Dr Anoop Enjeti

© 2017 Background Characterization of circulating microvesicles (MV) in healthy subjects in relation to various biological factors is not well studied. Objectives We evaluated the influence of age, gender, smoking status, lipid and hormone profiles on circulating MV in healthy subjects. Methods Platelet free plasma from 143 volunteer blood donors (males¿=¿80, females¿=¿63) was evaluated by standardized flow cytometry for MV expressing CD41 (platelet-derived), CD105 (endothelial-derived), CD235 (red cell-derived), TF (tissue factor) and phosphatidylserine (PS) MV. Procoagulant function was measured by the Xa based assay (XaCT) and endogenous thrombin potential (ETP) using thrombin generation assay. Results Those =¿29¿years and =¿60¿years had higher levels of MV subsets (CD41, CD235, TF and PS) compared to those aged 30¿59¿years. The median CD41, CD105, CD235, TF and PS expressing MV by flow cytometry were similar or lower in females, whilst procoagulant activity by the XaCT assay was higher (p¿=¿0.002). In smokers (n¿=¿21), certain MV subsets (CD41, TF and PS) and functional activity (ETP) was lower (p¿<¿0.05). Regression analysis showed that MV parameters of CD41, CD105, TF and ETP could be predicted independently by age, whilst smoking predicted for CD105, CD235, TF, PS and ETP. Certain MV parameters also correlated with BMI, lipid and hormone levels. The small RNA and miRNA levels did not differ by age group, smoking status or gender. Conclusions It is important to recognize that differences may arise depending on age, gender, BMI, lipid, hormone levels and smoking status in apparently healthy subjects when evaluating MV for pathogenic potential.

Copyright 2016 BMJ Publishing Group. All rights reserved. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive haematological malignancy in the elderly, with a high frequency of cutaneous and bone marrow involvement and poor prognosis. We report a case of BPDCN with classic presentation and discuss its treatment and the value of different investigation tools used in diagnosis and response assessment.

© 2016 Wolters Kluwer Health, Inc. All rights reserved. Treatment of acute myeloid leukaemia (AML) is challenging and emerging treatment options include protein phosphatase 2A (PP2A) activators. Fingolimod is a known PP2A activator that inhibits multiple signalling pathways and has been used extensively in patients with multiple sclerosis and other indications. The initial positive results of PP2A activators in vitro and mouse models of AML are promising; however, its safety for use in AML has not been assessed. From human studies of fingolimod in other indications, it is possible to evaluate whether the safety and toxicity profile of the PP2A activators will allow their use in treating AML. A literature review was carried out to assess safety before the commencement of Phase I trials of the PP2A activator Fingolimod in AML. From human studies of fingolimod in other indications, it is possible to evaluate whether the safety and toxicity profile of the PP2A activators will allow their use in treating AML. A systematic review of published literature in Medline, EMBASE and the Cochrane Library of critical reviews was carried out. International standards for the design and reporting of search strategies were followed. Search terms and medical subject headings used in trials involving PP2A activators as well as a specific search were performed for 'adverse events','serious adverse events', 'delays in treatment', ' side effects' and 'toxicity' for primary objectives. Database searches were limited to papers published in the last 12 years and available in English. The search yielded 677 articles. A total of 69 journal articles were identified as relevant and included 30 clinical trials, 24 review articles and 15 case reports. The most frequently reported adverse events were nausea, diarrhoea, fatigue, back pain, influenza viral infections, nasopharyngitis and bronchitis. Specific safety concerns include monitoring of the heart rate and conduction at commencement of treatment as cardiotoxicity has been reported. There is little evidence to suggest specific bone marrow toxicity. Lymophopenia is a desired effect in the management of multiple sclerosis, but may have implications in patients with acute leukaemia as it may potentially increase susceptibility to viral infections such as influenza. Fingolimod is a potential treatment option for AML with an acceptable risk to benefit ratio, given its lack of bone marrow toxicity and the relatively low rate of serious side effects. As most patients with AML are elderly, specific monitoring for cardiac toxicity as well as infection would be required.

