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December, 2007

Asterand Frozen Tissue Microarrays: Design, Construction and Application Tissue microarray technology has been used as a research tool, typically in the validation of oncology targets, for several years. Asterand supplies high quality tissue microarrays from formalin-fixed paraffin-embedded tissues in the form of ready-made arrays, or as arrays custom-made to meet clients' requirements. Using our expertise in this area, Asterand has recently developed frozen tissue microarrays suitable for use either immunohistochemical or in situ applications. In the course of developing the technology, core diameters have been reduced from 1.5mm to 0.6mm, thereby allowing the inclusion of larger numbers of samples on a single section and increasing the throughput from assay to analysis. The original format for multi-tumour tissue microarrays has been the pathology standard; formalin-fixed paraffin-embedded and there are several reasons why this has become the preferred approach; tissue morphology and antigenicity are well-preserved and array construction and analysis has been both standardised and automated. The resulting arrays are ideal tools for the analysis of protein expression by immunohistochemistry in large numbers of samples and can allow the analysis of protein expression through tumorigenesis in experiments designed with appropriate statistical power. One area where Asterand has recently expanded its product portfolio takes advantage of our expertise in tissue array construction, immunohistochemistry and the analysis of mRNA expression in tissue sections by in situ hybridization. Over the last twelve months, Asterand has developed a reliable protocol for the construction of frozen tissue arrays. Frozen tissue arrays have several advantages over paraffin embedded TMAs (a) RNA has been found to be less fragmented in frozen compared with formalin-fixed tissues thereby allowing optimal detection by in situ hybridization, and (b) proteins are maintained in their native state. For this reason, frozen tissues are recommended by the FDA for the analysis of cross-reactivity (‘off-target’) binding of therapeutic antibody candidates. Frozen arrays therefore offer the opportunity of greater efficiency in screening therapeutic antibodies. However, the construction of frozen tissue arrays poses a set of difficulties not encountered with paraffin-embedded arrays. The preservation of good tissue morphology and the detectability of RNA depends to a large degree on the maintenance of frozen tissue conditions. Our scientists have found a way to achieve this with frozen cores of 2mm depth by 1.5mm in diameter and at the same time ensure accurate placement and retention of cores. An initial series of prototype frozen TMAs was constructed and assessed and development of the technique led to the construction of FDArrayTM, a frozen array which contains the tissues recommended by the FDA for the analysis of antibody cross-reactivity (FIGURE 1). Sections from FDArrayTM were assessed histopathologically and immunohistochemically and while they lacked some of the well-preserved morphology of paraffin-embedded equivalents (FIGURE 2), they proved more than adequate for the localisation of bound antibodies by immunohistochemistry (FIGURE 3).      Figure 1. The tissue content, design and a photograph of a completed FDArray™ frozen tissue array. Figure 2. Comparison between the morphological preservation seen in frozen (panel A) and FFPE-embedded (panel B) sections of human stomach. Figure 3. Immunohistochemical detection of alpha-smooth muscle actin in selected cores from the FDArray™. Panels A-B show blood vessels in the submucosal cervix and C-D show seminiferous tubules. Panels B and D show the low levels of non-specific immunoreactivity detected following incubation with non-immune IgG. An additional use for frozen tissue arrays takes advantage of the preservation of mRNA within frozen tissue sections. Asterand’s standard in situ hybridization methodology involves the hybridization of 400-500b labelled riboprobes and for this to be achieved, target mRNAs must be present in fragments of at least that length. Abundant mRNA targets can be detected by in situ hybridization in formalin-fixed sections, but the sensitivity of the assay is compromised by the relatively poor preservation of the mRNA. The use of frozen tissue arrays allow moderate to high throughput analysis of cellular mRNA expression by in situ hybridization using tissues in which mRNAs are likely to be optimally preserved and can be most usefully used to explore target expression in circumstances where reliable antibodies are unavailable (Figure 4). Figure 4. Expression of a target mRNA in normal colon and colon adenocarcinoma within a frozen tissue microarray. The purple-blue chromagen indicates the presence of the target mRNA following detection with alkaline-phosphatase conjugated anti-DIG antibodies. In establishing a novel approach to the construction of frozen tissue microarrays, Asterand has developed a method with which to analyse both the on- and off-target reactivity of therapeutic antibody candidates and the expression of specific mRNAs by in situ hybridization. Through the combined use of the tissue array format and frozen human tissues, both types of studies can now be performed at maximal efficiency and sensitivity. Asterand will construct these frozen TMAs for our clients on a custom-project service basis for both research and FDA studies. For further information, please e-mail: advantage@asterand.com. ASTERAND SUPPORTS HUMAN DRUG DISCOVERY AND TRANSLATIONAL MEDICINE THROUGH THE PROVISION OF HIGH QUALITY WELL CHARACTERIZED HUMAN BIOMATERIALS AND PRECLINICAL RESEARCH SERVICES Visit us at www.Asterand.com