Improved Detection of the KIT D816V Mutation in Patients with Systemic Mastocytosis Using a Quantitative and Highly Sensitive Real-Time qPCR Assay Thomas.

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Improved Detection of the KIT D816V Mutation in Patients with Systemic Mastocytosis Using a Quantitative and Highly Sensitive Real-Time qPCR Assay Thomas Kristensen, Hanne Vestergaard, Michael Boe Møller The Journal of Molecular Diagnostics Volume 13, Issue 2, Pages 180-188 (March 2011) DOI: 10.1016/j.jmoldx.2010.10.004 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Real-time qPCR amplification plots. A: Amplification of a fourfold dilution series of KIT D816V mutation-positive DNA into wild-type DNA by the mutation-specific assay. The undiluted sample contained genomic DNA from approximately 13,800 mutation-positive heterozygous cells. The number of mutation-positive cells in each dilution is indicated at the curve. The average PCR efficiency of four standard curve experiments was 94%. B: Amplification of a KIT D816V mutation-negative donor sample by the control and mutation-specific assays. The mutation-specific assay produced amplification in a single replicate as a result of a weak nonspecific cross-reaction with the wild-type allele. The Journal of Molecular Diagnostics 2011 13, 180-188DOI: (10.1016/j.jmoldx.2010.10.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Reproducibility of the KIT D816V mutation detection method determined by repeated analysis of four PBMNC samples in five different qPCR runs. The percentage KIT D816V mutation-positive cells is indicated by a gray bar, with the corresponding assay sensitivity indicated by a white bar. The Journal of Molecular Diagnostics 2011 13, 180-188DOI: (10.1016/j.jmoldx.2010.10.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 The percentage KIT D816V mutation-positive PBMNCs in two cases of ASM, four cases of SM-AHNMD, and 15 cases of ISM. Case IDs refer to cases in Table 1. In mutation-positive samples, the percentage of mutation-positive cells in cases of ASM is indicated by a black bar, SM-AHNMD by a dark gray bar, and ISM by a light gray bar. The assay sensitivity of the individual samples is indicated by a white bar. Cases 3, 17, and 18, with no detectable KIT D816V mutation in PBMNCs, were all cases of ISM with the mutation detected in tissue, thus confirming that the patients carried the mutation. The Journal of Molecular Diagnostics 2011 13, 180-188DOI: (10.1016/j.jmoldx.2010.10.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions