| Abstract|| |
Background: Tissue microarray (TMA) is a novel and useful tool to efficiently analyze gene expression in histological tissues. Aim: Cost-efficient and easy to use automated tissue arrayers will provide a better instrumentation to generate TMAs. Thus, we designed and produced our tissue microarrayer to meet these needs. Materials and Methods: The HT-1 tissue microarrayer we designed and manufactured consists primarily of four parts, including an instrument to make array pores for the recipient paraffin blocks, a punch needle, an instrument for negative-pressure embedding, and a special manipulator. By using the HT-1, 14 different TMAs were made to accommodate 312 cases of tissues and TMA sections were tested by hematoxylin-eosin (H&E) staining, in situ hybridization, and immunohistochemistry. Results: Expand: Hematoxylin and eosin staining showed that the tissue cylinders were similar, even, and in order on the slides. Most importantly, the HT-1 microarrayer can make array pores in the recipient paraffin block with a single application in seconds. The HT-1 also contains a unique negative pressure system for embedding TMA blocks. In addition, HT-1 can make tissue cylinders with the same levels and depth for equally embedded and sectioning. Conclusions: The HT-1 tissue microarrayer is a device that is simple, economical and easy to use.
Keywords: Tissue microarrayer, tissue microarrays, tissue microarray blocks
|How to cite this article:|
Yang J, Zhang M, Su B, Chen X, Kang A. A novel tissue microarray instrumentation:The HT-1 tissue microarrayer. Indian J Pathol Microbiol 2012;55:314-8
|How to cite this URL:|
Yang J, Zhang M, Su B, Chen X, Kang A. A novel tissue microarray instrumentation:The HT-1 tissue microarrayer. Indian J Pathol Microbiol [serial online] 2012 [cited 2019 Aug 24];55:314-8. Available from: http://www.ijpmonline.org/text.asp?2012/55/3/314/101736
| Introduction|| |
Tissue microarray (TMA) is a novel and revolutionizing approach to allow multiplex histological analysis. TMA is a timesaving, high-throughput method used to study gene expression, which provides more reliable data. ,, Currently, TMAs have become a standard research platform for histopathological analysis of gene expression and correlations. ,, Prior to TMA, there were significant limitations involved with the analysis of gene expression in tissue specimens including the time-consuming process of making individual paraffin blocks, data consistency and reliability due to different spatial and timely generation of data from each tissue sections, and restricted patient sample size. Tissue microarray made it possible to hold up to 1000 separate tissue cores in array form, thus allowing multiplex histological analysis of gene expression.
The TMA technique involves the acquisition of core biopsies as small as 0.6 mm in diameter, from regions of interested paraffin-embedded tissues that are then inserted into recipient paraffin blocks in a precisely spaced, array pattern. Sections are then prepared from the array blocks. Particularly, TMAs is a "simple" logistics exercise consisting of the following steps (1): i) Collecting and selecting donor tissue blocks to be studied, and identifying the areas of interest and representative areas for arraying; ii) Preparing recipient paraffin blocks and making array pores in those blocks; iii) Punching tissue cylinders (core biopsy) from the marked representative areas of the donor paraffin block; iv) Inserting the tissue cylinders into the recipient paraffin block (block array) at high or low densities; v) Embedding the recipient block to get TMA paraffin blocks; and vi) Cutting TMA sections using a microtome. However, a key aspect of TMA technology involves the construction of tissue microarrays blocks (TMA block); a process now accomplished using commercially available TMA instruments. A number of such instruments are now on the market, which all share similar principle and structure and processing procedure. Nevertheless, to date, the using these instruments remains time-consuming and results in low efficiency. However, automated tissue arrayers and assisting instruments (http://www.alphelys.com, http://www.chemicon.com, and http://www.beecherinstruments.com, or other websites) have recently become available. ,,,, With current TMA arrayers the process of punching pores must be repeated in recipient blocks one by one until enough pores are attained, which is the first bottleneck of TMA technology. Expensive automated tissue arrayers (https://bioinfo.itc.it/TMA) and assisting instruments may alleviate such labor, but due to limited budgets these technologies may be out of reach for many research centers. We, therefore, invented a novel, easy to use, and low-cost HT-1 tissue microarrayer (Chinese patent ZL00207528.8 and ZL00262393.5). Using the HT-1 tissue microarrayer, the well-aligned array pores in recipient paraffin block can be punched by one action and higher quality TMA blocks can be constructed easily without any "locater" or assisting instruments.
