Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

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.

QUALITY IMPROVEMENT: Supporting a hospital in difficulty: -experience of a '-buddying' agreement to implement a new medical pathway.

Increased NHS regulation has identified many healthcare organisations with operational and/or financial difficulties. Although the causes are often complex, most cases are effectively managed internally with limited input from external agencies. How best to support the few organisations needing additional support has not been established. 'Buddying', in which senior clinical and managerial teams from a well performing organisation work with colleagues from an organisation in difficulty has been proposed as a potential solution. Previous reports suggest that these partnerships are generally valued by the organisation in difficulty but there is a paucity of measured operational benefit. In this article we present our experience of a 'buddying agreement' and its impact on the introduction of a new 'whole system' medical pathway (ie rotas, staffing, process) at an organisation in difficulty. We describe the process, problems, effect on operational performance, staff survey feedback six months post-implementation and the lessons learned. Factors critical to success were good communication; clear responsibilities, common values and strong governance; incorporation into an effective local improvement programme; targeting of specific issues; ability to influence people and foster relationships; adequate 'manpower' and gradual transition to local 'ownership'.

Management of Type 2B von Willebrand Disease during Pregnancy.

Type 2B von Willebrand disease is a rare bleeding condition resulting in thrombocytopenia and a reduction in large VWF multimers. It usually has an autosomal dominant pattern of inheritance. We report the management of a patient with type 2B von Willebrand disease, whose diagnosis was confirmed by demonstration of a R1306W mutation, through her first pregnancy. The patient's von Willebrand factor (VWF) antigen and VWF ristocetin cofactor levels rose throughout pregnancy, with an associated drop in the platelet count. The patient was successfully managed through labour to a surgical delivery with VWF concentrate, platelet transfusions and tranexamic acid. The patient delivered a male baby who was found to have inherited type 2B von Willebrand disease and had a significant cephalhaematoma at delivery. The baby was managed with VWF concentrate and platelet transfusions and made a full recovery. There is a lack of evidence to guide the best management of pregnant patients with type 2B von Willebrand disease. We adopted a pragmatic management plan, in keeping with other published case reports. To the best of our knowledge, this is the first case report in which the child was found to have inherited type 2B von Willebrand disease and encountered bleeding problems, making this case unique amongst the published literature.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

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.

Changing organisational leadership culture: focus on values changes culture.

In 2006 Guy's and St Thomas' NHS Foundation Trust not only had a financial challenge but also a cultural one: only just over half of staff would recommend the Trust as a place to work or receive treatment. This prompted the leadership team to embark on a journey spanning almost a decade, developing a values-based culture. The Trust values framework spans four levels of the organisation and sets out the values and behaviours expected of everyone, through to the strategic leaders in the organisation. This focus on leadership and behaviours at all levels has been underpinned with an appreciative inquiry approach to embedding the values and culture change. In this article, chief executive, Sir Ron Kerr, and OD colleagues, Sarah Morgan and Carolyn Norgate, detail the journey that Guy's and St Thomas' has undertaken, its impressive results, and the next stage in the Trust's journey.

The effect of social contingencies on nursing students' reactions during a rural clinical placement.

Students' feedback of their practicum experiences are typically documented only in terms of established nursing competencies and learning objectives. How nursing students cope with social contingencies (e.g., personal health) while away on clinical placement is not commonly reported in the literature. A sample of Australian student nurses was surveyed as a way of contributing new knowledge about what and how social contingencies could impact on a practicum experience. An analysis of the survey data provided by 244 students revealed that of the 14 contingencies used, financial pressure, accommodation, and geographic location, were rated as having the most influence. All of these social contingencies were examined by a principal components analysis. Three factors were identified and interpreted as professional organization, home organization, and personal organization. Three subscales were then derived using these factors and other measures were also calculated. Bivariate and multivariate relationships were subsequently determined. One key finding was that the first year students, compared to their more senior counterparts, expressed less stress during their practicum. The first year students, as opposed to their more experienced peers, also attached less importance to the professional organizational contingencies. The implications of the study for university administrators, nursing education faculty, and managers of clinical facilities conclude the paper.

Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study.

The presence of p53-pathway dysfunction in chronic lymphocytic leukemia (CLL) can be used to identify patients with chemotherapy-refractory disease. Therapeutic responses are known to vary between patients with chemosensitive CLL and may relate to differences in p53-pathway activity. We hypothesized that the magnitude or type of p53-pathway protein expression is heterogeneous in patients with chemosensitive disease and could associate with white cell responses. In this pilot study, changes in p53 and its transcriptional targets, p21/waf1 and MDM2 were analyzed by immunoblotting and densitometry in CLL cells from 10 patients immediately prior to the start of chemotherapy, and after culture for 24 hours (h) with fludarabine (n=7) or chlorambucil (n=3). The in vitro response was also compared to that in vivo in circulating cells pre-treatment, and at 24h and 96h of chemotherapy. Disease responses were evident in all patients after the first treatment-cycle. Significant p53 induction was observed in CLL cells treated in vitro and in vivo. Greater heterogeneity in the expression-intensity was observed in vivo (σ2=45.15) than in vitro (σ2=1.33) and the results failed to correlate (r(2)=0.18, p=0.22). p21/waf1 and MDM2 expression-profiles were also dissimilar in vitro and in vivo. Higher in vivo (but not in vitro) responses associated with changes in white cell count (p=0.026). Thus, heterogeneity of p53-pathway activity exists in chemosensitive CLL; in unselected patients, in vivo changes do not correlate with those in vitro, but may associate with post-treatment white cell responses.

New insights into haemostasis in liver failure.

The precise mechanisms leading to the coagulopathy of acute liver injury are unclear. To study this further, coagulation and immune changes have been compared in patients with acute liver injury secondary to paracetamol overdose, with chronic cirrhosis, and normal healthy controls. In acute liver injury, coagulation factors II, V, VII and X were reduced to a similar degree, and were significantly lower than factors IX and XI. In cirrhosis, by contrast, these coagulation factors were reduced to similar levels. Factor VIII increased in acute liver injury, but was normal in cirrhosis. Interleukin-6 and tumour necrosis factor-alpha levels increased in both patient groups, but were higher in paracetamol overdose. Thrombin-antithrombin and soluble tissue factor levels increased in those with acute liver injury, but were normal in patients with cirrhosis. Functional antithrombin was reduced in both acute liver injury and cirrhosis. It is hypothesized that in acute paracetamol-induced liver injury, immune activation leads to tissue factor-initiated consumption of factors II, V, VII and X, but that levels of factors IX and XI are better preserved because of inhibition of the thrombin-induced amplification phase of coagulation. These findings have implications for appropriate coagulation factor support for patients with acute liver injury.

A novel method for quantitation of human von Willebrand factor messenger RNA.

We have developed a novel assay for quantitation of human von Willebrand factor (vWF) messenger RNA (mRNA) using the ABI 7700 quantitative PCR system (Applied Biosystems, Warrington, Cheshire, UK). To allow accurate measurement of the vWF mRNA concentration in samples, we have used 18S ribosomal RNA as an internal control. The specificity of the assay was demonstrated by polyacrylamide gel electrophoresis and ABI Prism BigDye terminator cycle sequencing of the PCR product. We have also assessed the assay by using RNA from both a hepatocyte cell line which does not express vWF mRNA, and an endothelial cell line which does express vWF mRNA. By mixing varying amounts of these RNAs together, we were able to adjust the vWF quantity in known increments while keeping the 18S ribosomal RNA internal control constant. The quantitation of vWF mRNA correlated with the proportion of endothelial cell RNA (r(2)=0.912, p<0.001).