Crystal structure of a methimazole-based ionic liquid.

The structure of 1-methyl-2-(prop-2-en-1-ylsulfan-yl)-1H-imidazol-3-ium bromide, C7H11N2S(+)·Br(-), has monoclinic (P21/c) symmetry. In the crystal, the components are linked by N-H⋯Br and C-H⋯Br hydrogen bonds. The crystal structure of the title compound undeniably proves that methimazole reacts through the thione tautomer, rather than the thiol tautomer in this system.

Monitoring of chimerism following allogeneic haematopoietic stem cell transplantation (HSCT): technical recommendations for the use of short tandem repeat (STR) based techniques, on behalf of the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping Chimerism Working Group.

Analysis of short tandem repeats (STR) is the predominant method for post-transplant monitoring of donor engraftment. It can enable early detection of disease relapse, level of engraftment and provide useful information on the graft-versus-host disease (GVHD)/graft-versus-tumour (GVT) effect, facilitating therapeutic intervention. Harmonization and standardization of techniques and result interpretation is essential to reduce the impact of laboratory variability on both clinical management and the results of multi-centre clinical trials. However, the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) has highlighted significant issues inherent in STR testing that impact upon inter- and intra- laboratory variation. We present here consensus best practice guidelines and recommendations for STR chimerism testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 11 UK and Eire clinical laboratories. This document uses data obtained from the UK NEQAS LI Post-Stem Cell Transplant (SCT) Chimerism Monitoring Programme.

Standardizing leucocyte PNH clone detection: an international study.

BACKGROUND: Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol-deficient structures on red blood cells and white blood cells in paroxysmal nocturnal hemoglobinuria (PNH) were recently published. METHODS: UK NEQAS LI issued three stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific Standard Operating Procedures (SOPs) and pretitered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH External Quality Assessment (EQA) programs. RESULTS: Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their "in-house" methods, though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an interquartile range of 1.7% and this was demonstrated to be significantly different (P<0.001) to the standardized cohort. CONCLUSIONS: The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen among the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories.

Standardizing Leucocyte PNH clone detection: An international study.

Background: Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol (GPI)-deficient structures on Red Blood Cells (RBCs) and White Blood Cells (WBCs) in Paroxysmal Nocturnal Hemoglobinuria (PNH) were recently published. Methods: UK NEQAS LI issued 3 stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific SOPs and pre-titered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH EQA programmes. Results: Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their 'in-house' methods though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an inter-quartile range of 1.70 and this was demonstrated to be significantly different (P<0.001) to the standardized cohort. Conclusions: The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen amongst the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories. © 2014 Clinical Cytometry Society.

Comparison of methodological data measurement limits in CD4⁺ T lymphocyte flow cytometric enumeration and their clinical impact on HIV management.

UK NEQAS for Leucocyte Immunophenotyping, an ILAC G13:2000 accredited External Quality Assessment (EQA) organization, with over 3000 international laboratories participating in 14 programmes, issues 2 proficiency testing samples of stabilized whole blood to 824 participants in the Immune Monitoring (lymphocyte subset) programme every two months. We have undertaken a study of 58,626 flow cytometric absolute CD4⁺ T lymphocyte count data sets from these laboratories over a 12-year-period (2001-2012) to determine counting method variation in data measurement limits and how this could influence the clinical management of HIV patients. Comparison of relative error and 99.9% confidence limits for absolute CD4⁺ T lymphocyte values was undertaken using dual platform (DP) and single platform (SP) data and showed that the SP consistently outperformed DP, giving lower relative errors and confidence limits at clinically significant absolute CD4⁺ T lymphocyte counts. Our data shows that absolute CD4⁺ T lymphocyte counts should be obtained using single platform technology to reduce the variability at clinically relevant levels. On data where results (irrespective of platform) were below the international treatment threshold of 350 cells/µl, there was no significant misclassification between either SP or DP techniques meaning most patients would receive the correct treatment at the correct time. However, results that were above the treatment level of 350 cells/µl had a significant difference (P = 0.04) between DP and SP platforms, suggesting patients monitored using DP technology were 20% more likely to start therapy prematurely than those monitored with SP technology.

