Tissue factor pathway inhibitor is a potential modifier of bleeding risk in factor XI deficiency.

BACKGROUND: Factor (F) XI deficiency is associated with increased bleeding risk in some individuals. Neither FXI levels nor clinical clotting assays predict the bleeding risk. Compared with controls, FXI-deficient bleeders have reduced clot formation, decreased fibrin network density, and increased susceptibility to fibrinolysis. Tissue factor pathway inhibitor (TFPI) was recently implicated as a modifying factor in individuals with bleeding of unknown cause. OBJECTIVES: To determine the potential of TFPI in modifying the bleeding risk in FXI-deficient individuals. METHODS: The effects of TFPI on thrombin generation and clot formation, structure, and fibrinolysis in FXI-deficient plasma were measured in vitro in the absence or presence of inhibitory anti-TFPI antibody or exogenous recombinant TFPIα. Total plasma TFPI concentration was measured in 2 independent cohorts of controls and FXI-deficient individuals classified as bleeders or nonbleeders (cohort 1: 10 controls and 16 FXI-deficient individuals; cohort 2: 48 controls and 57 FXI-deficient individuals) and correlated with ex vivo plasma clot formation and fibrinolysis parameters associated with bleeding risk. RESULTS: In an in vitro FXI deficiency model, inhibition of TFPI enhanced thrombin generation and clot formation, increased the network density, and decreased fibrinolysis, whereas an increase in TFPI had the opposite effects. Compared with controls, plasma from FXI-deficient bleeders had higher TFPI concentration. Total plasma TFPI concentrations correlated with parameters from ex vivo clotting and fibrinolysis assays that differentiate FXI-deficient bleeders and nonbleeders. CONCLUSION: Coagulation and fibrinolysis parameters that differentiate FXI-deficient nonbleeders and bleeders were altered by plasma TFPIα. Total plasma TFPI was increased in FXI-deficient bleeders. TFPI may modify the bleeding risk in FXI-deficient individuals.

The Use and Effectiveness of an Online Diagnostic Support System for Blood Film Interpretation: Comparative Observational Study.

BACKGROUND: The recognition and interpretation of abnormal blood cell morphology is often the first step in diagnosing underlying serious systemic illness or leukemia. Supporting the staff who interpret blood film morphology is therefore essential for a safe laboratory service. This paper describes an open-access, web-based decision support tool, developed by the authors to support morphological diagnosis, arising from earlier studies identifying mechanisms of error in blood film reporting. The effectiveness of this intervention was assessed using the unique resource offered by the online digital morphology Continuing Professional Development scheme (DM scheme) offered by the UK National External Quality Assessment Service for Haematology, with more than 3000 registered users. This allowed the effectiveness of decision support to be tested within a defined user group, each of whom viewed and interpreted the morphology of identical digital blood films. OBJECTIVE: The primary objective of the study was to test the effectiveness of the decision support system in supporting users to identify and interpret abnormal morphological features. The secondary objective was to determine the pattern and frequency of use of the system for different case types, and to determine how users perceived the support in terms of their confidence in decision-making. METHODS: This was a comparative study of identical blood films evaluated either with or without decision support. Selected earlier cases from the DM scheme were rereleased as new cases but with decision support made available; this allowed a comparison of data sets for identical cases with or without decision support. To address the primary objectives, the study used quantitative evaluation and statistical comparisons of the identification and interpretation of morphological features between the two different case releases. To address the secondary objective, the use of decision support was assessed using web analytical tools, while a questionnaire was used to assess user perceptions of the system. RESULTS: Cases evaluated with the aid of decision support had significantly improved accuracy of identification for relevant morphological features (mean improvement 9.8%) and the interpretation of those features (mean improvement 11%). The improvement was particularly significant for cases with higher complexity or for rarer diagnoses. Analysis of website usage demonstrated a high frequency of access for web pages relevant to each case (mean 9298 for each case, range 2661-24,276). Users reported that the decision support website increased their confidence for feature identification (4.8/5) and interpretation (4.3/5), both within the context of training (4.6/5) and also in their wider laboratory practice (4.4/5). CONCLUSIONS: The findings of this study demonstrate that directed online decision support for blood morphology evaluation improves accuracy and confidence in the context of educational evaluation of digital films, with effectiveness potentially extending to wider laboratory use.

