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Bench A.J.,University of Cambridge | White H.E.,Salisbury NHS Foundation Trust | White H.E.,University of Southampton | Foroni L.,London Health Sciences Center | And 17 more authors.
British Journal of Haematology | Year: 2013

Molecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 1-3%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results. © 2012 Blackwell Publishing Ltd.


Peeling R.W.,London School of Hygiene and Tropical Medicine | Sollis K.A.,London School of Hygiene and Tropical Medicine | Glover S.,London School of Hygiene and Tropical Medicine | Crowe S.M.,Burnet Institute | And 10 more authors.
PLoS ONE | Year: 2015

Background: Measurement of CD4+ T-lymphocytes (CD4) is a crucial parameter in the management of HIV patients, particularly in determining eligibility to initiate antiretroviral treatment (ART). A number of technologies exist for CD4 enumeration, with considerable variation in cost, complexity, and operational requirements. We conducted a systematic review of the performance of technologies for CD4 enumeration. Methods and Findings: Studies were identified by searching electronic databases MEDLINE and EMBASE using a pre-defined search strategy. Data on test accuracy and precision included bias and limits of agreement with a reference standard, and misclassification probabilities around CD4 thresholds of 200 and 350 cells/μl over a clinically relevant range. The secondary outcome measure was test imprecision, expressed as % coefficient of variation. Thirty-two studies evaluating 15 CD4 technologies were included, of which less than half presented data on bias and misclassification compared to the same reference technology. At CD4 counts <350 cells/μl, bias ranged from -35.2 to +13.1 cells/μl while at counts >350 cells/μl, bias ranged from -70.7 to +47 cells/μl, compared to the BD FACSCount as a reference technology. Misclassification around the threshold of 350 cells/μl ranged from 1-29% for upward classification, resulting in under-treatment, and 7-68% for downward classification resulting in overtreatment. Less than half of these studies reported within laboratory precision or reproducibility of the CD4 values obtained. Conclusions: A wide range of bias and percent misclassification around treatment thresholds were reported on the CD4 enumeration technologies included in this review, with few studies reporting assay precision. The lack of standardised methodology on test evaluation, including the use of different reference standards, is a barrier to assessing relative assay performance and could hinder the introduction of new point-of-care assays in countries where they are most needed. © 2015, Public Library of Science. All rights reserved.


Whitby L.,UK NEQAS for Leucocyte Immunophenotyping | Whitby A.,UK NEQAS for Leucocyte Immunophenotyping | Fletcher M.,UK NEQAS for Leucocyte Immunophenotyping | Helbert M.,Royal Infirmary | And 2 more authors.
Cytometry Part B - Clinical Cytometry | Year: 2013

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. Copyright © 2013 International Clinical Cytometry Society.


PubMed | Centers for Disease Control and Prevention, Pangaea Global AIDS Foundation, World Health Organization, University of Witwatersrand and 7 more.
Type: Journal Article | Journal: PloS one | Year: 2015

Measurement of CD4+ T-lymphocytes (CD4) is a crucial parameter in the management of HIV patients, particularly in determining eligibility to initiate antiretroviral treatment (ART). A number of technologies exist for CD4 enumeration, with considerable variation in cost, complexity, and operational requirements. We conducted a systematic review of the performance of technologies for CD4 enumeration.Studies were identified by searching electronic databases MEDLINE and EMBASE using a pre-defined search strategy. Data on test accuracy and precision included bias and limits of agreement with a reference standard, and misclassification probabilities around CD4 thresholds of 200 and 350 cells/l over a clinically relevant range. The secondary outcome measure was test imprecision, expressed as % coefficient of variation. Thirty-two studies evaluating 15 CD4 technologies were included, of which less than half presented data on bias and misclassification compared to the same reference technology. At CD4 counts <350 cells/l, bias ranged from -35.2 to +13.1 cells/l while at counts >350 cells/l, bias ranged from -70.7 to +47 cells/l, compared to the BD FACSCount as a reference technology. Misclassification around the threshold of 350 cells/l ranged from 1-29% for upward classification, resulting in under-treatment, and 7-68% for downward classification resulting in overtreatment. Less than half of these studies reported within laboratory precision or reproducibility of the CD4 values obtained.A wide range of bias and percent misclassification around treatment thresholds were reported on the CD4 enumeration technologies included in this review, with few studies reporting assay precision. The lack of standardised methodology on test evaluation, including the use of different reference standards, is a barrier to assessing relative assay performance and could hinder the introduction of new point-of-care assays in countries where they are most needed.


Sack U.,University of Leipzig | Barnett D.,UK NEQAS for Leucocyte Immunophenotyping | Demirel G.Y.,Yeditepe University | Fossat C.,University Hospital Timone | And 6 more authors.
Cytometry Part B - Clinical Cytometry | Year: 2013

ISO 15189 has been introduced to enable any clinical laboratory, irrespective of geographic location, to be accredited against internationally recognized standards and therefore facilitate direct international comparison of laboratories. Together with increasing use of ISO 15189 for standardization and competition purposes, often triggered by demands of patients and clinicians, clinical flow cytometry laboratories are becoming increasingly challenged to introduce compliant quality management systems. Whilst in most countries, ISO 15189 accreditation is not yet compulsory, there is increasing evidence to suggest that the implementation of this standard is growing. As a result, the European Society of Clinical Cell Analysis (ESCCA) has analysed the impact of accreditation in clinical flow cytometry laboratories. It found, through a discussion forum, that staff qualification, adaptation of multicolour antibody panels, and implementation of a comprehensive quality system (including quality assessment) have been identified as major challenges. Copyright © 2013 International Clinical Cytometry Society.


Smit P.W.,London School of Hygiene and Tropical Medicine | Sollis K.A.,London School of Hygiene and Tropical Medicine | Fiscus S.,University of North Carolina at Chapel Hill | Ford N.,World Health Organization | And 11 more authors.
PLoS ONE | Year: 2014

Background: Dried blood spots (DBS) have been used as alternative specimens to plasma to increase access to HIV viral load (VL) monitoring and early infant diagnosis (EID) in remote settings. We systematically reviewed evidence on the performance of DBS compared to plasma for VL monitoring and EID. Methods and Findings: Thirteen peer reviewed HIV VL publications and five HIV EID papers were included. Depending on the technology and the viral load distribution in the study population, the percentage of DBS samples that are within 0.5 log of VL in plasma ranged from 52-100%. Because the input sample volume is much smaller in a blood spot, there is a risk of false negatives with DBS. Sensitivity of DBS VL was found to be 78-100% compared to plasma at VL below 1000 copies/ml, but this increased to 100% at a threshold of 5000 copies/ml. Unlike a plasma VL test which measures only cell free HIV RNA, a DBS VL also measures proviral DNA as well as cell-associated RNA, potentially leading to false positive results when using DBS. The systematic review showed that specificity was close to 100% at DBS VL above 5000 copies/ml, and this threshold would be the most reliable for predicting true virologic failure using DBS. For early infant diagnosis, DBS has a sensitivity of 100% compared to fresh whole blood or plasma in all studies. Conclusions: Although limited data are available for EID, DBS offer a highly sensitive and specific sampling strategy to make viral load monitoring and early infant diagnosis more accessible in remote settings. A standardized approach for sampling, storing, and processing DBS samples would be essential to allow successful implementation. Trial Registration: PROSPERO Registration #: CRD42013003621. © 2014 Smit et al.

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