Current treatment practices for essential thrombocythemia: survey results from European hematologists/oncologists.

Physicians from nine European countries were asked to complete a survey, which was conducted in two waves (Wave II, October 2009; Wave III, May 2010), based on their current treatment practices for essential thrombocythemia (ET). The aim of the study was to gain insight into physicians' criteria for treatment initiation and reasons for switching from one therapy option to another. The majority of patients receiving first-line cytoreductive therapy for ET were treated with hydroxycarbamide (HC; 63 and 71% in Waves II and III, respectively), while the majority of patients on second-line therapy received anagrelide (51 and 60% in Waves II and III, respectively). Efficacy was the main factor cited for switching therapies (cited by 47 and 58% of physicians in Waves II and III, respectively). Further studies are needed to determine whether current practices used by physicians for the treatment of ET are consistent with consensus guidelines.

JAK2V617F homozygosity arises commonly and recurrently in PV and ET, but PV is characterized by expansion of a dominant homozygous subclone.

Subclones homozygous for JAK2V617F are more common in polycythemia vera (PV) than essential thrombocythemia (ET), but their prevalence and significance remain unclear. The JAK2 mutation status of 6495 BFU-E, grown in low erythropoietin conditions, was determined in 77 patients with PV or ET. Homozygous-mutant colonies were common in patients with JAK2V617F-positive PV and were surprisingly prevalent in JAK2V617F-positive ET and JAK2 exon 12-mutated PV. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, we demonstrate that recurrent acquisition of JAK2V617F homozygosity occurs frequently in both PV and ET. PV was distinguished from ET by expansion of a dominant homozygous subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions. Our results suggest a model in which development of a dominant JAK2V617F-homzygous subclone drives erythrocytosis in many PV patients, with alternative mechanisms operating in those with small or undetectable homozygous-mutant clones.

Improving survival trends in primary myelofibrosis: an international study.

PURPOSE: Despite the lack of major improvements in the treatment of primary myelofibrosis (PMF), there are recent indications that the survival of patients might have increased over the years. This study was aimed at ascertaining whether survival prolongation has actually occurred in PMF. PATIENTS AND METHODS: A total of 802 patients diagnosed with PMF in four European countries were compared for the presentation of features and survival according to the diagnostic periods 1980 to 1995 (n = 434) and 1996 to 2007 (n = 368); relative survival was estimated for the two groups. RESULTS: Patients diagnosed between 1996 and 2007 more often had constitutional symptoms (31% v 23%) but a lower incidence of marked anemia (31% v 39%), leukocytosis greater than 25 × 10(9)/L (9% v 13%), and blood blasts (27% v 33%); risk distribution was comparable between the two groups. Median survival was 4.6 years (95% CI, 4.0 to 5.1) for patients from 1980 to 1995 and 6.5 years (95% CI, 5.5 to 7.4) for patients from 1996 to 2007 (P < .001). The latter group of patients showed improved relative survival, especially for women, patients younger than age 65 years, and patients with low or intermediate-1-risk disease. Rates of PMF-attributable mortality at 5 and 10 years were significantly lower in the second period; this reduction in disease-specific mortality occurred across all patient subgroups, except in intermediate-2-risk or high-risk patients. CONCLUSION: Survival of PMF is steadily improving, except in patients in poor-risk categories. This observation must be taken into account at the time of evaluating the survival impact of newer therapies for PMF, which are currently being tested in these patient subpopulations.

Guideline for the diagnosis and management of myelofibrosis.