© 2016 Introduction Circulating microvesicles (MV) can be analysed using a number of different techniques. The aim of this study was to evaluate the correlation between functional procoagulant based assays including thrombin generation, factor Xa activation test (XaCT), and phosphatidylserine factor Xa-activity by ELISA with optical MV enumeration by flow cytometry and nanoparticle tracking analysis. Methods Citrated blood samples were collected from 60 healthy volunteer blood donors after informed consent. Platelet free plasma was prepared using a standardized published protocol. MV subsets were enumerated by flow cytometry (BDFACS Canto) after staining with specific antibodies for platelets (CD41), endothelial cells (CD105), red cells (CD235) monocytes (CD14), tissue factor (CD142) and for phosphatidylserine expression by binding to annexin V. A standardized protocol using counting beads was employed. Nanotracking analysis was performed on both scatter and fluorescent settings after MV staining with quantum dot stain, Qdot 655. Procoagulant function was assessed by the XaCT assay on an automated coagulation analyser and by thrombin generation assay measuring endogenous thrombin potential (ETP), lagtime, peak (PEAK) and time to peak (ttPEAK) using a Calibrated Automated Thrombogram (CAT). The statistical analysis was carried out with Statistica 12 software using non-parametric tests (Spearman rank order correlations, with significance set at p¿<¿0.05). Results In normal healthy subjects, thrombin generation parameters correlated with levels of MV measured by flow cytometry. ETP, lagtime, ttPEAK and PEAK correlated with MV expressing phosphatidylserine (rs,Spearman rank order correlation was 0.29, 0.40, 0.31 and 0.34 respectively, p¿<¿0.05), and MV expressing tissue factor (rswas 0.29, 0.40, 0.31 and 0.34 respectively, p¿<¿0.05), whilst red cell derived MV correlated with lagtime, ttPEAK and PEAK (rs,was 0.35,0.30 and 0.3, respectively, p¿<¿0.05). Lagtime and ttPEAK negatively correlated with the clot based XaCT test (rs,was -¿0.34 and -¿0.30 respectively, p¿<¿0.05) and positively correlated with the ELISA MP-activity assay (rs¿=¿0.42 for both, p¿<¿0.05). In addition, endothelial MV levels weakly correlated with white cell counts (rs =0.27, p¿<¿0.05). Conclusions Thrombin generation and flow cytometry for phosphatidylserine or tissue factor expressing MV correlate well as markers for procoagulant activity. A combination of optical or non-optical enumeration as well as functional methods may be required for a complete profiling of circulating MV.

© 2016 Royal College of Pathologists of Australasia Most studies on the sensitivities of coagulation assays to direct oral anticoagulants (DOACs) are based on normal plasma spiked with anticoagulant in the laboratory. Recent studies have shown that reagent sensitivity varies significantly depending on whether spiked or patient samples are used. The aim of this study was to compare the sensitivities of routine coagulation assays in patient samples and commercial drug specific calibrators using commonly used activated partial thromboplastin time (APTT) and prothrombin time (PT) reagents (i.e., Actin FS and Neoplastine CI Plus for APTT and PT, respectively) in Australian laboratories. Samples collected at Pathology North Hunter (PN-H) for dabigatran (n¿=¿39), rivaroxaban, (n¿=¿56) or apixaban levels (n¿=¿22) between February 2013 and November 2015 were analysed and compared to two different commercial drug specific calibrators from different manufacturers for each DOAC. Our results show that dabigatran (Hyphen and Technoclone) and rivaroxaban (Stago) calibrators tend to overestimate the APTT but are similar to patient samples for PT. A cut-off DOAC level of 50¿ng/mL based on results from patient samples within the laboratory can be used as the lower limit which will result in prolongation of APTT for dabigatran (sensitivity 96%, n¿=¿25) and PT for rivaroxaban (sensitivity 97%, n¿=¿29), respectively. Individual laboratories should be familiar with the sensitivity of their coagulation reagents to different DOACs including differences between patient samples versus different commercial drug specific calibrators.

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML.

Copyright © 2015 Royal College of Pathologists of Australasia. All rights reserved. An automated cartridge-based detection system (GeneXpert; Cepheid) is being widely adopted in low throughput laboratories for monitoring BCR-ABL1 transcript in chronic myelogenous leukaemia. This Australian study evaluated the longitudinal performance specific characteristics of the automated system.The automated cartridge-based system was compared prospectively with the manual qRT-PCR-based reference method at SA Pathology, Adelaide, over a period of 2.5 years. A conversion factor determination was followed by four re-validations. Peripheral blood samples (n = 129) with international scale (IS) values within detectable range were selected for assessment. The mean bias, proportion of results within specified fold difference (2-, 3- and 5-fold), the concordance rate of major molecular remission (MMR) and concordance across a range of IS values on paired samples were evaluated.The initial conversion factor for the automated system was determined as 0.43. Except for the second re-validation, where a negative bias of 1.9-fold was detected, all other biases fell within desirable limits. A cartridge-specific conversion factor and efficiency value was introduced and the conversion factor was confirmed to be stable in subsequent re-validation cycles. Concordance with the reference method/laboratory at >0.1-=10 IS was 78.2% and at =0.001 was 80%, compared to 86.8% in the >0.01-=0.1 IS range. The overall and MMR concordance were 85.7% and 94% respectively, for samples that fell within ± 5-fold of the reference laboratory value over the entire period of study.Conversion factor and performance specific characteristics for the automated system were longitudinally stable in the clinically relevant range, following introduction by the manufacturer of lot specific efficiency values.