Analysis of minimal sample volumes from head and neck cancer by laser scanning cytometry.
ABSTRACT 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.
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 microl full blood are stained for CD antigens by direct immunolabelling and for DNA by 7-aminoactinomycin D.
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.
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.
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ABSTRACT: Flow cytometry (FCM) is the gold standard for immunophenotyping of peripheral blood leukocytes (PBLs). Slide-based cytometry (SBC) systems, for example the laser scanning cytometer (LSC(R), CompuCyte), can give additional information (repeated staining and scanning, morphology). In order to adequately judge the clinical usefulness of LSC for immunophenotyping it is obligatory to compare it with FCM. The aim of this study was to systematically compare immunophenotyping by both FCM and LSC methods and to test the correlation of the results. PBLs were stained with directly labeled monoclonal antibodies with the whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in parallel by a FACScan (BD-Biosciences) using standard protocols and by LSC with different triggers (forward scatter, CD45 FITC, or 7-AAD). For 7-AAD measurements by LSC, slides were additionally fixed with acetone before 7-AAD staining. Calculating the percentage distribution of PBLs obtained by LSC and by FCM showed very good correlation with regression coefficients close to 1.0 for the major populations and the lymphocyte sub-populations (neutrophils, monocytes, and lymphocytes; T-helper-, T-cytotoxic-, B-, NK-cells). The best trigger for LSC was 7-AAD. LSC can be recommended for immunophenotyping of PBLs especially in cases where only limited sample volumes are available or where additional analysis of the cells' morphology is important. The detection of rare leukocytes or weak antigens is limited; in these cases appropriate amplification steps for immunofluorescence should be engaged.Journal of Immunological Methods 05/2006; 311(1-2):130-8. · 2.23 Impact Factor
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ABSTRACT: One hundred and fifty years after Virchow introduced his fundamental concept of cellular pathology, we now have tools that allow us to unravel the mechanisms of single living cells on a previously unprecedented level of detail. By exploring the molecular cellular phenotype, multiparametric cytometry not only detects specific cellular functions in general but also offers insights into the interaction of single subunits of proteins (e.g., growth factor receptors). Several quantitative and objective techniques allow analysis of single-cell preparations as well as tissue sections to obtain data on different cellular parameters. This opens the way to quantitative and objective histology, which in the future may be possible even without blood or the need to make an incision. To use this huge amount of data for treatment decisions in an individual patient, novel bioinformatic concepts are needed in order to predict the individual course of a disease. The concept of cytomics centers on the cell as the integral unit of all life and explores diseases starting from the cell and going to subcellular units (top-down analysis).HNO 05/2008; 56(4):383-8. · 0.42 Impact Factor
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ABSTRACT: Polychromatic analysis of biological specimens has become increasingly important because of the emerging new fields of high-content and high-throughput single cell analysis for systems biology and cytomics. Combining different technologies and staining methods, multicolor analysis can be pushed forward to measure anything stainable in a cell. We term this approach hyperchromatic cytometry and present different components suitable for achieving this task. For cell analysis, slide based cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed following restaining of the object. We demonstrate various approaches for hyperchromatic analysis on a SBC instrument, the Laser Scanning Cytometer. The different components demonstrated here include (1) polychromatic cytometry (staining of the specimen with eight or more different fluorochromes simultaneously), (2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), (3) differential photobleaching (differentiating fluorochromes by their different photostability), (4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and (5) photodestruction (destruction of FRET dyes). Based on the ability to relocate cells that are immobilized on a microscope slide with a precision of approximately 1 microm, identical cells can be reanalyzed on the single cell level after manipulation steps. With the intelligent combination of several different techniques, the hyperchromatic cytometry approach allows to quantify and analyze all components of relevance on the single cell level. The information gained per specimen is only limited by the number of available antibodies and sterical hindrance.Cytometry Part A 08/2006; 69(7):691-703. · 3.71 Impact Factor