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Quantitative DNA-image cytometry (ICM) is used to diagnose malignancy via detecting changes in DNA content. We aimed to estimate the value of cytology, DNA-ICM, and their combination in diagnosing pancreatic malignancy.One hundred twenty-one endoscopic ultrasound-guided fine-needle aspiration samples from 116 patients suspected for pancreatic malignancy were examined by cytology and DNA-ICM. Their results and the final diagnoses (malignancy or not) were collected. Diagnostic values were compared among cytology, DNA-ICM, and their combination.The DNA-ICM had a lower sensitivity and accuracy than cytology (64.2% vs 81.1%; 71.9% vs 85.1%). The combination of the techniques significantly improved the diagnostic accuracy compared with that of cytology or DNA-ICM alone (0.932 vs 0.905, P = 0.02; 0.932 vs 0.821, P < 0.0001). Using the Youden index, we determined that one cell with DI (DNA index) ≥2.5 is the optimal cutoff value for DNA-ICM to diagnose pancreatic malignancy. After adoption of this criterion, the sensitivity and accuracy were improved to 74.7% and 80.2% with DNA-ICM and 90.5% and 92.6% with the combined method.The DNA-ICM is an effective complementary method to cytology in diagnosing pancreatic malignancy. Although the diagnostic value for DNA-ICM is lower than that of cytology, an improved value was obtained after their combination.