Clinical fluorescence cytometry : improvements to preparation methods and instrumentation

Abstract

Fluorescence cytometry, which can be carried out by flow or image cytometry (FCM, ICM), provides a highly sensitive and specific tool for quantitative analysis of cell material. It is emerging from being a pure research method and becoming an important adjunct to clinical practice. To better realize its potential, more research is needed. This thesis focuses on improvements to preparation methods and instrumentation.

DNA flow cytometry of cells from solid tumors can be used to analyze ploidy and proliferation in tumor cell populations and it appears to hold a considerable potential in terms of predicting the aggressiveness of the lesions. In this thesis, a technique for enucleation of tissues is presented that is based on formalin fixation and protease treatment. In fresh tissues, the procedure generates minimal amounts of debris and, in both embedded and fresh tissues, it produces suspensions of cell nuclei with good stainability of DNA and extremely low levels of aggregation. Low debris and aggregate levels are advantageous for high sensitivity in the analysis. The new method was applied to the preparation of fresh tumor tissues and compared with the widely used Vindelöv technique. We found that it is preferable to use the new method since it produces less debris and aggregates. Furthermore, different algorithms for post-acquisition subtraction of "background" were compared. The best correction was obtained with a combined cut-nuclei aggregate correction algorithm. To give a quantitative estimate of the certainty of S-phase measurement for proliferation analysis, a procedure was developed, that takes into account the magnitude of the "background", the resolution of the analysis, the ploidy level and the number of measured cells. A significant correlation was found between S-phase values measured by FCM and ICM in paired clinical samples. The low coefficient of correlation in this comparison was attributed to the high random error due to low cell numbers in ICM histograms.

In the analysis of cells labeled with multiple fluorophores, the sensitivity of the fluorescence analysis can be reduced as a result of spectral overlap of the dyes; such overlap often gives contributions of one fluorochrome's fluorescence in the other's analysis. To obtain specific isolation of fluorochrome emission in double fluorescence FCM, the comparatively simple and inexpensive two-wavelength optics that uses a single mercury arc lamp was implemented. With this optical system, correlated measurements of cellular DNA and protein were done. Furthermore, another low-budget optical system for specific fluorescence analysis by FCM was developed employing a combination of mercury arc lamp and argon-ion laser. This system was used in an assay for analysis of ploidy, proliferation and apoptosis in paraffin-embedded material. In retrospective investigations, such analysis has the potential to provide integrated information about tumor cell gain and cell loss, i.e. tumor growth. Furthermore, a double fluorescence microscope was developed for simultaneous visual perception of two specific fluorescence images in real-time and quantitative measurement.