Laboratory misdiagnosis of von Willebrand disease in post- menarchal females: A multi- center study

Increased awareness of von Willebrand Disease (VWD) has led to more frequent diagnostic laboratory testing, which insurers often dictate be performed at a facility with off- site laboratory processing, instead of a coagulation facility with onsite processing. Off- site processing is more prone to preanalytical variables causing falsely low levels of von Willebrand Factor (VWF) due to the additional transport required. Our aim was to determine the percentage of discordance between off- site and onsite specimen processing for VWD in this multicenter, retrospective study. We enrolled females aged 12 to 50- years who had off- site specimen processing for VWF assays, and repeat testing performed at a consulting institution with onsite coagulation phlebotomy and processing. A total of 263 females from 17 institutions were included in the analysis. There were 251 subjects with both off- site and onsite VWF antigen (VWF:Ag) processing with 96 (38%) being low off- site and 56 (22%) low onsite; 223 subjects had VWF ristocetin co- factor (VWF:RCo), 122 (55%) were low off- site and 71 (32%) were low onsite. Similarly, 229 subjects had a Factor VIII (FVIII) assay, and 67 (29%) were low off- site with less than half, 29 (13%) confirmed low with onsite processing. Higher proportions of patients demonstrated low VWF:Ag, VWF:RCo, and/or FVIII with off- site processing compared to onsite (McNemarʼs test P- value <.0005, for all assays). These results emphasize the need to decrease delays from sample procurement to processing for VWF assays. The VWF assays should ideally be collected and processed at the same site under the guidance of a hematologist.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156476/2/ajh25869.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156476/1/ajh25869_am.pd

Hemophilia A mice have a shortened lifespan compared to wild type controls.

<p><b>(A)</b> Hemophilia A mice have a median survival of 254 days. n = 138 (total from both backgrounds), p < 0.0001 (log-rank [Mantel-Cox] test) compared to wild type mice, n = 96. <b>(B)</b> C57Bl/6 F8 null mice have a median survival of 236 days. n = 67, p < 0.0001 (log-rank [Mantel-Cox] test) compared to wild type mice, n = 54. <b>(C)</b> B6129 F8 null mice have a median survival of 288 days. n = 71, p < 0.0001 (log-rank [Mantel-Cox] test) compared to wild type mice, n = 39. <b>(D)</b> Factor VIII deficient animals have decreased median lifespan regardless of gender. Median survival of F8 null female mice (361.5 days) was significantly longer than that of F8 null male mice (207 days). p = 0.0009, log-rank [Mantel-Cox] test. <b>(E)</b> Female C57Bl/6 F8 null mice have a median survival of 236 days, n = 29 (p < 0.0001 when compared to wild type controls, n = 31). Male C57Bl/6 F8 null mice have a median survival of 245.5 days, n = 38 (p < 0.0001 when compared to wild type controls, n = 23). <b>(F)</b> Female B6129 F8 null mice have a median survival of 654 days, n = 26 (p = 0.022 when compared to wild type controls, n = 16). Male B6129 F8 null mice have a median survival of 173 days, n = 45 (p < 0.0001 when compared to wild type controls, n = 23).</p

Hemophilia A mice sustain lethal bleeding events.

<p><b>(A</b>) Illustration depicts sites of lethal bleeds which include intracranial, joint/soft tissue, thoracic, and intra-abdominal. <b>(B)</b> and <b>(C)</b> are representative pictures of gross bleeding observed during the study. <b>(B)</b> Mouse discovered dead in cage with swollen joint visibly noticeable (arrow indicates the affected joint). <b>(C)</b> Grossly visible joint/soft tissue bleed noted on necropsy (arrow indicates same joint as in (B)).</p

Successful Management of Blue Rubber Bleb Nevus Syndrome (BRBNS) with Sirolimus

Blue rubber bleb nevus syndrome (BRBNS) is a rare disease with vascular malformations in several systems of the body, most commonly the skin and gastrointestinal tract. Bleeding from the gastrointestinal (GI) tract is a major complication, which may lead to chronic iron deficiency anemia and the need for frequent blood transfusions due to ongoing gastrointestinal blood loss. In this case report, we describe a now 19-year-old female with BRBNS who required six blood transfusions per year and after starting sirolimus is symptom- and transfusion-free

piggyBac-mediated phenotypic correction of factor VIII deficiency

Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB), to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII null mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model

A Hyperactive Transposase Promotes Persistent Gene Transfer of a piggyBac DNA Transposon

Nonviral vector systems are used increasingly in gene targeting and gene transfer applications. The piggyBac transposon represents an alternative integrating vector for in vivo gene transfer. We hypothesized that this system could achieve persistent gene transfer to the liver when administered systemically. We report that a novel hyperactive transposase generated higher transposition efficiency than a codon-optimized transposase in a human liver cell line. Hyperactive transposase-mediated reporter gene expression persisted at levels twice that of codon-optimized transposase in the livers of mice for the 6-month study. Of note, expression persisted in mice following partial hepatectomy, consistent with expression from an integrated transgene. We also used the hyperactive transposase to deliver the human α1-antitrypsin gene and achieved stable expression in serum. To determine the integration pattern of insertions, we performed large-scale mapping in human cells and recovered 60,685 unique hyperactive transposase-mediated insertions. We found that a hyperactive piggyBac transposase conferred an altered pattern of integration from that of insect piggyBac transposase, with a decreased frequency of integration near transcription start sites than previously reported. Our results support that the piggyBac transposon combined with the hyperactive transposase is an efficient integrating vector system for in vitro and in vivo applications

Real- world data of immune tolerance induction using recombinant factor VIII Fc fusion protein in patients with severe haemophilia A with inhibitors at high risk for immune tolerance induction failure: A follow- up retrospective analysis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/166393/1/hae14192_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/166393/2/hae14192.pd