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Strategic Insights 
· Introduction
· Proteomics Goes Cellular
· Pathology Goes Molecular
February 18, 2004 | Now that the human genome has been sequenced, and composition analysis of the most commonly used prokaryotic and eukaryotic experimental models is well under way, drug research and clinical development efforts are increasingly focused on protein function, subcellular localization, and tissue distribution. New technologies combining the simultaneous analysis of protein expression with a broad spectrum of target tissue specimens are required for further progress in proteomics.

It is widely accepted that comprehensive analysis of functional proteins in the context of their tissue environment will lead to the rapid identification, characterization, and development of novel diagnostic markers and therapeutic targets. Enormous amounts of data are generated during preclinical and clinical trials, so innovative high-throughput technology platforms are needed to characterize and quantify the effects of novel pharmaceuticals on a variety of human tissues and organs.

Enter tissue microarrays (TMAs). With technological advances developed in the late 1990s, TMA technology represents one of the most promising in the field of functional proteomics.

Before TMAs, a pathologist placed individual tissue sections by hand on separate glass slides for further staining and analysis. A tissue microarrayer essentially automates this process. The instrument obtains cylindrical specimens (tissue cores) from standard histological paraffin blocks, arranges them into a single paraffin block in a precise pattern, and prepares sections of the block containing the arrayed tissue (typically 4 to 5 microns thick) on histological glass slides for further microscopic examination.

Hundreds of individual tissue specimens can be arrayed on a single glass slide. The arrayed sections can be further probed and analyzed microscopically using standard laboratory methods, such as hematoxylin/eosin (H&E), immunohistochemistry (IHC), or in situ hybridization (ISH). These techniques allow simultaneous analysis of protein and RNA expression patterns in their tissue environment, as well as DNA profiling.

Tissue microarrays enable researchers to perform high-throughput microscopic studies on multiple tissue specimens with less sample material and less reagent material needed to visualize the target. 
TMA technology enables researchers and pathologists to perform high-throughput microscopic studies on multiple tissue specimens with less sample material and less reagent material needed to visualize the target. For example, a typical IHC analysis on a single tissue slide requires approximately 100µl of diluted antibody. However, by placing 100 tissue sections on a single slide, the amount of antibody required to generate a single data point is reduced hundredfold.

Another important advantage is that TMAs produce uniform staining of multiple tissue sections on one slide, resulting in enhanced reproducibility.

Powerful Diagnostic-Marker Tool 
TMA vendors currently take two approaches to marketing and distribution, similar to DNA microarray vendors: Commonly used tissues are combined in "standard" arrays and sold as separate catalog items; custom tissue arrays are made "in house" using tissues provided by the customer or tissue banks maintained by the company offering the service.

TMAs may be produced using a wide range of spot sizes, and they are often arrayed in 60-spot or 180-spot formats. In some instances, though, spot densities as high as 400 to 500 spots per slide can be achieved.

Building a Tissue Microarray - TMA slides are constructed by obtaining cylindrical specimens (tissue cores) from standard histological paraffin blocks and arranging them into a single microarray paraffin block. The new block is then cut into 4- to 5-micron-thick sections that contain 60 to 180 tissue specimens. These sections can then be stained using standard laboratory methods.  

Slides are often prepared with normal and diseased tissues together to enable simultaneous analysis and comparison of tissue-expression patterns in one experiment. In addition, some applications call for arrays of normal tissues from different organs or diseased tissues from different patients.

But it is also possible to build arrays of tissues from different organs, different structures within an organ, and tissues from different animals for research use.

TMAs can be used in several different applications, including diagnostic marker validation, protein and RNA-expression profiling, drug-target validation, and clinical controls. For example, one of the most powerful methods for characterizing potential diagnostic markers and therapeutic targets involves IHC analysis of protein expression in diseased tissue and normal tissue.

Although TMAs are useful for many different studies, cancer biomarker analysis is perhaps the most popular application of this technology today. The 10 most common tumor types incorporated in TMAs are breast cancer, prostate cancer, lung cancer, colon cancer, head and neck cancer, melanoma, non-Hodgkin's lymphoma, kidney cancer, ovarian cancer, and brain cancer.

Normal tissue samples, which serve as controls when determining biomarker relevance, are also included in the TMAs. Analysis of existing literature in the cancer arena shows that TMA technology has demonstrated significant potential as a method of increasing throughput in diagnostic marker and therapeutic target validation.

An essential characteristic of TMA-based diagnostic testing is compatibility with various image-analysis techniques, including automated and remote pathology diagnosis. This feature helps link the expression of the given protein to a specific tissue, developmental stage, and tumor type. In addition, the quantitative nature of modern microscopic image-analysis lets researchers quickly obtain, classify, archive, and electronically transfer vast amounts of graphic information on the patterns of a biomarker's expression.

Another important application for TMAs is developing controls for pathology laboratories. Clinical tests performed daily in diagnostic facilities require the simultaneous performance of positive and negative controls during testing. TMA technology represents a simple method for developing consistent controls with minimum reagent costs and without wasting precious control material.

To fully realize the high-throughput potential of TMA technology, however, it's necessary to have an automated computerized imaging system at the back end of microscopic analysis. Several TMA imaging systems have been designed to acquire, process, and archive multiple images of stained tissue sections.

A good TMA imaging system should meet performance metrics that include high reproducibility of image capture, fast and accurate digital analysis of the degree of tissue staining, standardized scoring of gene or protein expression, portable format of resulting files, and compatibility with standard laboratory test reports and quality assurance/quality control procedures.

Patrick Schneider is vice president of business development and scientific affairs for Chemicon International, a division of Serologicals. He may be reached at 

> Pathology Goes Molecular 


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