| Materials and Methods|| |
Design of the HT-1 tissue microarrayer and work principle
To make TMAs, punching array pores in recipient blocks and embedding multi-tissue cylinders into recipient blocks are divided into two key steps. The novel HT-1 tissue microarrayer has been designed and manufactured to make these two steps much easier in the following ways:
The instrumentation to make recipient block
We have designed a novel multiple recipient block maker, called the recipient block-molding machine, which includes three types (such as 46, 67, and 78 for row and column) to accommodate 24, 42, and 56 tissue cylinders, respectively. The spacing between the cores in one TMA is fixed. Using one type of recipient block-molding machine, we can make one type of TMA blocks [Figure 1]a, while different types of the recipient block-molding machine can make different types of recipient paraffin blocks with one single action within several seconds. This instrument is comprised of three parts: An array pores forming metal stamp and a series of metal embedding boxes and a well-arranged array hollow punch needles with corresponding inner cores. The array pores-metal stamp consists of a pair of metal plates and a bracket. The lower plate is fixed with a well-arranged array hollow punch needles, while the upper plate is fixed with corresponding inner cores to array hollow punch needles, which can slide up and down according to array hollow punch needles. The inner diameters of the punch needles are designed from 0.5 to 2.5 mm, depending on the desired cut diameter. The instrument for making array recipient blocks is fixed in the special manipulator by the bracket. The lower plate with hollow array punch needles can move up-and-down by controlling the handle. Simultaneously, the residual paraffin in array hollow punch needles is cleared automatically by the piston action of the inner core in array hollow punch needles. In addition, the metal embedding box is used to make 0.5-1.0 cm depth of the recipient paraffin blocks.
|Figure 1: The HT-1 tissue microarrayer. (a) The recipient paraffi n block module and punch needle (left); Illustration of working principle (right). (b) The special manipulator and the negative-pressure embedding module (left). Illustration of working principle (right)|
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The punch needle [Figure 1]a was designed to make identically cylindrical tissue columns from donor blocks and then transfer them to the corresponding receiver pores of the recipient paraffin block manually. The punch needle contains two parts: A hollow punch needle and the inner core, which can be fixed in the special manipulator. The hollow punch needles with a hard and sharp tip are used to take tissue columns and the plunges are used to push the tissue out of the steel tube into array cavities in the recipient block. The hollow punch needle can move up and down by controlling the handle. The tissue cylinders taken from the donor block can be pushed out automatically by the inner core of hollow punch needles.
The special manipulator
The special manipulator was designed to install the instrument for making recipient blocks or punch needles [Figure 1]b. By controlling the handle of the special manipulator, the recipient block molding machine or the punch needle installed on the special manipulator can move up and down to punch different array pores in recipient paraffin block or to take identical cylindrical tissue columns from donor blocks.
