The Application of Molecular Diagnostics to Stained Cytology Smears

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Presentation transcript:

The Application of Molecular Diagnostics to Stained Cytology Smears Maja H. Oktay, Esther Adler, Laleh Hakima, Eli Grunblatt, Evan Pieri, Andrew Seymour, Samer Khader, Antonio Cajigas, Mark Suhrland, Sumanta Goswami The Journal of Molecular Diagnostics Volume 18, Issue 3, Pages 407-415 (May 2016) DOI: 10.1016/j.jmoldx.2016.01.007 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic representation of the molecular approach used for mutation and translocation detection. A: qPCR clamp was used for EGFR, KRAS, and BRAF mutation detection. This technology uses a probe constructed using modified nucleotides [xenonucleic acid (XNA)] that perfectly match the wild-type sequence. The XNA probe does not separate from the sample DNA containing the wild-type sequence during the amplification phase, resulting in no PCR product. In the presence of mutation in the DNA sample the loosely bound XNA probe separates from the sample, allowing amplification to proceed. The sample was considered positive when the threshold cycle (CT) difference in reactions with and without the XNA clamp was less than 5 CTs. B: Quantitative RT-PCR was used for echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK) translocation detection. Primers were used along the 3′ and 5′ ends of the ALK gene. EML4–ALK translocation was diagnosed if there were more than 10 CTs difference between 3′ and 5′ during RT-qPCR amplification. POL, polymerase. The Journal of Molecular Diagnostics 2016 18, 407-415DOI: (10.1016/j.jmoldx.2016.01.007) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Examples of control and positive PCR amplification curves. Quantitative PCR (qPCR) clamp amplification curves using a control, wild-type DNA sequence (A), a sample DNA containing a mutation (B), and RT-qPCR amplification curves using a control sample negative for echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK) translocation showing less that 10 threshold cycles (CTs) difference between 3′ and 5′ primers during RT-qPCR amplification (C). D: RT-qPCR amplification curves using a sample positive for EML4–ALK translocation showing more that 10 CTs difference between the 3′ and 5′ ends during RT-qPCR amplification. SYBR green is from Thermo Fisher Scientific. The Journal of Molecular Diagnostics 2016 18, 407-415DOI: (10.1016/j.jmoldx.2016.01.007) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions