A practical method to determine the amount of tissue to analyze using laser scanning cytometry

Abstract

Background: Laser scanning cytometry (LSC) is a new technology similar to flow cytometry but generates data from analysis of successive microscopic fields. Unlike its use in other applications, LSC‐generated data are not random when used for tissue sections, but are dependent on the microanatomy of the tissue and the distribution and expression of the protein under investigation. For valid LSC analysis, the data generated requires the evaluation of a sufficient tissue area to ensure an accurate representation of expression within the tissue of interest. Methods: In this report, we describe a simple and common sense method for determining the area of tissue required for sound LSC analysis by tracking the variation in the measure of target expression with increasing number of fields until it approaches zero. Results: This approach was used to evaluate the expression of immunohistochemical markers with differing tissue distributions in liver (PMP70, CYP1A2, and Ki67 positive macrophages) and a colorectal adenocarcinoma (activated caspase‐3 positive cells), which exhibited diffuse, regional (centrilobular), random, and irregular distribution patterns respectively. Conclusions: Analyses of these markers demonstrated that the amount of tissue area required to reach a steady measure of a parameter increased with increasing variability of the tissue distribution. © 2007 International Society for Analytical Cytology

Laser Scanning Cytometry: Principles and Applications—An Update

“…The unique capabilities of LSC that make it possible to analyze tissue sections in which cells are stained either with fluorochromes, absorption dyes, or with both at the same time contributed to another area of wide LSC application in recent years (131–136): Mapping tissue sections with respect to localization of particular proliferation or metabolic markers and quantification of their expression yielded much information in different branches of cell physiology and in oncology that could not be obtained by other means.…”

Clinical applications of slide‐based cytometry – an update

“…There have been novel ways to analyze slide-based fluorescence data relevant for tissue sections, e.g. multiple threshold scanning (Gerstner et al [18]), multicolor tissue cytometry (Ecker et al [19]), and phantom contouring (Megyeri et al [20]), and guidelines have been developed regarding the minimal tissue area to be analyzed in order to obtain representative data (Wijsman et al [21]). However, most clinical assays so far make use of the fact that only minimal sample volumes are needed for unequivocal data (Gerstner & Tà rnok [22]; Tá rnok & Gerstner [23]).…”

Basics of standardization and calibration in cytometry – a review

“…Therefore, it would be optimal to measure the entire sample, in particular if histologic sections are analyzed in a representative way. Wijsman et al [86] suggest a method to determine the amount of tissue required to obtain representative data. If the measure of a parameter of interest reaches a steady state over continued sampling, sampling has been adequate.…”

“…However, the required scan area differs for markers with differing expression patterns. In general, the lower the frequency of a parameter and the more heterogeneous its distribution the more extensive the sampling procedure must be [86].…”

“…Therefore, the size of the analyzed tissue has to be considered in SBC analyses. Given that cells are unevenly distributed within solid tissue a sufficient area of the tissue must be analyzed to obtain representative data [86,87]. Cell cultures are not as much affected by this as tissue sections since cells (of a single cell culture) on the plate are usually very similar in different areas of the well.…”