The guideline group regarding the diagnosis and management of myelofibrosis was selected to be representative of UK-based medical experts, together with a contribution from a single expert from the USA. MEDLINE and EMBASE were searched systematically for publications in English from 1966 until August 2011 using a variety of key words. The writing group produced the draft guideline, which was subsequently revised by consensus of the members of the General Haematology and Haemato-oncology Task Forces of the British Committee for Standards in Haematology (BCSH). The guideline was then reviewed by a sounding board of UK haematologists, the BCSH and the British Society for Haematology Committee and comments incorporated where appropriate. The criteria used to state levels and grades of evidence are as outlined in the Procedure for Guidelines commissioned by the BCSH; the 'GRADE' system was used to score strength and quality of evidence. The objective of this guideline is to provide healthcare professionals with clear guidance on the investigation and management of primary myelofibrosis, as well as post-polycythaemic myelofibrosis (post-PV MF) and post-thrombocythemic myelofibrosis (post-ET MF) in both adult and paediatric patients.

VERITAS?: A time for VERIQAS™ and a new approach to training, education, and the quality assessment of CD4+ T lymphocyte counting (I).

BACKGROUND: The aim of clinical laboratories is to produce accurate and reproducible results to enable effective and reliable clinical practice and patient management. The standard approach is to use both internal quality control (IQC) and external quality assessment (EQA). IQC serves, in many instances, as a "go, no go" tool to provide real time assurance that instruments and reagent or test systems are performing within defined specifications. EQA however, takes a snapshot at a specific point in time of the full testing process, results are compared to other laboratories performing similar testing but inevitably has some built in delay from sample issue to performance data review. In addition, if IQC or EQA identify areas of concern it can be difficult to determine the exact nature of the problem. In an attempt to address this problem, we have developed an instant QA panel that we have termed VERIQAS™, specifically for CD4(+) T lymphocyte counting, and have undertaken a "proof of principle" pilot study to examine how the use of VERIQAS™ could result in improvement of laboratory performance. In addition, we have examined how this approach could be used as a training and education tool (in a domestic/international setting) and potentially be of value in instrument validation/switch studies (a switch study being defined as a laboratory changing from one method/instrument to a new method/instrument with the VERIQAS™ panel being used as an adjunct to their standard switch study protocol). METHODS: The basic panel consists of 20 stabilized samples, with predefined CD4(+) T lymphocyte counts, that span low clinically relevant to normal counts, including some blinded replicates (singlet up to quadruplicate combinations). The CD4(+) T lymphocyte target values for each specimen is defined as the trimmed mean ± 2 trimmed standard deviations, where the trimmed values are derived from the CD4(+) T lymphocyte counts reported by the participating centers (~780 laboratories) that receive each UK NEQAS for Leucocyte Immunophenotyping send out. Results for the VERIQAS™ panel were returned online, via a specially designed website, and the participant was provided with an immediate assessment (pass or fail). RESULTS: To date, the panel has been preliminary trialed by eight laboratories to (i) assess pre-EQA qualification (two laboratories); (ii) address performance issues (two laboratories); or (iii) validate new instruments or techniques (four laboratories). Interestingly, even in this pilot study, the panel has been instrumental in identifying specific technical problems in laboratories with EQA performance issues as well as confirming that implementation of new techniques or instruments have been successful. CONCLUSION: We report here a new and novel "proof of principle" pilot study to quality assessment, that we have termed VERIQAS™, designed to provide instant feedback on performance. Participating laboratories receive 20 "blinded" samples that are in singlet up to quadruplicate combinations. Once a centre reports its results via a website, immediate feedback is provided to both the participant and the EQA organizers, enabling, if required, the initiation of targeted remedial action. We have also shown that this approach has the potential to be used as a tool for prequalification, troubleshooting, training and instrument verification. Pilot phase field trials with VERIQAS™ have shown that the panel can highlight laboratory performance problems, such as suboptimal instrument set up, pipetting and gating strategies, in a rapid and efficient manner. VERIQAS™ will now be introduced, where appropriate, as a second phase study within UK NEQAS for Leucocyte Immunophenotyping to assist those laboratories that have performance issues and also made available to laboratories for training and education of staff and instrument validation studies.

