Two unrelated families were recruited in the French Reference Center for von Willebrand Disease with moderate bleeding symptoms associated with low von Willebrand factor (VWF) antigen levels, decreased collagen binding assay, and no or partial response to desmopressin. Genetic analysis showed the presence of heterozygous mutations in the A3 domain away from the collagen-binding surface: 1 never reported previously (p.L1696R) and another (p.P1824H) described in a Spanish family. The mutations were reproduced by site-directed mutagenesis and mutant VWF was expressed in different expression systems, COS-7 cells, baby hamster kidney cells, and in VWF-deficient mice through hydrodynamic injection. p.L1696R and p.P1824H were associated with very low expression levels both in vitro and in vivo, with intracellular retention for p.P1824H. Both homozygous mutants displayed decreased binding to collagen types I and III but also decreased binding to platelet glycoproteins Ib and IIbIIIa. Co-transfections with wild-type VWF partially corrected these defects, except that collagen binding remained abnormal. The in vivo thrombosis response was severely reduced for both heterozygous mutants. In conclusion, we report 2 VWF A3 domain mutations that induce a combined qualitative and quantitative defect.

With increasing recognition of the complications related to coagulopathies, it is of paramount importance for all orthopedic surgeons to possess a basic knowledge of common bleeding disorders. The evaluation of the coagulopathic patient requires a careful history, physical examination, and laboratory evaluation. Bleeding disorders commonly include quantitative and qualitative platelet and coagulation factor disorders and coagulation inhibitors. The management of these coagulopathies that can be encountered in elective and nonelective practice is often ignored. With appropriate knowledge and a multidisciplinary approach with hematologists and cardiologists, surgeons can perform minor and major orthopedic procedures safely and effectively.

Glanzmann thrombasthenia (GT) is characterized by mucocutaneous bleeding due to platelets that fail to aggregate in response to physiologic stimuli. GT, a rare inherited disease, is caused by quantitative or qualitative deficiencies of αIIbβ3, an integrin receptor for adhesive proteins. Coded by the ITGA2B and ITGB3 genes, αIIbβ3 mediates platelet-to-platelet attachment, aggregation and clot retraction. Despite widespread mutation analysis, the reason for the extensive variation in both the severity and intensity of bleeding among affected individuals remains poorly understood. Although genetic defects of ITGB3 affect other tissues where β3 is present as αvβ3 (the vitronectin receptor), the bleeding phenotype continues to dominate. The authors now examine the relationship between genotype and phenotype in classic and variant forms of GT, and reassess if the nature of the gene mutation influences bleeding and treatment aimed at restoring hemostasis.

Even though mutations in epigenetic regulators frequently occur in myeloproliferative neoplasms, their effects on the epigenome have not been well studied. Furthermore, even though primary myelofibrosis (PMF) has a markedly worse prognosis than essential thrombocytosis or polycythemia vera, the molecular distinctions between these subgroups are not well elucidated. We conducted the HELP (HpaII tiny fragment enriched by LM-PCR) assay to study genome-wide methylation in polycythemia vera, essential thrombocytosis, and PMF samples compared with healthy controls. We determined that polycythemia vera and essential thrombocytosis are characterized by aberrant promoter hypermethylation, whereas PMF is an epigenetically distinct subgroup characterized by both aberrant hyper- and hypomethylation. Aberrant hypomethylation in PMF was seen to occur in non-CpG island loci, showing further qualitative differences between the disease subgroups. The differentially methylated genes in polycythemia vera and essential thrombocytosis were involved predominantly in cell signaling pathways and were enriched for binding sites of GATA1 and other transcription factors. In contrast, aberrantly methylated genes in PMF were involved in inflammatory pathways and were enriched for NF1, LEF1, and other transcription factors. Within the PMF subgroup, cases with ASXL1 disruptions formed an epigenetically distinct subgroup with relatively increased methylation. Cases of myeloproliferative neoplasms (MPN) with TET2 mutations showed decreased levels of hydroxymethylation and distinct set of hypermethylated genes. In contrast, the JAK2V617F mutation did not drive epigenetic clustering within MPNs. Finally, the significance of aberrant methylation was shown by sensitivity of MPN-derived cell lines to decitabine. These results show epigenetic differences between PMF and polycythemia vera/essential thrombocytosis and reveal methylomic signatures of ASXL1 and TET2 mutations.

