A novel diagnostic algorithm for heparin-induced thrombocytopenia.

INTRODUCTION: While diagnostic algorithm using PF4-heparin enzyme-linked immunosorbent assay (ELISA) optical density (OD), and heparin neutralization assay (HNA), or 4T score have been proposed to replace serotonin-release assay (SRA) for heparin-induced thrombocytopenia (HIT), their performance against SRA is unclear. In this study, we proposed and validated the performance of a new algorithm combining PF4-heparin ELISA optical density (OD), HNA and 4T score against SRA for HIT diagnosis. METHODS: Heparin neutralization assays were performed on specimens submitted for HIT testing with positive PF4-heparin ELISA from December 2015 to September 2017, which were separated into a "training" and a "validation" data set. 4T scores were calculated for ELISA positive cases. RESULTS: A total of 123 consecutive unique patient samples had positive PF4-heparin ELISA with also HNA data, SRA data, and 4T scores available. Compared to SRA, a "laboratory criteria" (ELISA OD ≥ 1.4 and HNA ≥ 70%) had a sensitivity of 88% (14/16) and specificity of 91% (42/46), and a "combined criteria" (4T score = 8, or 4T score = 6 or 7 and ELISA OD ≥ 1.0, or 4T score = 4 or 5 and ELISA OD ≥ 2.0) had a sensitivity of 75% (12/16) and specificity of 98% (45/46) in the training data set. Laboratory and combined criteria had 90% (56/62) concordance rate. Importantly, for these concordant cases, the diagnostic specificity is 100% (46/46). Based on the data, a novel diagnostic algorithm combining these 2 criteria was proposed and validated prospectively. CONCLUSION: A novel algorithm has high diagnostic accuracy and potentially could eliminate the need for SRA testing in at least 90% patients with suspected HIT.

First report on the safety and efficacy of an extended half-life glycoPEGylated recombinant FVIII for major surgery in severe haemophilia A.

BACKGROUND: N8-GP (turoctocog alfa pegol) is an extended half-life glycoPEGylated recombinant factor VIII (FVIII) product developed for the prevention and treatment of bleeds in haemophilia A patients. AIM: This is a planned interim analysis of pathfinder™3, an international, open-label, Phase 3 trial evaluating the efficacy and safety (including immunogenicity) of N8-GP administered before, during and after major surgery in severe haemophilia A patients aged ≥12 years. METHODS: Sixteen patients who underwent 18 major surgical procedures (including synovectomy, joint replacement and ankle arthrodesis) were included here. Postoperative assessments were conducted daily for days 1-6, and once for days 7-14. Primary endpoint was N8-GP haemostatic efficacy, assessed after completion of surgery using a four-point scale ('excellent', 'good', 'moderate', 'none'). RESULTS: Haemostasis was successful (rated 'excellent' or 'good') on completion of surgery in 17 (94.4%) procedures and rated as 'moderate' (5.6%) for one surgery in a patient with multiple comorbidities who needed an intraoperative N8-GP dose (20.7 IU kg-1 ). In the postoperative period, three bleeds occurred (one during days 1-6; two during days 7-14); all were successfully treated with N8-GP. Mean N8-GP consumption on day of surgery was 80.0 IU kg-1 ; patients received a mean of 1.7 doses (median: 2, range: 1-3). No safety concerns were identified. CONCLUSION: The data showed that N8-GP was effective and well tolerated for the prevention and treatment of bleeds during major surgery; such FVIII products with extended half-lives may modify current treatment schedules, enabling fewer infusions and earlier patient discharge.

Retrospective cohort study comparing activated partial thromboplastin time versus anti-factor Xa activity nomograms for therapeutic unfractionated heparin monitoring in pediatrics.

BACKGROUND: Unfractionated heparin (UFH) is widely used to treat thromboembolic disease, but monitoring in children is challenging. Both activated partial thromboplastin time (aPTT) and anti-factor Xa activity (anti-Xa) are utilized, but a comparison of dosing nomograms has not been reported in pediatrics. OBJECTIVE: To compare the performance of aPTT and anti-Xa for UFH monitoring in pediatric patients. DESIGN/METHODS: A retrospective cohort study was conducted in patients ≤ 21 years old treated with UFH at Johns Hopkins Hospital from January 2009 to May 2011. For monitoring, an aPTT nomogram was used for the initial 15 months, and an anti-Xa nomogram was used for the subsequent 12 months. Clinical characteristics, laboratory data and outcomes were analyzed. RESULTS: Thirty-four patients were monitored with aPTT and 26 patients with anti-Xa. There was no significant difference in median time to therapeutic range (11.6 h aPTT, 95%CI = 6.0-17.0; 9.9 h anti-Xa, 95%CI = 7.3-20.7) or per cent of patients achieving therapeutic measurements at 24 (79% aPTT, 95%CI = 62-91; 73% anti-Xa, 95%CI = 52-88) and 48 h (88% aPTT, 95%CI = 73-97; 96% anti-Xa, 95%CI = 80-100). However, anti-Xa measurements were more frequently therapeutic than aPTT (74% [95%CI = 69-78] vs. 54% [95%CI = 50-59]). Variance between anti-Xa and aPTT measurements was high (R(2)  = 0.236). No significant difference was seen in bleeding incidence (9% aPTT, 95%CI = 2-24; 15% anti-Xa, 95%CI = 4-35). CONCLUSION: The time to achieve therapeutic measures and bleeding outcomes were not significantly different between anti-Xa and aPTT nomograms. However, a small study size limits the power to detect clinically relevant differences. The results warrant larger prospective studies of UFH monitoring in children with thromboembolic disease.

