web database alongside the FIXa protein structure., Methods: The mutations database was built using MySQL and structural analyses were based on a homology model for the human FIXa structure based on closely-related crystal structures., Results: Mutations have been found in 336 (73%) out of 461 residues in FIX. There were 812 unique point mutations, 182 deletions, 54 polymorphisms, 39 insertions and 26 others that together comprise a total of 1113 unique variants. The 64 unique mild severity mutations in the mature protein with known circulating protein phenotypes include 15 (23%) quantitative type I mutations and 41 (64%) predominantly qualitative type II mutations. Inhibitors were described in 59 reports (1.6%) corresponding to 25 unique mutations., Conclusion: The interactive database provides insights into mechanisms of hemophilia B. Type II mutations are deduced to disrupt predominantly those structural regions involved with functional interactions. The interactive features of the database will assist in making judgments about patient management., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Retinopathy of prematurity (ROP) is a multifactorial disease, but little is known about its relationships with neonatal nutritional policies. Human, maternal milk is the best possible nutritional option for all premature infants, including those at high risk for severe complications of prematurity, such as ROP., Objective: This is a secondary analysis of data collected during two multicenter RCTs performed consecutively (years 2004 through 2008) by a network of eleven tertiary NICUs in Italy. The two trials aimed at assessing effectiveness of fluconazole prophylaxis (Manzoni et al., N Engl J Med 2007 Jun 14;356(24):2483-95), and of bovine lactoferrin supplementation (Manzoni et al., JAMA 2009 Oct 7;302(13):1421-8), in prevention of invasive fungal infection, and of late-onset sepsis in VLBW infants, respectively. We tested the hypothesis that exclusive feeding with fresh maternal milk may prevent ROP of any stage - as defined by the ETROP study - in VLBW neonates, compared to formula feeding., Methods: We analyzed the database from both trials. Systematic screening for detection of ROP was part of the protocol of both studies. The definition of threshold ROP was as defined by the ETROP study. Univariate analysis was performed to look for significant associations between ROP and several possible associated factors, and among them, the type of milk feeding (maternal milk or formula for preterms). When an association was indicated by p < 0.05, multiple logistic regression was used to determine the factors significantly associated with ROP., Results: In both trials combined, 314 infants received exclusively human maternal milk (group A), and 184 a preterm formula because their mothers were not expected to breastfeed. The clinical, demographical and management characteristics of the neonates did not differ between the two groups, particularly related to the presence of the known risk factors for ROP. Overall, ROP incidence (any stage) was significantly lower in infants fed maternal milk (11 of 314; 3.5%) as compared to formula-fed neonates (29 of 184; 15.8%) (RR 0.14; 95% CI 0.12-0.62; p = 0.004). The same occurred for threshold ROP (1.3% vs. 12.3%, respectively; RR 0.19; 95% CI 0.05-0.69; p = 0.009). At multivariate logistic regression controlling for potentially confounding factors that were significantly associated to ROP (any stage) at univariate analysis (birth weight, gestational age, days on supplemental oxygen, systemic fungal infection, outborn, hyperglycaemia), type of milk feeding retained significance, human maternal milk being protective with p = 0.01., Conclusions: Exclusive human, maternal milk feeding since birth may prevent ROP of any stage in VLBW infants in the NICU., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Investigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wild-type recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecular-weight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that isolated collagen-binding defects are classified as a distinct subtype of von Willebrand disease.