ISHAGE protocol: are we doing it correctly?

BACKGROUND: Flow cytometric CD34(+) stem cell enumeration is routinely performed to optimize timing of peripheral blood stem cell collections and assess engraftment capability of the apheresis product. While a number of different flow methodologies have been described, the highly standardized ISHAGE protocol is currently the most widely employed, with 204/255 (81%) international participants in the UK NEQAS CD34(+) stem cell enumeration program indicating their use of this method. Recently, two laboratories were identified as persistent poor performers, a fact attributed to incorrect ISHAGE protocol usage/setup. This prompted UK NEQAS to question whether other laboratories were making similar errors and, if so, how this might affect individual EQA performance. METHODS AND RESULTS: In send out 0801, where two stabilized samples were issued, the EQA center surveyed 255 participants with flow analysis data and subsequent results collected. One hundred and ninety-six laboratories returned results with 103 returning dot plots. Eighty-three out of one hundred and three stated that they used the ISHAGE protocol gating strategy but 43% (36/83) were incorrectly set-up. Analysis of the data showed those incorrectly using single platform ISHAGE gating strategy were twice as likely to fail an EQA exercise compared to those using the protocol correctly. This failure rate increased two fold when incorrect ISHAGE protocol was used in a dual platform setting. CONCLUSION: This study suggests a widespread fundamental lack of understanding of the ISHAGE protocol and the need to deploy it correctly, potentially having significant clinical implications and highlights the need to monitor participants rigorously in their deployment of the ISHAGE protocol. It is hoped that once these findings have been disseminated, performance can be improved.

Inhibition of histidine ammonia lyase by 8-methoxypsoralen and psoralen-oxidized photoproducts.

The effect of 8-methoxypsoralen-UVA therapy on the catalysis of histidine to trans-urocanic acid by histidine ammonia lyase (HAL, EC 4.3.1.3) was examined using an enzymatic assay from Sigma-Aldrich where the growth of the trans-urocanic acid peak at 277 nm was monitored. A Rayonet Photochemical Mini Reactor (Model RMR-600) equipped with eight, 3500 Å light sources and a custom UVA filter (Model S-BAL3 2.9 mm), from the Solar Light Company, were used to expose various reaction mixtures to broadband UVA light and UVA/UVB light. A UV-Vis spectrophotometer (Model Shimadzu UV 2540) with a temperature-controlled cell holder (Model TCC240) was used to monitor the growth of the trans-urocanic peak. Results of dark-binding experiments of 8-methoxypsoralen in denatured ethanol indicate no inhibition of enzyme activity due to ethanol but noncompetitive inhibition due to 8-methoxypsoralen. The effects of preirradiated 8-methoxypsoralen, with both broadband UVA and UVA/UVB, indicate that inhibition was due to psoralen-oxidized photoproducts. Inhibition of HAL was found when exposed to broadband UVA/UVB and to a lesser extent when exposed to broadband UVA.

Molecular mechanisms associated with leukemic transformation of MPL-mutant myeloproliferative neoplasms.

Somatic activating mutations in MPL, the thrombopoietin receptor, occur in the myeloproliferative neoplasms, although virtually nothing is known about their role in evolution to acute myeloid leukemia. In this study, the MPL T487A mutation, identified in de novo acute myeloid leukemia, was not detected in 172 patients with a myeloproliferative neoplasm. In patients with a prior MPL W515L-mutant myeloproliferative neoplasm, leukemic transformation was accompanied by MPL-mutant leukemic blasts, was seen in the absence of prior cytoreductive therapy and often involved loss of wild-type MPL by mitotic recombination. Moreover, clonal analysis of progenitor colonies at the time of leukemic transformation revealed the presence of multiple genetically distinct but phylogenetically-related clones bearing different TP53 mutations, implying a mutator-phenotype and indicating that leukemic transformation may be preceded by the parallel expansion of diverse hematopoietic clones.