Constructing Tissue Microarrays Without Prefabricating Recipient

Anatomic Pathology / CONSTRUCTING TISSUE MICROARRAYS

Constructing Tissue Microarrays Without Prefabricating Recipient Blocks A Novel Approach Ni Chen, MD, and Qiao Zhou, MD, PhD Key Words: Tissue microarray; Construction; Technique; Method; Application DOI: 10.1309/LHCJRFBUH8Q6QD3N

Abstract Tissue microarray (TMA) is a powerful research tool and is applied in such diverse areas as tumor marker validation and laboratory quality control. Existing TMA construction techniques require an essential step of prefabricating recipient paraffin blocks on which holes are punched so that tissue cores can be inserted. This procedure has several disadvantages, such as accidental block breakage during hole punching and difficulty ensuring that the cores are flush with the block surface. We developed a novel TMA construction technique without prefabricating recipient blocks. We used double-sided adhesive tape attached to x-ray film as an adhesive platform on which the tissue cores were placed securely. The array of tissue cores then was embedded in an embedding mold. We have been making high-quality TMAs with up to 220 cores within 2 to 3 hours using this highly dependable, efficient, versatile, and cost-effective technique, which can be adopted by pathology laboratories and researchers with minimal investment.

Tissue microarray (TMA) technology has emerged as a powerful tool in biomedical research,1-4 finding use in such diverse areas as tumor marker validation and expression profiling,1-8 molecular classification and prognostication of tumors,7,9-12 and interlaboratory and intralaboratory quality control,13-17 among other applications.18-20 Together with DNA microarrays and emerging protein and antibody microarrays, TMA is considered one of the essential tools in large-scale, high-throughput analysis.18-20 Whereas DNA and protein microarrays aim at simultaneous assay of hundreds to tens of thousands of genes or proteins in a cell or tissue sample, TMA is the ideal approach to assaying, for example, the expression status of a gene (eg, by in situ hybridization) or protein (eg, by immunohistochemical analysis) in up to hundreds of samples simultaneously.18-20 In principle, the construction of a TMA consists of transferring tissue cores from various donor blocks to a recipient block, which then is used to cut sections containing all the tissue cores embedded in the recipient block.1-4 Techniques described in the literature1-4,21 use an essential step of prefabricating recipient paraffin blocks on which holes are punched so that tissue cores can be inserted. This approach has several disadvantages. First, block breakage during hole punching will result in an unusable recipient block. This is particularly serious in procedures in which hole punching and core insertion were performed one by one, because breakage of a half-finished recipient block with dozens of tissues cores is difficult to handle and might result in waste of precious tissue samples. Second, for manual techniques that are adopted by many laboratories, it is difficult to punch well-aligned and closely packed holes. Third, tissue cores, which frequently are not of the same length, can Am J Clin Pathol 2005;124:103-107

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hardly be placed in the recipient block so that all cores always are flush with the surface. Thus, to cut sections carrying all the cores, it often is necessary to trim the blocks, resulting in loss of valuable tissue samples. Fourth, any tiny space between the cores and the paraffin around them, despite a paraffin filling step, might adversely influence appropriate merging of the two during later procedures, resulting in unsatisfactory blocks and sections. We developed an apparently simple, yet highly dependable, efficient, and adaptable technique of TMA construction based on an adhesive platform without prefabricating recipient blocks, which requires no special instruments and, thus, little investment. We believe this novel approach to TMA construction will be useful and applicable in all laboratories interested in TMA construction.

Materials and Methods Materials and Instruments A 1-mm diameter core biopsy needle with stylet (Shanghai SA Medical & Plastic Instruments, Shanghai, China) was used for coring ❚Image 1A❚. Needles of different gauge could be used according to researchers’ needs. The needle was blunted. Double-sided adhesive tape, a pair of stainless steel tweezers with curved tips, razor blades, discarded x-ray film, and embedding molds (such as the one from Leica, Heidelberg, Germany, shown in ❚Image 1C❚ or any similar molds) were the basic materials needed (Image 1).

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Locating the Coring Site The H&E-stained sections from donor blocks were reviewed and coring site marked on the block according to H&E histologic findings. Preparing the Adhesive Platform A piece of x-ray film (or similar supporting material) was cut according to the size of the embedding mold. The piece we used was 3.5 × 2.5 cm. A piece of double-sided adhesive tape was cut to size and attached to the x-ray film, and the protective paper on top of the adhesive tape was removed to expose the adhesive surface. The platform was now ready to receive tissue cores. Alternatively, to help align the tissue cores, various formats of lanes could be created by cutting with a razor blade and removing strips of the protective paper, as shown in ❚Image 2❚. Retrieving Tissue Cores The core biopsy needle was drilled 3 to 4 mm into the donor block at the prelocated site. After pulling the needle out, the stylet was inserted to carefully push out the tissue core, which could be placed temporarily in a well of a 96-well plate, in the planned order of placement according to array layout to avoid errors, if it was not attached immediately to the adhesive tape platform. Arraying the Tissue Cores Each retrieved tissue core was trimmed minimally at one end with a sharp razor blade to make the end flat and wax-free. The tissue core then was attached securely on the adhesive platform by using the tweezers according to the

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❚Image 1❚ A, Basic materials for tissue microarray construction without prefabricating recipient blocks: a modified core biopsy needle with stylet, donor blocks (shown with holes resulting from coring), a pair of tweezers for handling the cores, and an optional tissue cassette cover (to help align the cores as a module when attaching the tissue cores to the tape). Also shown is a finished platform with tissue cores in place and a close-up view (B). The tissue cores are attached securely to the adhesive platform, resembling columns on a plate. C, The platform is fit into an embedding mold, ready for embedding.

