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Fletcher M.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI | Sutherland D.R.,Laboratory Medicine Program | Whitby L.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI | Whitby A.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI | And 7 more authors.
Cytometry Part B - Clinical Cytometry | Year: 2014

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. © 2014 International Clinical Cytometry Society.

Whitby L.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI | Whitby A.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI | Fletcher M.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI | Barnett D.,UK NEQAS for Leucocyte Immunophenotyping UK NEQAS LI
Cytometry Part B - Clinical Cytometry | Year: 2015

Background CD4+ T-lymphocyte subset enumeration is routinely used for monitoring HIV disease progression, with approximately 300,000 tests performed annually in the UK alone. Technical variables can impact upon any laboratory test and therefore the final result obtained. Here, we report the findings of a survey questionnaire issued to 1,587 clinical flow cytometry laboratories to: (a) determine if the UK NEQAS for Leucocyte Immunophenotyping (UK NEQAS LI) lymphocyte subset external quality assessment (EQA) programme was suitable for current laboratory needs and practices; and (b) assess the impact of these responses on clinical practice where CD4+ T-lymphocyte subsets analysis is undertaken. The survey covered areas not traditionally examined by EQA such as: staffing numbers, flow cytometer age and service intervals, plus six test specific sections covering: leukaemia immunophenotyping, CD4+ T-lymphocyte subsets analysis (reported here), CD34+ stem cell testing, low level leucocyte enumeration, minimal residual disease testing and PNH testing. Results The responses revealed major methodological variations between centres undertaking CD4+ T-lymphocyte subset analysis. Significant differences existed in basic laboratory practices such as: normal range derivation; pipetting techniques; instrument maintenance and units of reporting, all of which results in non-adherence to international guidelines. Discussion Despite the availability of international guidelines our survey highlighted a lack of concordance amongst laboratory techniques. Such variation could adversely impact on patient care and clinical trial data. Therefore, it is recommended centres undertaking flow cytometric CD4+ T-lymphocyte subsets analysis urgently review their methodologies and normal ranges to ensure they are fit for purpose and meet current international guidelines. © 2015 International Clinical Cytometry Society.

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