Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis
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- Georg Greiner
- Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
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- Michael Gurbisz
- Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
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- Franz Ratzinger
- Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
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- Nadine Witzeneder
- Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
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- Ingrid Simonitsch-Klupp
- Department of Pathology, Medical University of Vienna, Vienna, Austria
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- Gerlinde Mitterbauer-Hohendanner
- Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
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- Matthias Mayerhofer
- Ludwig Boltzmann Institute of Osteology, Hanusch Hospital, Vienna, Austria
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- Leonhard Müllauer
- Department of Pathology, Medical University of Vienna, Vienna, Austria
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- Wolfgang R Sperr
- Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria
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- Peter Valent
- Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria
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- Gregor Hoermann
- Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
説明
<jats:title>Abstract</jats:title> <jats:sec> <jats:title>BACKGROUND</jats:title> <jats:p>The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations.</jats:p> </jats:sec> <jats:sec> <jats:title>METHODS</jats:title> <jats:p>We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR).</jats:p> </jats:sec> <jats:sec> <jats:title>RESULTS</jats:title> <jats:p>dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR (P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival (P < 0.001).</jats:p> </jats:sec> <jats:sec> <jats:title>CONCLUSIONS</jats:title> <jats:p>dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis.</jats:p> </jats:sec>
収録刊行物
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- Clinical Chemistry
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Clinical Chemistry 64 (3), 547-555, 2018-03-01
Oxford University Press (OUP)