The negative-pressure embedding instrument
The negative-pressure embedding instrument was designed to re-embed multi-tissue cylinders into recipient block to construct a TMA block. It consists of a vacuum pump and embedding box and negative-pressure room [Figure 1]b right. The embedding box is situated in the negative-pressure room, which is connected to the vacuum pump via a tube. The bottom of the embedding box consists of a metal mesh, which can adjust negative-pressure so that air bubbles between the tissue cylinders and array pores can be drained out. In addition, the screen mesh can also be used to adjust temperature for the embedding process. Melted paraffin can enter the gap between tissue core and receiver pores to connect tissue cylinders and paraffin in a body [Figure 1]b right.
| Results|| |
By using the HT-1 tissue microarrayer and according the workflow [Figure 2], we made 14 TMA blocks that contained 312 cases of paraffin-embedded tissues (i.e., 82 cases breast cancer, 10 cases normal tissues, 13 cases benign tumors, and 207 cases different cancer tissues). All of the paraffin blocks were obtained from The Department of Pathology, The Second Hospital of Medical School at Xi`an Jiaotong University. The size of TMA blocks were 78 (rowcolumn) for accommodating 56 tissue cylinders, respectively, with a 1.5-mm diameter tissue core [Figure 3]a and b. While constructing a TMA a few control tissues are included as orientation marker of the block, or a blank core is placed in one corner. After identified the control tissue or blank core and the row or column, we can we could easily ensure the correct orientation of the control tissues while evaluating ISH and IHC results. The TMA paraffin blocks were then sectioned to obtain 5 μm consecutive tissue sections,  which were then mounted onto glass slides.  Sections were then subjected to H&E staining  and in situ hybridization  and immunohistochemical analysis  of gene expression. The TMA sections were stained for H&E and the data showed that the arrayed-1.5 mm tissue cylinders were similar, even, and in order on the slides [Figure 3]c and d. Moreover, some of these TMA sections underwent in situ hybridization analysis of hTER mRNA and hTERT mRNA expression with in situ hybridization kit (Department of Pathology, Beijing Medical University, Beijing, China) [Figure 3]f and h. In addition, these TMA sections were immunostained for estrogen receptor (ER), progesterone receptor (PR), and p53 proteins with SP kit and Dako REAL TM Elivision TM detection kit as described previously. , The monoclonal mouse anti-human ER, PR, and p53 antibodies and the SP kit and Dako REAL TM Elivision TM detection kit were from Dako Company (Carpinteria, CA). The data showed that H&E staining and in situ hybridization and immunostaining procedures of these TMA sections resulted in only a few folded or missing tissues and dystopia was uncommon. Furthermore, the color reaction was very clear and the background was weak [Figure 3]c-J.
|Figure 2: Working fl ow of the HT-1 tissue microarrayer. Steps 1-3, Construction of array pores on the recipient paraffi n blocks. Steps 4-6, Preparation of tissue cylinders and transplantation into the array holes in the recipient paraffin block. Step 7, Preparation of TMA sections. The prepared TMA paraffi n block can be sectioned by a routine microtome. Step 8, Use of TMA sections for gene expression or histology evaluation after staining|
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|Figure 3: TMAs recipient paraffi n block, TMAs paraffi n block, and TMAs sections. (a) The recipient paraffin block. (b) TMA paraffin block made by the HT-1 tissue microarrayer. (c, d) H and E staining of TMA sections. (e) Immunohistochemical staining of TMA sections. (f) Expression of hTER mRNA expression in ovarian serous cystadenocarcinoma detected by ISH. (g) Expression of hTERT mRNA in esophageal squamous cell carcinoma detected by ISH. (h, i, j) Immunohistochemical analysis of ER (h) and PR (i) and p53 (j) expression in breast cancer|
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| Discussion|| |
In the current study, we have designed and produced a much improved and easy to use HT-1 tissue microarrayer. The data from this study demonstrated that the HT-1 tissue microarrayer is a simple, economical, and easy to use personal device. For example, using one type of recipient block molding machine, we can make one type of TMA block [Figure 1],[Figure 3]a and b. Different types of recipient paraffin blocks can be made by recipient block molding machine with one single action within several seconds. Furthermore, the H&E stained TMA sections showed that tissue cylinders were similar in size, even, and in order on the slides. The tissue structure was well preserved during in situ hybridization and immunohistochemical analysis, and these procedures resulted in only a few tissues that were folded, missing, or that had dystopia. These data together suggest that the HT-1 tissue microarrayer is not just an alternative tissue microarrayer, but is more efficient, economical, and easy to use device.