A diagnostic evaluation of single screen testing for malaria in the returning traveler: A large retrospective cohort study.

BACKGROUND: Screening for malaria in the returning traveler has often required repeat testing; however, audit data suggest that patients have not been reattending. We sought to ascertain if this was safe by examining the diagnostic efficacy of a single screen consisting of a rapid diagnostic test (RDT) and a thin film. METHODS: We conducted a retrospective cohort study of patients with suspected malaria who attended in the past 5 years from two large teaching hospitals. We assessed the diagnostic accuracy of a single screen, reporting measures of sensitivity and specificity. To establish a reference standard, we cross-linked data with the national malaria registry held at Public Health England and regional centers. RESULTS: The cohort consisted of 1365 patients, of whom 33 opted out of the research and one did not have a complete initial screen. Of those 1331 screens there were 74 cases of Plasmodium falciparum (prevalence of 5.6%) and 104 of any malaria species (prevalence of 7.8%). Sensitivity for the detection of P. falciparum was 100.00% (95% confidence interval [CI] = 95.1 to 100), with a specificity of 99.4% (95% CI = 98.9 to 99.8). For the detection of any species of malaria the sensitivity was slightly lower due to the presence of one false negative; sensitivity was 99.0% (95% CI = 94.8 to 100) and specificity was 99.5% (95% CI = 98.9 to 99.8). CONCLUSIONS: A single thin film and RDT is likely to be sufficient as a first screen for falciparum malaria in the returning traveler with important caveats. For those sent home from emergency departments, appropriate safety netting must be provided. Further prospective study is required to investigate this approach.

Switchable foldamer ion channels with antibacterial activity.

Synthetic ion channels may have applications in treating channelopathies and as new classes of antibiotics, particularly if ion flow through the channels can be controlled. Here we describe triazole-capped octameric α-aminoisobutyric acid (Aib) foldamers that "switch on" ion channel activity in phospholipid bilayers upon copper(ii) chloride addition; activity is "switched off" upon copper(ii) extraction. X-ray crystallography showed that CuCl2 complexation gave chloro-bridged foldamer dimers, with hydrogen bonds between dimers producing channels within the crystal structure. These interactions suggest a pathway for foldamer self-assembly into membrane ion channels. The copper(ii)-foldamer complexes showed antibacterial activity against B. megaterium strain DSM319 that was similar to the peptaibol antibiotic alamethicin, but with 90% lower hemolytic activity.

Dual-action CXCR4-targeting liposomes in leukemia: function blocking and drug delivery.

CXC chemokine receptor 4 (CXCR4) is overexpressed by a broad range of hematological disorders, and its interaction with CXC chemokine ligand 12 (CXCL12) is of central importance in the retention and chemoprotection of neoplastic cells in the bone marrow and lymphoid organs. In this article, we describe the biological evaluation of a new CXCR4-targeting and -antagonizing molecule (BAT1) that we designed and show that, when incorporated into a liposomal drug delivery system, it can be used to deliver cancer therapeutics at high levels to chronic lymphocytic leukemia (CLL) cells. CXCR4 targeting and antagonism by BAT1 were demonstrated alone and following its incorporation into liposomes (BAT1-liposomes). Antagonism of BAT1 against the CXCR4/CXCL12 interaction was demonstrated through signaling inhibition and function blocking: BAT1 reduced ERK phosphorylation and cell migration to levels equivalent to those seen in the absence of CXCL12 stimulation (P < .001). Specific uptake of BAT1-liposomes and delivery of a therapeutic cargo to the cell nucleus was seen within 3 hours of incubation and induced significantly more CLL cell death after 24 hours than control liposomes (P = .004). The BAT1 drug-delivery system is modular, versatile, and highly clinically relevant, incorporating elements of proven clinical efficacy. The combined capabilities to block CXCL12-induced migration and intracellular signaling while simultaneously delivering therapeutic cargo mean that the BAT1-liposome drug-delivery system could be a timely and relevant treatment of a range of hematological disorders, particularly because the therapeutic cargo can be tailored to the disease being treated.

