Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies
Authors Mairinger F, Walter R, Vollbrecht C, Hager T, Worm K, Ting S, Wohlschläger J, Zarogoulidis P, Zarogoulidis K, Schmid K
Received 29 March 2014
Accepted for publication 13 May 2014
Published 13 August 2014 Volume 2014:7 Pages 1441—1447
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 7
Fabian D Mairinger,1 Robert FH Walter,2 Claudia Vollbrecht,3 Thomas Hager,1 Karl Worm,1 Saskia Ting,1 Jeremias Wohlschläger,1 Paul Zarogoulidis,4 Konstantinos Zarogoulidis,4 Kurt W Schmid1
1Institute of Pathology, 2Ruhrlandklinik, West German Lung Center, University Hospital Essen, Essen, 3Institute of Pathology, University Hospital Cologne, Cologne, Germany; 4Pulmonary Department, Oncology Unit, G Papanikolaou General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece
Background and methods: Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection.
Results: A total of 250 µg DNA (concentration 5 µg/µL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings.
Conclusion: We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.
Keywords: isothermal multiple displacement amplification, isothermal, whole-genome amplification, routine diagnostics, biopsies, microcarcinomas, microdissection