Female factor IX deficiency due to maternally inherited X-inactivation.

X-chromosome inactivation is normally a random event that is regulated by the X chromosome itself. Rarely, females are affected by X-linked disorders from extremely skewed X-chromosome inactivation. Here, we report a family with hemophilia B with female expression through inherited X skewing that appears to be independent of either X chromosome. This finding suggests the possibility of a dominant autosomal contribution to inherited skewed X inactivation.

Linkage of M-CSF signaling to Mitf, TFE3, and the osteoclast defect in Mitf(mi/mi) mice.

Osteoclasts are multinucleated hematopoietic cells essential for bone resorption. Macrophage colony-stimulating factor (M-CSF) is critical for osteoclast development and function, although its nuclear targets in osteoclasts are largely unknown. Mitf and TFE3 are two closely related helix-loop-helix (HLH) transcription factors previously implicated in osteoclast development and function. We demonstrate that cultured Mitf(mi/mi) osteoclasts are immature, mononuclear, express low levels of TRAP, and fail to mature upon M-CSF stimulation. In addition, M-CSF induces phosphorylation of Mitf and TFE3 via a conserved MAPK consensus site, thereby triggering their recruitment of the coactivator p300. Furthermore, an unphosphorylatable mutant at the MAPK consensus serine is specifically deficient in formation of multinucleated osteoclasts, mimicking the defect in Mitf(mi/mi) mice. These results identify a signaling pathway that appears to coordinate cytokine signaling with the expression of genes vital to osteoclast development.

c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi.

Microphthalmia (Mi) is a bHLHZip transcription factor that is essential for melanocyte development and postnatal function. It is thought to regulate both differentiated features of melanocytes such as pigmentation as well as proliferation/survival, based on phenotypes of mutant mouse alleles. Mi activity is controlled by at least two signaling pathways. Melanocyte-stimulating hormone (MSH) promotes transcription of the Mi gene through cAMP elevation, resulting in sustained Mi up-regulation over many hours. c-Kit signaling up-regulates Mi function through MAP kinase phosphorylation of Mi, thereby recruiting the p300 transcriptional coactivator. The current study reveals that c-Kit signaling triggers two phosphorylation events on Mi, which up-regulate transactivation potential yet simultaneously target Mi for ubiquitin-dependent proteolysis. The specific activation/degradation signals derive from MAPK/ERK targeting of serine 73, whereas serine 409 serves as a substrate for p90 Rsk-1. An unphosphorylatable double mutant at these two residues is at once profoundly stable and transcriptionally inert. These c-Kit-induced phosphorylations couple transactivation to proteasome-mediated degradation. c-Kit signaling thus triggers short-lived Mi activation and net Mi degradation, in contrast to the profoundly increased Mi expression after MSH signaling, potentially explaining the functional diversity of this transcription factor in regulating proliferation, survival, and differentiation in melanocytes.

alpha-Melanocyte-stimulating hormone signaling regulates expression of microphthalmia, a gene deficient in Waardenburg syndrome.

The pituitary peptide alpha-melanocyte-stimulating hormone (alpha-MSH) stimulates melanocytes to up-regulate cAMP, but the downstream targets of cAMP are not well understood mechanistically. One consequence of alpha-MSH stimulation is increased melanization attributable to induction of pigmentation enzymes, including tyrosinase, which catalyzes a rate-limiting step in melanin synthesis. The tyrosinase promoter is a principle target of the melanocyte transcription factor Microphthalmia (Mi), a factor for which deficiency in humans causes Waardenburg syndrome II. We show here that both alpha-MSH and forskolin, a drug that increases cAMP, stimulate a rapid increase in Mi mRNA and protein levels in both melanoma cell lines and primary melanocytes. This up-regulation requires a cAMP-responsive element within the Mi promoter, and the pathway leading to Mi stimulation is subject to tight homeostatic regulation. Although cAMP signaling is ubiquitous, the Mi promoter was seen to be cAMP-responsive in melanocytes but not in nonmelanocytes. Moreover, dominant negative interference with Mi impeded successful alpha-MSH stimulation of tyrosinase. The regulation of Mi expression via alpha-MSH thus provides a direct mechanistic link to pigmentation. In addition, because the human melanocyte and deafness condition Waardenburg syndrome is sometimes caused by haploinsufficiency of Mi, its modulation by alpha-MSH suggests therapeutic strategies targeted at up-regulating the remaining wild type Mi allele.

Age-resolving osteopetrosis: a rat model implicating microphthalmia and the related transcription factor TFE3.

Microphthalmia (Mi) is a basic helix-loop-helix-leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells, and bone development. Two dominant-negative mi alleles (mi/mi and Mior/Mior) in mice cause osteopetrosis. In contrast, osteopetrosis has not been observed in a number of recessive mi alleles, suggesting the existence of Mi protein partners important in osteoclast function. An osteopetrotic rat of unknown genetic defect (mib) has been described whose skeletal sclerosis improves dramatically with age and that is associated with pigmentation defects reminiscent of mouse mi alleles. Here we report that this rat strain harbors a large genomic deletion encompassing the 3' half of mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts. Mi is not detectable in primary osteoblasts. In addition TFE3, a b-HLH-ZIP transcription factor related to Mi, was found to be expressed in osteoclasts, but not osteoblasts, and to coimmunoprecipitate with Mi. These results demonstrate the existence of members of a family of biochemically related transcription factors that may cooperate to play a central role in osteoclast function and possibly in age-related osteoclast homeostasis.