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Master Thesis

Développement d'un test standardisé de quantification de l'expression de la myéloperoxydase des polynucléaires neutrophiles du sang périphérique par cytométrie en flux pour le diagnostic d'exclusion des syndromes myélodysplasiques : évaluation de différentes méthodes de perméabilisation

Abstract : Myelodysplastic syndromes (MDS) are clonal bone marrow neoplasms that predominate in the elderly whose diagnostic work-up relies on bone marrow aspiration. To prevent the risks and discomfort associated with bone marrow aspiration, a peripheral blood assay based on the semi-quantification of myeloperoxidase (MPO) in neutrophils by flow cytometry was jointly developed by three university-affiliated hospitals (Grenoble, Clermont-Ferrand and Saint-Etienne). An intra-individual robust coefficient of variation (RCV) value for peripheral blood neutrophil MPO expression lower than 30% accurately rules out MDS with a sensitivity estimate of 100%. The concept has been validated at Grenoble University Hospital and an inter-regional project has been accepted for multicenter validation. To facilitate the use of the test in other laboratories, it is important to ensure the possibility of using reagents other than those we have used, especially for membrane permeabilization. Indeed, many commercial kits are available in the commercial market.The aim of this study was to examine performances of two permeabilization kits (Dako Intrastain™ and BD FACS™ Permeabilizing Solution 2) compared to the method reference (BD Intrasure™ kit). We compared the RCV values for peripheral blood neutrophil MPO expression obtained with the 3 permeabilization methods for 10 controls and 10 cases. Controls were adults with unconfirmed suspicions of MDS. Cases were adults with established diagnosis of MDS or CMML, as defined by current guidelines. The RCV values with BD FACS™Permeabilizing Solution 2 were quantitatively comparable to the reference protocol (mean relative change, -1%). All cases with established MDS diagnosis had RCV values higher than 30%. However, RCV of neutrophil MPO expression using Dako InstraStain Kit appears to be different (mean relative change, +64%) and without discrimination between cases and controls. In conclusion, our findings suggest that BD FACS™ Permeabilizing Solution 2 represents a good technique for RCV of neutrophil MPO expression in peripheral blood compare with reference protocol. A standardization of different fixation/permeabilization methods is needed in order to use the published cut off 30%.
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Master Thesis
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https://dumas.ccsd.cnrs.fr/dumas-03215744
Contributor : Jean-Hugues Morneau <>
Submitted on : Monday, May 3, 2021 - 3:07:53 PM
Last modification on : Wednesday, May 5, 2021 - 3:23:33 AM

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  • HAL Id : dumas-03215744, version 1

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Laura Scheffen. Développement d'un test standardisé de quantification de l'expression de la myéloperoxydase des polynucléaires neutrophiles du sang périphérique par cytométrie en flux pour le diagnostic d'exclusion des syndromes myélodysplasiques : évaluation de différentes méthodes de perméabilisation. Médecine humaine et pathologie. 2021. ⟨dumas-03215744⟩

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