ABSTRACT
The aim of the present study is to propose alternative automatic methods to time consuming interactive sorting of elements for DNA ploidy measurements. One archival brain tumour and two archival breast carcinoma were studied, corresponding to 7120 elements (3764 nuclei, 3356 debris and aggregates). Three automatic classification methods were tested to eliminate debris and aggregates from DNA ploidy measurements (mathematical morphology (MM), multiparametric analysis (MA) and neural network (NN)). Performances were evaluated by reference to interactive sorting. The results obtained for the three methods concerning the percentage of debris and aggregates automatically removed reach 63, 75 and 85% for MM, MA and NN methods, respectively, with false positive rates of 6, 21 and 25%. Information about DNA ploidy abnormalities were globally preserved after automatic elimination of debris and aggregates by MM and MA methods as opposed to NN method, showing that automatic classification methods can offer alternatives to tedious interactive elimination of debris and aggregates, for DNA ploidy measurements of archival tumours.
Subject(s)
DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Image Cytometry/methods , Ploidies , Aneuploidy , Astrocytoma/chemistry , Astrocytoma/genetics , Brain Neoplasms/chemistry , Brain Neoplasms/genetics , Breast Neoplasms/chemistry , Breast Neoplasms/genetics , Diploidy , Evaluation Studies as Topic , Female , Humans , Image Cytometry/statistics & numerical data , Neural Networks, ComputerABSTRACT
BACKGROUND: Image cytometry has proved to provide a good alternative to flow cytometry for DNA ploidy measurement of archival tumors. However, when interactively done this technique is unable to give statistically valuable results within an acceptable time for clinical oncology. METHODS: An image cytometer was developed for fully automatic DNA ploidy quantitation, focusing efforts on speed and accuracy. Software functionalities include systematic acquisition of fields on a microscopic slide, detection, localization and sorting of nuclei, computation of the DNA content together with post-processing tools, for a deeper analysis of the DNA ploidy diagram. RESULTS: DNA ploidy analysis of archival breast carcinoma samples illustrates the accuracy of DNA ploidy measurements and the sensitivity in the detection of DNA ploidy abnormalities as a result of cell sorting. CONCLUSIONS: Fully automatic image cytometry is able to combine qualities of flow cytometry (automatic analysis of a statistically significant collection of cell nuclei) with additional advantages: sorting of unwanted events (debris, stromal and inflammatory cell nuclei) and facilities for an a posteriori control of the quality of cell selection. This method is well suited to DNA ploidy analysis of archival cancer samples.
Subject(s)
Aneuploidy , Breast Neoplasms/diagnosis , DNA, Neoplasm/analysis , Image Cytometry/methods , Image Processing, Computer-Assisted/methods , Software , Breast Neoplasms/genetics , Cell Nucleus/pathology , Expert Systems , Female , Flow Cytometry , Humans , Image Cytometry/instrumentation , Image Processing, Computer-Assisted/instrumentation , Paraffin Embedding , Ploidies , Sensitivity and Specificity , Time FactorsABSTRACT
An automatic machine, dedicated to solid tumor DNA ploidy quantitation has been built in order to provide pathologists with a tool usable in clinical practice. Main efforts were focused on an automation of each step of the analysis and on an elimination of any subjective choice, while preserving the quality of measurement. As the software is independent of the machine architecture, it offers performances which increase in parallel with the rapid evolution of the computers. An illustration of the various functionalities of the automaton is proposed through the study of deparaffined breast cancer samples.
Subject(s)
DNA, Neoplasm/analysis , Image Processing, Computer-Assisted , Software , Breast Neoplasms/chemistry , Breast Neoplasms/pathology , Cell Nucleus/chemistry , Cell Nucleus/pathology , Female , Flow Cytometry , Humans , Image Processing, Computer-Assisted/methods , Ploidies , Reproducibility of ResultsABSTRACT
Biomembranes can be stained on large semi-thin sections of epoxy embedded tissue with silver nitrate after iodine impregnation and without resin removal.
