Multiparameter absorption measurements in automated microscopy. Simultaneous quantitative determination of DNA and nuclear antigen.

OBJECTIVE: To test a dual DNA-nuclear antigen staining method for multiparameter absorption image analysis. STUDY DESIGN: MCF 7 cells, grown on glass slides, served as a model to test the staining technique. For DNA, Feulgen-based CAS quantitative DNA staining, and for nuclear antigen, alkaline phosphatase-based immunocytochemical staining with CAS Red as the chromogen, were used. MIB-1, estrogen and progesterone receptors were used as examples of nuclear antigen staining. Measurements were performed with the DISCOVERY image analyzer. RESULTS: Scatterplots, in which the nuclear antigen content was plotted against the DNA content, were obtained. Immunostain-positive and -negative populations could be discriminated. These cells were visualized in image galleries. The DNA histograms of the positive and negative cells showed no change in coefficient of variation or integrated optical density ratio of the G0, G1 and G2 + M peaks as compared to single DNA staining. The intensity of the immunostain increased as compared to the single immunostaining result. CONCLUSION: This staining technique allows the simultaneous accurate measurement of costained DNA and antigen within the same nucleus. This opens the possibility for studies in which nuclear antigen expression is monitored during the cell cycle or in cells of different ploidy classes. Identified cells can also be visualized by presentation in an image gallery or by relocation on the slide. This can support the analysis of clinical samples, where cytometric data can be correlated with and confirmed by visual diagnosis.

CytosafePLUS. A workstation for screening, supervision, reviewing, quality assurance and education in cytopathology.

In pathology, the subjective cytologic and histologic diagnosis suffers from large observer variability. Effective methods of reducing the effects of this observer variability in cytologic diagnosis are close supervision of the diagnostic process and multiple screening by different observers of cases that have a higher risk of abnormalities as well as closely supervised follow-up procedures for early lesions. Accurate supervision of screening completeness has become possible with the development of computer programs and microscope instrumentation to register the path of the objective over the slide and therefore the fields that have been inspected by the screener (Axio-Home, Carl Zeiss, Oberkochen, Germany; Navigator, Becton Dickinson Cellular Imaging Systems, Leiden, the Netherlands). Based on these technologies, all objects of interest on the microscope slide are marked electronically, and the X,Y coordinates of these marks are stored in the computer. With these coordinates, a motor-driven microscope stage can be redirected to the selected objects, enabling the observer to quickly review the objects. The reviewer can thus reinspect cells or groups of cells, previously identified, in a much more accurate way than is presently possible, by marking objects of interest with ink dots. To these marked objects, specific diagnoses can be attached, as can comments or questions, which are also stored in the computer and are made part of the cytology report. After analysis, all marked objects can be relocated automatically with the help of the computer-directed microscope stage. Using this concept, a subsequent observer or a supervisor can easily and effectively reply to the remarks attached to these objects. It is also possible to reedit the comments made or to attach answers to specific questions. In an interobserver study, encompassing 50 cases of columnar cell abnormalities screened by five observers using the Navigator microscope stage and software, it proved possible to reduce reviewing time significantly. On the basis of marked cytologic characteristics discriminating cellular features in the diagnosis, endocervical columnar abnormalities could be identified. The coordinates of the objects of interest are made part of the laboratory record of the cytology findings (Cytosafe, Omnisys, Hoevelaken, the Netherlands). This development largely facilitates the review of previous findings when evaluating follow-up specimens. With the use of a Navigator motor-driven stage and CCD camera, microscope images can be made of the specimen under study. These images can be stored in the computer in a digitized format, labeled with their X,Y coordinates on the specimen. The CytosafePLUS workstation, encompassing the Navigator automated cell positioning system and CCD attachment, is a highly effective tool in cytologic screening, reviewing and reporting, in surveillance of follow-up, in intralaboratory and interlaboratory consultation, in cytology training, in proficiency testing and in quality assurance.