In general, automatic and manual tissue microarrayers consist of a punch needle to make array pores in a recipient block, a punch needle (coring needle) to punch tissue cylinders from a donor paraffin block, and an operation platform. These tissue microarrayers all use a single punch needle to punch array pores in a recipient block. In order to get a well-arranged recipient block, the users have to punch the pores one by one in the recipient block, which often results in cracking during the course of punching array pores.  The reason may be due to the hardness and plasticity of paraffin, which changes with temperature. Moreover, preparation of the recipient paraffin block is a time-consuming, arduous and onerous procedure. Nevertheless, the HT-1 tissue microarrayer solves this problem by just using one action to punch array pores in the recipient paraffin block. And this will help to preserve the donor and recipient blocks when punching during these processes.
Furthermore, preparation of tissue cylinders from donor paraffin blocks and embedding the TMA block are other critical steps in the TMA-making process. Recently, tissue microarrayers have significantly improved these steps with a series of automations (see the details in website of http://www.beecherinstruments.com). However, in fact, these automations only improve approximately 5% efficiency of the entire TMA-making process.  Therefore, despite the increased cost of advanced arrayers, efficiency and quality of TMA blocks has improved very little. Although the HT-1 machine does not involve automation, it dramatically reduces the time to prepare a recipient block, leading to more time to precisely make tissue cores and embedding procedure. In addition, our machine significantly improved the embedding process, which generates more quality TMA blocks. As a whole, the HT-1 tissue microarrayer improved the deficiencies of the currently available tissue microarrayers, improved technical difficulties, and reduced labor intensity. For example, this microarrayer avoids cracking of the recipient block when making the recipient paraffin block, and markedly preserves and improves the efficiency and quality of the recipient block.
In addition, because of the gravity difference between the tissue cylinder and paraffin, some tissue cylinders float and shift during the embedding process leaving some tissue cylinders. Moreover, during transplanting tissue cylinders into array pores in the recipient block, air bubbles between tissue cylinders and array pores also prevent the tissue cylinders from fusing tightly with the recipient block tightly during embedding. In the HT-1 tissue microarrayer, a negative pressure-embedding technique is employed. The air bubbles between the tissue cylinders and array pores can be drained by adjusting the embedding temperature and negative pressure during embedding process, and the space between tissue cylinders and receiving paraffin block is filled with melted paraffin; so all tissue cylinders and recipient paraffin block can be firmly combined together.
We built 14 TMAs paraffin blocks with a size of 46, 67, or 78 with the HT-1 tissue microarrayer. Twenty to thirty minutes was required to process each TMA paraffin block. Furthermore, it was possible to make 230±30 sections from TMA paraffin block built by HT-1 tissue microarrayer. The results showed that using HT-1 tissue microarrayer, we were able to efficiently preserve recipient blocks. In addition, HE staining of the TMA sections showed very good integrity of tissue structure. In situ hybridization and immunohistochemistry analysis induce only few tissue folding, missing, or dystopia and that tissue structure was well preserved. Together, these data suggest that this HT-1 tissue microarrayer is a novel, more efficient, economical, and easy to use device, than devices currently available.
| Acknowledgment|| |
We thank Medjaden Bioscience Limited for assisting in the preparation of this manuscript.
