Introduction: Approximately one fifth of stage I and II colon cancer patients will suffer from recurrent disease. This is partly due to the presence of small nodal tumour infiltrates, which are undetected by standard histopathology using Haematoxylin & Eosin (H&E) staining on one slice and thus may not receive beneficial adjuvant therapy. A new diagnostic, semi-automatic system, called one-step nucleic acid amplification (OSNA), was recently designed for the detection of cytokeratin 19 (CK19) mRNA as a surrogate for lymph node metastases. The objective of the present investigation was to compare the performance of OSNA with both standard H&E as well as intensive histopathologic analyses in the detection of colon cancer lymph node micro- and macro-metastases.Methods: In this prospective study 313 lymph nodes from 22 consecutive stage I - III colon cancer patients were assessed. Half of each lymph node was analysed initially based on one slice of H&E followed by an intensive histologic work-up (5 levels of H&E and immuno-histochemistry staining for each slice), the other half was analysed using OSNA.Results: All OSNA results were available after less than 40 minutes. Fifty-one lymph nodes were positive and 246 lymph nodes negative with both OSNA and standard H&E. OSNA was more sensitive to detect small nodal tumor infiltrates compared to H&E (11 OSNA pos. /H&E neg.). Compared to intensive histopathologic analyses, OSNA had a sensitivity of 94.5% and a specificity of 97.6% to detect lymph node micro- and macro-metastases with a concordance rate of 97.1%. An upstaging due to OSNA was found in 2/13 (15.3%) initially node negative colon cancer patients.Conclusion: OSNA appears to be a powerful and promising molecular tool for the detection of lymph node macro- and micro-metastases in colon cancer patients. OSNA has a similar performance in the detection of micro- and macro-metastases compared to intensive histopathologic investigations and appears to be superior to standard histology with H&E. Since the use of OSNA allows the analysis of the whole lymph node, the problem of sampling bias and undetected tumor deposits due to uninvestigated material will be overcome in the future and OSNA may thus improve staging in colon cancer patients. It is hoped that this improved staging will lead to better patient selection for adjuvant therapy and consecutively improved local and distant control as well as better overall survival.