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P Tekola

Publications and source records attributed to P Tekola.

5 recordsLinked to original sources
PubMed1996

Three-dimensional confocal laser scanning DNA ploidy cytometry in thick histological sections.

DNA ploidy measurement by flow (FCM) or image cytometry (ICM) of single cell suspensions of solid tumour has prognostic value, but it would be a definite advantage if the assessment could be done on histological sections. However, this is usually not possible by means of standard ICM, due to the capping of nuclei in thin sections, or overlap in thick sections. Three-dimensional (3D) microscopy by means of confocal laser scanning microscopy (CLSM) could solve this problem in theory but the results published so far are not very satisfactory. A new method has been developed in which the DNA content of haploid (human testis spermatozoa), diploid, tetraploid, octaploid (human and rat liver and human spermatogonia), and near-triploid (human breast cancer) nuclei stained with YOYO-1 iodide has been measured by a newly developed 3D image cytometry method (3DICM) in 20 microns thick histological sections. YOYO-1 iodide is a new highly sensitive, specific, stoichiometric, and stable fluorescent dye for DNA. DNA ploidy of a breast cancer which was near-triploid with FCM and ICM was also assessed with 3DICM in a tissue section adjacent to the section used for FCM and ICM and the results were compared. The integrated 3DICM fluorescence intensity showed good linearity (r = 0.99) with the real DNA content of all nuclei analysed. In human tissue, the coefficient of variation of 3DICM for haploid (n = 12), diploid (n = 63), triploid (n = 13), tetraploid (n = 12), and octaploid (n = 3) ploidy distributions was 5.1, 6.6, 4.2, 4.0, and 0.6 per cent, respectively (n = the number of nuclei). For the rat liver, the CV of the diploid (n = 21), tetraploid (n = 31), and octaploid (n = 3) peaks was 6.7, 4.8, and 1.6 per cent, respectively. Repeated "blind' measurements of nuclei with different DNA indices showed excellent reproducibility between different observers (r = 0.98). It is concluded that the 3DICM method used is accurate, reproducible, and clinically feasible in thick histological sections. This is especially important in small lesions, or if the results of DNA ploidy measurement of single cell suspensions (by FCM) or imprints (by ICM) are inadequate.

Animals↗
PubMed1994

Highly sensitive, specific, and stable new fluorescent DNA stains for confocal laser microscopy and image processing of normal paraffin sections.

The area, volume, shape, DNA content, and chromatin pattern of nuclei can be important for the diagnosis and prognosis of cancers. Confocal laser scan microscopy can be useful to obtain such information by optical slicing and three-dimensional (3-D) reconstruction of nuclei in thick paraffin sections. To retrieve individual quantitative features from the section, highly sensitive, specific, and stable fluorescent stains are required. Two new nuclei acids stains (TOTO-1 iodide and YOYO-1 iodide) can detect picogram amounts of nucleic acids in gels. Despite their high sensitivity to detect DNA, they have not been used to stain nuclei in paraffin embedded tissue sections (as routinely applied in surgical cancer pathology). We have developed a technique to stain nuclei in 4% buffered formaldehyde fixed paraffin tissue sections using TOTO-1 iodide and YOYO-1 iodide. The technique developed gives bright specific staining of the nuclei (with nearly zero background intensity, much less than with acriflavine). Moreover, TOTO-1 iodide and YOYO-1 iodide stains both give fluorescent signals only when they interact with DNA. Thus washing off the excess stain left on the stained specimen is not necessary (washing of excessive stain is necessary with acriflavine). Care has to be taken that the deparaffinizing liquids (xylol etc.) are not polluted with eosin (a frequently used counterstain in surgical pathology specimens), as this gives undesired fluorescence of cytoplasm and connective tissue at the same wavelength as TOTO-1 iodide and YOYO-1 iodide. Contrary to acriflavine, bleaching of TOTO-1 iodide and YOYO-1 iodide fluorescence is minimal, even after 30 min continuous laser light excitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acriflavine↗
PubMed1994

Measurement by confocal laser scanning microscopy of the volume of epidermal nuclei in thick skin sections.

The mean volume and shape of nuclei assessed in standard tissue sections by means of stereologic and morphometric methods are associated with prognosis in tumors of different sites. Thus, accurate quantification of the volume and shape of cell nuclei can be important for cancer patients and also may be useful for a better understanding of basic cellular events, such as growth and differentiation. Confocal laser scan microscopy (CLSM) makes it possible to obtain image sets consisting of very thin serial optical slices from thick tissue sections. These images can be used with digital image processing to construct a three-dimensional (3-D) model of individual nuclei. We used CLSM to study a 50-micron-thick paraffin section of a skin biopsy. Image processing was applied to the CLSM images to precisely segment epidermal nuclei from the background, and serial two-dimensional (2-D) binary images were created. Alignment of the 2-D images that form the 3-D model of the original nuclei was carefully controlled. The series of 2-D binary images was connected with an algorithm to form the 3-D model of each complete nucleus and organized for the 3-D visualization and analysis using a 3-D volume rendering method. In this way we measured the volume and surface area of 22 intact epidermal nuclei. The mean nuclear volume was 174.7 micron3, the standard deviation of the volume 26.47 micron3, the mean surface area 168.0 micron2 and the standard deviation of the surface area 22.00 micron2.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus↗
PubMed1993

Quantitative microscopical and confocal laser scanning microscopy for intermediate endpoint biomarkers in breast cancer: potential and reproducibility.

Diagnostic quantitative pathological (QP) determinations are increasingly used in our hospital. The number of requests for QP for reference materials is rising rapidly. This is understandable; quantitative assessments have a strong prognostic value and can be very reproducible, depending on the care taken with a number of factors including cell and tissue processing, application of the appropriate stains, and the measurement protocol used. As to the latter, systematic random sampling gives the best intra- and interobserver agreement (with correlation coefficients between observers for certain features > or = 0.94). Flow cytometric determinations are often regarded as more reproducible than interactive morphometry due to the high speed of the assessments, the large number of objects measured per specimen, and the lack of observer interaction. Indeed, flow cytometrically assessed DNA ploidy is very reproducible, even though the % S-phase fraction is much more variable. Unlike image cytometry (ICM), visual inspection of cells is not easily accomplished with flow cytometry (FCM). With ICM, the fully automated measurement of DNA in thousands of cells is possible in 3-5 minutes, with a very low coefficient of variation (< or = 2% for the diploid and tetraploid peak of liver cell nuclei). ICM also allows measurement of texture features. However, quantitative immunohisto/cytochemical determinations may not always be as reproducible as sometimes believed. Recently, we found large variations in the measurements, made by a commercially available image processing instrument, of the estrogen and progesterone receptors, Ki-67, cathepsin D, and neu protein overexpression in breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