Quantitative Assay of Mutated Nucleophosmin in Acute Myeloid Leukemia.

BACKGROUND: In our previous work, we provided strong evidence that nucleophosmin (NPM) gene mutation has an important role in leukemogenesis of primary acute myeloid leukemia (AML). Furthermore, we speculated a new targeted therapy in patients with primary AML and bearing mutated NPM (mNPM). Based on these results together with findings of other researchers, it was essential to develop a method for accurate detection of mNPM. METHODS: Our method based on utilizing the most recent flow cytometeric techniques and instruments in measuring mNPM. Attributed to their availability and technical feasibility, we used human leukemia cell lines to validate our method. RESULTS: The main findings were differential expression of wild-type NPM (wtNPM) within the same sample. Furthermore flow cytometry (FCM) was a simple straightforward tool for quantitative assay of mNPM. CONCLUSIONS: In this work we developed an innovative technique that could enable quantitative assay of mNPM, and ease its use as a biomarker in cytogenetic and molecular prognostication of primary AML. In addition the study suggested that FCM could differentiate mNPM expression within cells of the same patient thus could be used for monitoring of minimal residual disease.

Synthesis and biological activity of a CXCR4-targeting bis(cyclam) lipid.

A bis(cyclam)-capped cholesterol lipid designed to bind C-X-C chemokine receptor type 4 (CXCR4) was synthesised in good overall yield from 4-methoxyphenol through a seven step synthetic route, which also provided a bis(cyclam) intermediate bearing an octaethyleneglycol-primary amine that can be easily derivatised. This bis(cyclam)-capped cholesterol lipid was water soluble and self-assembled into micellar and non-micellar aggregates in water at concentrations above 8 µM. The bioactivity of the bis(cyclam)-capped cholesterol lipid was assessed using primary chronic lymphocytic leukaemia (CLL) cells, first with a competition binding assay then with a chemotaxis assay along a C-X-C motif chemokine ligand 12 (CXCL12) concentration gradient. At 20 µM, the bis(cyclam)-capped cholesterol lipid was as effective as the commercial drug AMD3100 for preventing the migration of CLL cells, despite a lower affinity for CXCR4 than AMD3100.

Abnormal plasma clot formation and fibrinolysis reveal bleeding tendency in patients with partial factor XI deficiency.

Individuals with factor XI (FXI) deficiency have a variable bleeding risk that cannot be predicted from plasma FXI antigen or activity. This limitation can result in under- or overtreatment of patients and risk of bleeding or thrombosis. Previously, plasma clot fibrinolysis assays showed sensitivity to bleeding tendency in a small cohort of patients with severe FXI deficiency. Here, we determined the ability of plasma clot formation, structure, and fibrinolysis assays to predict bleeding tendency in a larger, independent cohort of patients with severe and partial FXI deficiency. Patients were characterized as nonbleeders or bleeders based on bleeding after tonsillectomy and/or dental extraction before diagnosis of FXI deficiency. Blood was collected in the absence or presence of the contact pathway inhibitor corn trypsin inhibitor (CTI). Clotting was triggered in platelet-poor plasma with tissue factor, CaCl2, and phospholipids in the absence and presence of thrombomodulin or tissue plasminogen activator. Clot formation and fibrinolysis were assessed by turbidity and confocal microscopy. CTI-treated plasmas from bleeders showed significantly reduced clot formation and decreased resistance to fibrinolysis compared with plasmas from controls or nonbleeders. Differences were enhanced in the presence of CTI. A model that combines activated partial thromboplastin time with the rate of clot formation and area under the curve in fibrinolysis assays identifies most FXI-deficient bleeders. These results show assays with CTI-treated platelet-poor plasma reveal clotting and clot stability deficiencies that are highly associated with bleeding tendency. Turbidity-based fibrinolysis assays may have clinical utility for predicting bleeding risk in patients with severe or partial FXI deficiency.

