Analysis of minimal sample volumes from head and neck cancer by laser scanning cytometry

Background: The increasing diversity in therapeutic strategies in head and neck oncology is dependent on the development of equally appropriate diagnostic tools. A growing number of diagnostic procedures is intended to be performed on an out-patient basis. In this context, analyses of hypocellular specimens such as fine-needle aspirate biopsies (FNABs) or swabs are very important: There are minimal side-effects, and they can be analysed within hours. Material and Methods: Laser scanning microscopy (LSC) is a microscope-based method combining the advantages of flow cytometry and image analysis: In addition to the fluorescence data of each individual cell, its morphology can be documented by re-staining with a conventional cytological staining. Any cell can then be re-localised in the microscope for direct observation. FNABs and swabs are incubated in PBS, erythrocytes are lysed, and cells are mounted on slides. After fixation in ethanol, cells are stained for cytokeratin by indirect immunolabelling and for DNA by propidium iodide. Analysis by LSC is performed to determine the ploidy of the epithelial cells. For immunophenotyping of peripheral blood in cancer patients by LSC 20 Id full blood are stained for CD antigens by direct immunolabelling and for DNA by 7-aminoactinomycin D. Results: FNABs and swabs were taken from 150 malignancies of different sites in total; all specimens yielded sufficient cells (>5,000). 30 tumours of the parotid gland were analysed in detail: Out of 9 malignant tumours 8 showed aneuploidy, whereas all 21 benign tumours were diploid. Immunophenotyping in 23 tumour patients showed a significant reduction of lymphocytes in the peripheral blood as compared to healthy individuals. Conclusions: Further studies have to be performed to validate the analysis of hypocellular specimens by LSC and to determine its role in routine clinical work. Its potential is most evident in tumours that are not accessible for open biopsy such as those of the parotid gland or the larynx.