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Anatomic Pathology / ORIGINAL ARTICLE

planned layout. The completed array looks like columns of tissue cores standing on their flat ends on the platform ❚Image 1B❚. A tissue cassette cover (such as the yellow cover shown in Image 1A or any similar covers) could be used optionally to help align the cores by laying the cassette cover on top of the adhesive platform, which resulted in the 7 × 9 array shown in Image 1B. The alternative and preferred approach of creating lanes, as shown in Image 2, could be used to construct other layouts. Moreover, using this lane-based platform also helped to avoid tissue core tipping during embedding, and it facilitated peeling the adhesive tape from the paraffin block after embedding. Embedding Another piece of double-sided adhesive tape was applied to the bottom of the x-ray film, and the adhesive platform with tissue cores in situ was placed into an embedding mold

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(Image 1C), which then was preheated briefly at 60°C on the heat plate of an embedding station (eg, Leica EG 1140, Leica) or, alternatively, in an oven, just to slightly soften the tissue cores. Paraffin at approximately 70°C was injected carefully to fill the mold and then cooled to room temperature. Arrays with higher core density (usually >100 cores) were placed on the heat plate briefly to achieve complete merging of the tissue cores with injected paraffin before cooling to room temperature. Finishing the Block The block was cooled further at 4°C for 10 minutes. The x-ray film and the adhesive tape at the bottom of the block were peeled off. The exposed surface was the cutting surface ready for sectioning, with all tissue cores flush with the block surface, and minimal trimming was needed.

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❚Image 2❚ More examples of array platforms under construction to show various formats that can be adopted. A, 6 × 16 array. B, Two sectors of a 5 × 10 array. C, An H&E-stained section (7 × 13 array). Alignment was facilitated by creating lanes on the adhesive platform (A and B, viewed from top of the platform). This preferred alternative circumvented the restrictions imposed when using the cassette cover, and offered more closely packed cores, less intercore spacing, and superior alignment of the cores.

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Results and Discussion The procedures (Images 1 and 2) and a TMA block ❚Image 3❚ made by this technique are illustrated, as are H&Estained (Images 2C and 3) ❚Image 4A❚ and immunostained ❚Image 4B❚ sections from the block. Although we illustrated the procedure with 7 × 9 arrays, other formats can be adopted easily as desired (Image 2). We have been routinely making 60- to 220-core TMAs by this technique. It is desirable to

❚Image 3❚ Finished tissue microarray (TMA) block and sections (H&E-stained) made from the block (left). Close-up of a TMA section, illustrating the quality of the cores (right). Use of the cassette cover to line up the core resulted in wider but even spacing between the cores. A

divide the cores into sectors (as shown in Image 2B). Each laboratory may use a format that best fits its needs. The method we describe is highly dependable. Because the cutting surface is flat with tissue cores flush with the block surface when embedding is finished, minimal trimming is needed before sectioning. TMAs with 3-mm-long tissue cores typically can cut hundreds of 4-µm serial sections with all cores represented, and only a few core losses or displacements will be encountered. Thus, rare and valuable tissue samples are conserved more effectively. If donor blocks with thicker tissue chunks were secured, a much larger number of sections could be obtained. The technique also is very efficient. We are able to routinely make a 100-core TMA within just 2 hours, starting from coring and ending with finishing the blocks. Personnel training also is minimal, and a technician can learn the procedure essentially in 1 day. The technique we describe is straightforward, and we have encountered few problems. Although it is conceivable that tissue cores could be broken during handling, we have not encountered this problem during transferring from the donor block to the adhesive platform or during embedding. Nevertheless, it is advisable to pay attention to the following points to obtain the best results with the technique: (1) Planning of the layout is essential before coring and arraying. Core-core spacing must be considered before starting. (2) Before the cores are placed onto the adhesive platform, make sure one end of the core is flat so that it will stand vertically on end. (3) This end also should be essentially wax-free to be attached securely to the tape. The presence of paraffin at the core end could result in tipping of cores during embedding as

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❚Image 4❚ A, A representative tissue core of malignant melanoma (H&E). B, Streptavidin-alkaline phosphatase immunostaining for HMB45 (DAKO, Carpinteria, CA) in a pigmented malignant melanoma. Visualization was with AP red (Zymed, South San Francisco, CA), resulting in positive signals of red contrasting well with the melanin pigment.

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Anatomic Pathology / ORIGINAL ARTICLE

paraffin melts, although this can be remedied by using the tweezers. (4) The mold preheating step (brief preheating at 60°C before injecting 70°C paraffin, just to slightly soften the cores) was very helpful to achieve superb merging of the injected paraffin and the tissue cores, preventing any tiny space that could result from imperfect merging and consequent sectioning problems. However, it is important to avoid overheating or prolonged heating because this could result in core tipping. (5) On the embedding station, after the preheating step, the embedding temperature of approximately 70°C generally gives superior results. To achieve complete merging of the tissue cores and the injected paraffin, which is vital for high-quality array blocks, as well as sections, arrays with higher tissue core density might need to be heated briefly on the heat plate after paraffin injection and then cooled to room temperature. This novel TMA construction method, which is versatile, dependable, efficient, and cost-effective, offers numerous advantages and avoids problems associated with procedures that require prefabricating recipient blocks. Although we described a manual procedure, instrumentation based on the principles is conceivable. From the Pathology Department, West China Hospital, West China Medical School, Sichuan University, Chengdu, China. Supported in part by grant 30125023 from the Natural Science Funding Committee and grant 2002CCA01400 from the Ministry of Science and Technology of China, Beijing. Address reprint requests to Dr Zhou: Pathology Dept, West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041, China.

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