ASP2215 in the treatment of relapsed/refractory acute myeloid leukemia with FLT3 mutation: background and design of the ADMIRAL trial.

Acute myeloid leukemia (AML) is a heterogeneous disease with cure rates of only 30-40% in patients <60 years old. Cytogenetic and molecular markers have improved our understanding of the different prognostic entities in AML. FLT3 mutations are present in 30-40% of AML cases, conferring a poor prognosis with reduced survival. AXL activates FLT3, impacting adversely on outcome. Both FLT3 and AXL constitute promising molecular targets. ASP2215 (gilteritinib) is a novel, dual FLT3/AXL inhibitor with promising early phase trial data (NCT02014558). A Phase III randomized multicenter clinical trial, comparing ASP2215 to salvage chemotherapy in relapsed/refractory AML with FLT3-mutations is now open to recruitment (NCT02421939). Trial design and objectives are discussed here.

Role of Nucleophosmin Gene Mutation in Leukemogenesis of Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder that carries very poor prognosis. Understanding molecular basis of AML leukemogenesis could lead to the emergence of effective targeted therapies for AML. AML bearing nucleophosmin (NPM) gene mutation has distinct features. This study was conducted to investigate the role of mutated (m) NPM in pathogenesis of de novo AML through studying its contribution in proliferation of AML cell line cells. METHODS: Two types of human leukemia cell lines were used. One of them was a model for AMLs with mNPM and the other for AMLs with wild type (wt) NPM. Assessment of the proliferative role of mNPM in AML was carried out using cell culture and viability studies. The obtained results were reaffirmed by immunocytochemical and immunoblotting techniques. RESULTS: Analysis of results was done with the appropriate computer software. It showed higher proliferative potential of cells with mNPM compared to those bearing wtNPM only. Furthermore, the immunocytochemical studies demonstrated subcellular localization of NPM isoforms during various phases of mitosis. Mitosis was associated with cytoplasmic translocation of wtNPM in certain phases, while localization of mNPM remained unchanged throughout the cell cycle. Results of immunoblotting showed little or no change in protein expression of either NPM moieties during mitosis. CONCLUSIONS: The current study demonstrated important contribution of NPM gene mutation in enhancing proliferation of AML cell lines. These results confirmed the role of mNPM in AML leukemogenesis, and highlighted the importance of targeting mNPM in new evolving AML therapies.

Web-Based Virtual Microscopy of Digitized Blood Slides for Malaria Diagnosis: An Effective Tool for Skills Assessment in Different Countries and Environments.

BACKGROUND: Morphological examination of blood films remains the reference standard for malaria diagnosis. Supporting the skills required to make an accurate morphological diagnosis is therefore essential. However, providing support across different countries and environments is a substantial challenge. OBJECTIVE: This paper reports a scheme supplying digital slides of malaria-infected blood within an Internet-based virtual microscope environment to users with different access to training and computing facilities. The feasibility of the approach was established, allowing users to test, record, and compare their own performance with that of other users. METHODS: From Giemsa stained thick and thin blood films, 56 large high-resolution digital slides were prepared, using high-quality image capture and 63x oil-immersion objective lens. The individual images were combined using the photomerge function of Adobe Photoshop and then adjusted to ensure resolution and reproduction of essential diagnostic features. Web delivery employed the Digital Slidebox platform allowing digital microscope viewing facilities and image annotation with data gathering from participants. RESULTS: Engagement was high with images viewed by 38 participants in five countries in a range of environments and a mean completion rate of 42/56 cases. The rate of parasite detection was 78% and accuracy of species identification was 53%, which was comparable with results of similar studies using glass slides. Data collection allowed users to compare performance with other users over time or for each individual case. CONCLUSIONS: Overall, these results demonstrate that users worldwide can effectively engage with the system in a range of environments, with the potential to enhance personal performance through education, external quality assessment, and personal professional development, especially in regions where educational resources are difficult to access.