| References|| |
|1.||Kononen J, Bubendorf L, Kallioniemi A, Bärlund M, Schraml P, Leighton S, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998;4:844-7. |
|2.||Shergill IS, Shergill NK, Arya M, Patel HR. Tissue microarrays: A current medical research tool. Curr Med Res Opin 2004;20:707-12. |
|3.||Skacel M, Skilton B, Pettay JD, Tubbs RR. Tissue microarrays: A powerful tool for high-throughput analysis of clinical specimens: A review of the method with validation data. Appl Immunohistochem Mol Morphol 2002;10:1-6. |
|4.||Avninder S, Ylaya K, Hewitt SM. Tissue microarray: A simple technology that has revolutionized research in pathology. J Postgrad Med 2008;54:158-62. |
|5.||Sauter G, Simon R, Hillan K. Tissue microarrays in drug discovery. Nat Rev Drug Discov 2003;2:962-72. |
|6.||Nocito A, Kononen J, Kallioniemi OP, Sauter G. Tissue microarrays (TMAs) for high-throughput molecular pathology research. Int J Cancer 2001;94:1-5. |
|7.||Gillett CE, Springall RJ, Barnes DM, Hanby AM. Multiple tissue core arrays in histopathology research: A validation study. J Pathol 2000;192:549-53. |
|8.||Hidalgo A, Piña P, Guerrero G, Lazos M, Salcedo M. A simple method for the construction of small format tissue arrays. J Clin Pathol 2003;56:144-6. |
|9.||Pan CC, Chen PC, Chiang H. An easy method for manual construction of high-density tissue arrays. Appl Immunohistochem Mol Morphol 2004;12:370-2. |
|10.||Vogel UF, Bueltmann BD. Simple, inexpensive, and precise paraffin tissue microarrays constructed with a conventional microcompound Table and a drill grinder. Am J Clin Pathol 2006;126:342-8. |
|11.||Wang L, Deavers MT, Malpica A, Silva EG, Liu J. Tissue macroarray: A simple and cost-effective method for high-throughput studies. Appl Immunohistochem Mol Morphol 2003;11:174-6. |
|12.||Zhang X, Li L, Chen S, Yang D, Wang Y, Zhang X, et al. Rapamycin treatment augments motor neuron degeneration in SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Autophagy 2011;7:412-25. |
|13.||Wang P, Xu TY, Guan YF, Su DF, Fan GR, Miao CY. Perivascular adipose tissue-derived visfatin is a vascular smooth muscle cell growth factor: Role of nicotinamide mononucleotide. Cardiovasc Res 2009;81:370-80. |
|14.||Sivridis E, Giatromanolaki A, Zois C, Koukourakis MI. The "stone-like" pattern of autophagy in human epithelial tumors and tumor-like lesions: An approach to the clinical outcome. Autophagy 2010;6:830-3. |
|15.||Moussay E, Kaoma T, Baginska J, Muller A, Van Moer K, Nicot N, et al. The acquisition of resistance to TNFa in breast cancer cells is associated with constitutive activation of autophagy as revealed by a transcriptome analysis using a custom microarray. Autophagy 2011;7:760-70. |
|16.||Tanabe F, Yone K, Kawabata N, Sakakima H, Matsuda F, Ishidou Y, et al. Accumulation of p62 in degenerated spinal cord under chronic mechanical compression: Functional analysis of p62 and autophagy in hypoxic neuronal cells. Autophagy 2011;7:1462-71. |
|17.||Wang P, Xu TY, Guan YF, Tian WW, Viollet B, Rui YC, et al. Nicotinamide phosphoribosyltransferase protects against ischemic stroke through SIRT1-dependent adenosine monophosphate-activated kinase pathway. Ann Neurol 2011;69:360-74. |
|18.||Xue LY, Chiu SM, Oleinick NL. Atg7 deficiency increases resistance of MCF-7 human breast cancer cells to photodynamic therapy. Autophagy 2010;6:248-55. |
|19.||Datta MW, Kahler A, Macias V, Brodzeller T, Kajdacsy-Balla A. A simple inexpensive method for the production of tissue microarrays from needle biopsy specimens: Examples with prostate cancer. Appl Immunohistochem Mol Morphol 2005;13:96-103. |
Department of Pathology, Second Hospital of Medical College, Xi'an Jiaotong University, 157 West 5th Road, Xi'an, Shaan'xi 710004
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]