Deficiency of or defects in the plasma protein von Willebrand factor (VWF) lead to bleeding and von Willebrand disease (VWD), which may be congenital or acquired. VWD is considered the most common inherited bleeding disorder and laboratory testing for VWF level and activity is critical for appropriate diagnosis and management. We have designed and established a novel Flow Cytometry (FC) based method for measuring VWF antigen (VWF:Ag) and collagen binding (VWF:CB), together in the same tube and at the same time. The results of the novel FC method have been compared against existing reference methods using a range of normal and pathological material. Methods correlated well (VWF:Ag, r=0.866; VWF:CB, r=0.888) and generally permitted similar discrimination of quantitative versus qualitative VWD types (e.g. type 1 vs type 2A or 2B VWD). The novel procedure is expected to permit future streamlined performance of VWD screening, either using stand-alone FC systems or potentially incorporated into FC-capable automated blood cell and particle counters to allow for improved, automated and faster identification or exclusion of VWD.

Serum thrombopoietin in thrombocytopenic infants is largely related to the cause of thrombocytopenia and the underlying disease. Many perinatal factors can affect thrombopoietin level.A prospective cross-sectional study on 119 thrombocytopenic neonates: 54 full term and 65 preterm had been conducted. Thrombopoietin assay was done using a qualitative enzyme-linked immunosorbent assay technique. The test was repeated on the change of clinical status (recovery or deterioration).Lowering of thrombopoietin level was noted on reversal of platelet count to normal (P<0.001). Survival is significantly related to platelet count in full term (P = 0.04), but insignificant among thrombocytopenic preterms. Platelet count is negatively correlated to thrombopoietin level in neonates both in full term and preterm (r = -0.59, -0.69, respectively, P<0.001). Platelet count was found to be the best predictor for duration of recovery of thrombocytopenia in neonates compared with other factors including thrombopoietin level.Thrombocytopenic neonates had high levels of thrombopoietin. Despite the high thrombopoietin level in neonates died with severe thrombocytopenia, yet, mortality is related to the cause and outcome of thrombocytopenia rather than the serum thrombopoietin level. It is recommended to diagnose and treat the underlying cause of thrombocytopenia rather than to generalize the therapy based on thrombopoietin level.

Quantitative and qualitative platelet abnormalities of neonates must be defined using evidence-based reference ranges, constructed according to gestational and postnatal age.Platelet counts, and demographic and outcome data, were obtained from neonates in the Intermountain Healthcare system in the western USA and template bleeding times were determined from neonates in Italy.Reference ranges were constructed by excluding values from neonates with diagnoses associated with abnormal platelet counts (small for gestational age (SGA), pregnancy-induced hypertension (PIH), infection and necrotizing enterocolitis (NEC)). Values remaining in the database after excluding these diagnoses were organized into 5th to 95th percentile ranges. At 23-25 weeks gestation, thrombocytopenia (<5th percentile) was defined by a platelet count <100,000/µl. Severe thrombocytopenia (platelet count <50,000/µl) occurred in 2.4% of neonatal intensive care unit (NICU) admissions and was largely due to acquired consumptive causes (bacterial and fungal sepsis, NEC and extracorporeal membrane oxygenation). No correlation was found between platelet count and subsequent central nervous system (CNS), pulmonary or gastrointestinal (GI) bleeding. The mortality rate did not correlate with the lowest platelet count but was proportionate to the number of platelet transfusions received. Platelet transfusions, administered according to guidelines, were given to 7% of NICU admissions, but a change in the guidelines from "count-based" to "mass-based" was associated with a reduction to 4%, with no increase in CNS, pulmonary, GI or cutaneous haemorrhage. Bleeding times were twice as long in neonates <33 weeks gestation as in term neonates, and shortened to term values by day of life ten.When reference ranges for platelets, appropriate to gestational and postnatal ages, are used, more uniformity occurs in definitions. This uniformity will foster consistency in diagnosis, treatment and outcomes-reporting.