Graphene in therapeutics delivery: Problems, solutions and future opportunities.

Graphene based nanomaterials are being used experimentally to deliver therapeutic agents to cells or tissues both in vitro and in vivo. However, substantial challenges remain before moving to safe and effective use in humans. In particular, it is recognised that graphene molecules undergo complex interactions with solutes, proteins or cellular systems within the body, and that these interactions impact significantly on the behaviour or toxicity of the molecule. Approaches to overcome these problems include modification of the graphene or its combination with other molecules to accentuate favourable characteristics or modify adverse interactions. This has led to an emerging role for graphene as one part of highly-tailored multifunctional delivery vehicles. This review examines the knowledge that underpins present approaches to exploit graphene in therapeutics delivery, discussing both favourable and unfavourable aspects of graphene behaviour in biological systems and how these may be modified; then considers the present place of the molecule and the challenges for its further development.

Do We Know Why We Make Errors in Morphological Diagnosis? An Analysis of Approach and Decision-Making in Haematological Morphology.

BACKGROUND: The laboratory interpretation of blood film morphology is frequently a rapid, accurate, and cost-effective final-stage of blood count analysis. However, the interpretation of findings often rests with a single individual, and errors can carry significant impact. Cell identification and classification skills are well supported by existing resources, but the contribution and importance of other skills are less well understood. METHODS: The UK external quality assurance group in haematology (UK NEQAS(H)) runs a Continued Professional Development scheme where large digital-images of abnormal blood smears are presented using a web-based virtual microscope. Each case is answered by more than 800 individuals. Morphological feature selection and prioritisation, as well as diagnosis and proposed action, are recorded. We analysed the responses of participants, aiming to identify successful strategies as well as sources of error. FINDINGS: The approach to assessment by participants depended on the affected cell type, case complexity or skills of the morphologist. For cases with few morphological abnormalities, we found that accurate cell identification and classification were the principle requirements for success. For more complex films however, feature recognition and prioritisation had primary importance. Additionally however, we found that participants employed a range of heuristic techniques to support their assessment, leading to associated bias and error. INTERPRETATION: A wide range of skills together allow successful morphological assessment and the complexity of this process is not always understood or recognised. Heuristic techniques are widely employed to support or reinforce primary observations and to simplify complex findings. These approaches are effective and are integral to assessment; however they may also be a source of bias or error. Improving outcomes and supporting diagnosis require the development of decision-support mechanisms that identify and support the benefits of heuristic strategies while identifying or avoiding associated biases. FUNDING: The CPD scheme is funded by participant subscription.

Sample conditions determine the ability of thrombin generation parameters to identify bleeding phenotype in FXI deficiency.

Individuals with Factor XI (FXI) deficiency have a variable bleeding tendency that does not correlate with FXI:C levels or genotype. Comparing a range of sample conditions, we tested whether the thrombin generation assay (TGA) could discriminate between control subjects (n = 50) and FXI-deficient individuals (n = 97), and between those with bleeding tendency (n = 50) and without (n = 24). The comparison used platelet-rich plasma (PRP) and platelet-poor plasma (PPP), either with or without corn trypsin inhibitor (CTI) to prevent contact activation, over a range of tissue factor (TF) concentrations. When contact activation was inhibited and platelets were absent, FXI:C levels did not correlate with thrombin generation parameters, and control and FXI-deficient individuals were not distinguished. In all other sample types, the best discrimination was obtained using TF 0.5 pM and assay measures: endogenous thrombin potential (ETP) and peak height. We showed that although a number of conditions could distinguish differences between the groups tested, TGA measured in PRP with CTI best differentiated between bleeders and nonbleeders. These measures provided high sensitivity and specificity (peak height receiver operating characteristic [ROC] area under the curve [AUC] = 0.9362; P < .0001) (ETP ROC AUC = 0.9362; P < .0001). We conclude that by using sample conditions directed to test specific pathways of FXI activation, the TGA can identify bleeding phenotype in FXI deficiency.

