BACKGROUNDThe liquid-based cytology Papanicolaou (Pap) test has gradually replaced the conventional Pap test, as it seems to be more efficient, in part because it uses computer assistance. However, Pap test diagnoses are still subjective and are cost-consuming or time-consuming. The authors describe a new digital holographic microscopy instrument and accompanying software. This technology provides an instantaneous, 3-dimensional image reconstruction of cells directly from the uterine cervical sample vial. METHODSAnalyses were performed using the proprietary digital holographic microscopy (DHM) instrument, computer, and software. Residual materials from 3 randomly chosen laboratories were analyzed and subjected to DHM, and the sensitivity and specificity of DHM were calculated for the detection neoplasia. RESULTSIn 41 specimens that yielded normal Pap test results, 1333 cells were analyzed using DHM; and, in 30 specimens that yielded abnormal Pap test results, 494 cells were analyzed using DHM. Available histologic correlation was as follows: 4 histologic samples were diagnosed as grade 1 cervical intraepithelial neoplasia (corresponding to 2 cytologic diagnoses of low-grade squamous intraepithelial lesion and 2 cytologic diagnoses of atypical squamous cells of undetermined significance), 4 histologic samples were diagnosed as grade 3 cervical intraepithelial neoplasia (corresponding to 2 cytologic diagnoses of low-grade squamous intraepithelial lesion and 2 cytologic diagnosis of atypical squamous cells, cannot rule out high-grade squamous intraepithelial lesion), and 11 lesions were diagnosed as benign. Receiver operating characteristic curve analysis indicated that the best criteria were the nuclear-to-cytoplasmic ratio (area under the curve, 0.88; 95% confidence interval, 0.68-1.00) and the greatest nuclear dimension (area under the curve, 0.85; 95% confidence interval, 0.66-1.00). CONCLUSIONSThis preliminary study demonstrated for the first time that the DHM technique is suitable for the processing of gynecologic cervical samples. Nevertheless, DHM criteria and parameters could be better defined. Hopefully, holographic analysis will be performed automatically and will provide an instantaneous, cost-effective diagnosis from a closed vial with the preservation of all cellular material. Cancer Cytopathol 2016;124:573-80. (c) 2016 American Cancer Society. This preliminary study demonstrates for the first time that digital holographic microscopy is a suitable technique for screening gynecologic cervical samples. Although the criteria and parameters for holographic analysis need better definition, holographic analysis eventually may be performed automatically and could provide an instantaneous, cost-effective diagnosis from a closed vial with the preservation of all cellular material.