Von Willebrand disease (VWD) is an inherited bleeding disorder caused by the quantitative or qualitative deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived VWF/factor VIII (FVIII) concentrates is required in patients unresponsive to desmopressin. To assess the efficacy, safety and ease of use of a new, volume-reduced (VR) formulation of VWF/FVIII concentrate Haemate(®) P in patients requiring treatment for bleeding or prophylaxis for recurrent bleeding or for invasive procedures. Pharmacoeconomic variables were also recorded. Data were analysed using descriptive statistics. This was a multicentre, prospective, observational study. Consecutively enrolled patients received Haemate(®) P VR according to their needs, and were followed for 24 months. Of the 121 patients enrolled, 25.6% had type 3 VWD and more than 40% had severe disease. All patients were followed for 2 years, for a total of 521 visits. On-demand treatment was given to 61.9% of patients, secondary long-term prophylaxis to 25.6% and prophylaxis for surgery, dental or invasive procedures to 45.5%. The response to treatment was rated as good to excellent in >93-99% of interventions. The new formulation was well tolerated by all patients with no report of drug-related adverse events. The switch to volume-reduced Haemate(®) P was easy to perform and infusion duration was decreased twofold compared with the previous formulation. Volume-reduced Haemate(®) P was at least as effective and well-tolerated as the previous formulation.

The pseudohyperkalemia in thrombocytosis is assumed to be due to potassium released from blood cells during blood clotting as reported previously, but its mechanisms remain to be cleared. Although plasma potassium measurements with blood collection tubes containing heparin are performed in many hospitals to avoid pseudohyperkalemia, the burden on patients may come out with further blood sampling by another heparinized tube. Taken together, we investigated laboratory data possibly involved in pseudohyperkalemia in 184 samples from patients with myeloproliferative neoplasma (MPN), and studied estimation capability for true values of serum potassium, driving a correction formula by means of several laboratory data to explain the difference of measured potassium values (K-difference: serum value minus plasma value). Platelet count and mean corpuscular volume (MCV) were adopted as significant variables correlated to K-difference as a result of multiple regression analysis. A correction formula was driven by multiple regression equation with these two variables as follows: y = 0.0006 x 1+0.0004 x 2-0.177 (r= 0.885; x 1, platelet count; x 2, MPV). The correction formula was considered to be useful for estimating the true value of serum potassium in samples from patients with MPN because the corrected serum potassium value correlated highly with plasma potassium value (r = 0.885). These results propose that true values of serum potassium can be estimated by the correction of measured serum potassium values with platelet count and MCV, suggesting that not only quantitative factors but also qualitative factors may be involved in pseudohyperkalemia.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of the von Willebrand factor (VWF). VWD is classified into three types--type 1 (partial quantitative deficiencies), type 2 (qualitative defects) and type 3 (complete deficiency of VWF). In this study we explored genotype and phenotype characteristics of patients with VWD with the aim of dissecting the distribution of mutations in different types of VWD. One hundred fourteen patients belonging to 78 families diagnosed to have VWD were studied. Mutation analysis was performed by direct sequencing of the VWF . Large deletions were investigated by multiplex ligation-dependent probe amplification (MLPA) analysis. The impact of novel candidate missense mutations and potential splice site mutations was predicted by in silico assessments. We identified mutations in 66 index patients (IPs) (84.6%). Mutation detection rate was 68%, 94% and 94% for VWD type 1, 2 and 3, respectively. In total, 68 different putative mutations were detected comprising 37 missense mutations (54.4%), 10 small deletions (14.7%), two small insertions (2.9%), seven nonsense mutations (10.3%), five splice-site mutations (7.4%), six large deletions (8.8%) and one silent mutation (1.5%). Twenty-six of these mutations were novel. Furthermore, in type 1 and type 2 VWD, the majority of identified mutations (74% vs. 88.1%) were missense substitutions while mutations in type 3 VWD mostly caused null alleles (82%). Genotyping in VWD is a helpful tool to further elucidate the pathogenesis of VWD and to establish the relationship between genotype and phenotype.