Monocyte-derived dendritic cells from chronic myeloid leukaemia have abnormal maturation and cytoskeletal function that is associated with defective localisation and signalling by normal ABL1 protein.

OBJECTIVES: Mature dendritic cells (DCs) may be derived from the BCR/ABL1 expressing monocytes in chronic myeloid leukaemia. These cells have potential therapeutic applications, but are recognised to have defective function. In normal DCs, activation and maturation depend on ABL1 dependent signals. We therefore tested the hypothesis that in the DCs of chronic myeloid leukaemia, the presence of the BCR/ABL1 molecule disrupts normal ABL1 signal pathways, and contributes to the observed functional defects of the cells. METHODS: We employed in vitro culture of clinical samples, combining microscopic and biochemical techniques with a phosphoproteomic approach to compare and characterise DCs from normal individuals and chronic myeloid leukaemia patients. RESULTS AND CONCLUSIONS: We identified an altered intracellular localisation for ABL1 within DCs derived from the monocytes of chronic myeloid leukaemia. The protein was found in the perinuclear region co-distributed with the adapter-protein CRKL and the BCR/ABL1 protein. This altered distribution was associated with defective generation of ABL1-dependent maturation signals, and a dislocation of ABL1 from the F-actin cytoskeleton. We suggest that abnormal ABL1-dependent signals contribute to the recognised functional defects affecting chronic myeloid leukaemia DCs.

Lymphocytes from chronic lymphocytic leukaemia undergo ABL1-linked amoeboid motility and homotypic interaction as an early adaptive change to ex vivo culture.

BACKGROUND: Those stimuli that together promote the survival, differentiation and proliferation of the abnormal B-lymphocytes of chronic lymphocytic leukaemia (CLL) are encountered within tissues, where together they form the growth-supporting microenvironment. Different tissue-culture systems promote the survival of the neoplastic lymphocytes from CLL, partly replicating the in vivo tissue environment of the disorder. In the present study, we focussed on the initial adaptive changes to the tissue culture environment focussing particularly on migratory behaviour and cellular interactions. METHODS: A high-density CLL culture system was employed to test CLL cell-responses using a range of microscopic techniques and flow cytometric analyses, supported by mathematical measures of cell shape-change and by biochemical techniques. The study focussed on the evaluation of changes to the F-actin cytoskeleton and cell behaviour and on ABL1 signalling processes. RESULTS: We showed that the earliest functional response by the neoplastic lymphocytes was a rapid shape-change caused through rearrangement of the F-actin cytoskeleton that resulted in amoeboid motility and promoted frequent homotypic interaction between cells. This initial response was functionally distinct from the elongated motility that was induced by chemokine stimulation, and which also characterised heterotypic interactions between CLL lymphocytes and accessory cells at later culture periods. ABL1 is highly expressed in CLL lymphocytes and supports their survival, it is also recognised however to have a major role in the control of the F-actin cytoskeleton. We found that the cytoplasmic fraction of ABL1 became co-localised with F-actin structures of the CLL lymphocytes and that the ABL1 substrate CRKL became phosphorylated during initial shape-change. The ABL-inhibitor imatinib mesylate prevented amoeboid movement and markedly reduced homotypic interactions, causing cells to acquire a globular shape to rearrange F-actin to a microvillus form that closely resembled that of CLL cells isolated directly from circulation. CONCLUSION: We suggest that ABL1-induced amoeboid motility and homotypic interaction represent a distinctive early response to the tissue environment by CLL lymphocytes. This response is separate from that induced by chemokine or during heterotypic cell-contact, and may play a role in the initial entry and interactions of CLL lymphocytes in tissues.

Single-cell analysis of K562 cells: an imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein.

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.