An alternative high output tissue microarray technique
- Yuan Shi†1,
- Deming He†1,
- Yingyong Hou1Email author,
- Qin Hu1,
- Chen Xu1,
- Yalan Liu1,
- Dongxian Jiang1,
- Jieakesu Su1,
- Haiying Zeng1 and
- Yunshan Tan1Email author
© Shi et al; licensee BioMed Central Ltd. 2013
Received: 27 November 2012
Accepted: 8 January 2013
Published: 21 January 2013
Tissue microarray (TMA) is a high throughput research tool, which has greatly facilitated and accelerated in situ tissue analyses. However, its productivity has been restricted due to the confined thickness of traditional donor block. Here, we introduce an improved high output TMA method that is applicable to a broader range of tissue samples.
In this method, a 3.6 cm long and 2.7 cm wide recipient block with 88 square lattices (3 mm in width) was first prepared using several commercial instruments. A 2 mm wide and 6 mm long tissue rod was then prepared using a self-made blade-shaped knife from each paraffin embedded donor block of gastrointestinal stromal tumors. These rods were manually arrayed one by one into the corresponding lattices of the 60°C pre-softened recipient block with the guide of holes drilled with a steel needle. A 70-rod TMA was made to testify this method.
The prepared TMA had well defined array configurations, good tissue morphology and fully preserved proteins and DNA. A total of 500–1000 TMA sections could be easily obtained from a TMA block.
This low-cost and time-saving method provides an alternative sampling tool for high output TMA.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1979605867857990
KeywordsTissue rod Tissue microarray Technology Sampling tool High output
In 1986, Battifora proposed an ingenious idea for combining multiple tissue samples into a single “sausage” composite held together with a wrapper of intestinal casing. Wan further modified the sausage technique by assembling the tissue cores manually extracted from parent tissue blocks without further deparaffinization. Several years after these pioneering and prototypical works of the modern tissue microarrays (TMAs), Kononen et al. proposed and developed a novel high throughput TMA technique in 1998 to precisely re-embed up to 1,000 cores of donor tissue cylinders from a formalin-fixed paraffin-embedded “donor” block into a “recipient” paraffin block with the help of highly precise punching instruments. As a high throughput research tool, TMA, also known as tissue chip, is considered as a recent innovation in the field of pathology and has greatly facilitated and accelerated in situ tissue analyses. Sections from TMA can be stained for protein, DNA or RNA targets using in situ immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or mRNA in situ hybridization (RNA-ISH), respectively. The utility of TMA is generally found to be comparable to that of large sections. TMA technique has dramatically changed traditional tissue sampling process of the above-mentioned in situ technologies, which are extremely tedious, time consuming, labor intensive and costly. For example, Schraml and coworkers completed 3 FISH experiments on amplifications of three oncogenes in 3 × 397 tumors within a week.
TMA technology is extremely powerful, providing researchers the potential to derive extensive gene expression profiles invaluable particularly in the areas of tumor biology, clinical oncology and diagnostic test development[4, 8–10]. In addition, TMA technology also plays an important role in educating pathologists about quality control in IHC and tissue banking.
Up to now, arrayers used for TMA are generally designed and supplied by several companies. However, they are quite expensive. To achieve cheap TMA, Shebl et al. introduced an inexpensive mechanical pencil tip method for small paraffin tissue microarrays. Although up to 1,000 different tissues can be analyzed in one TMA block, all TMAs are restricted by the thickness of paraffin embedded block because they are cylindrical cores obtained by punching the donor block. As a result, only about 100–300 sections are available from one TMA block. In this study, we describe an alternative method using various tools including several in-house instruments to obtain rods of tissues from donor blocks and to create the recipient block with 88 small lattices. The rods of tissues were vertically embedded in the recipient block, which is fundamentally different from previous methods and could greatly improve the output of sections in one TMA block. Thus, it could be used as an alternative TMA approach for appropriate cases.
Selection of appropriate paraffin blocks with gastrointestinal stromal tumors (GISTs)
70 cases of GISTs were retrospectively collected from the department of pathology, Zhongshan Hospital, Fudan University. Prior written informed consent was signed by all patients and the study protocol was approved by the Ethics Committee Board at Zhongshan Hospital, Fudan University.
Preparation of tissue “rod”
Preparation of recipient block and plantation of the donor tissue rods into the recipient block
For the IHC, all incubations were performed at room temperature and washes were performed with TBST. Tissues sectioned at 5 μm on slides were dewaxed and rehydrated. Antigen retrieval was performed in a pressure cooker at 110°C for 5 min in a retrieval buffer (S2367, DAKO). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (S2023, DAKO). Sections were then incubated with CD117 antibody (DAKO) for 30 min and the signals were visualized with diaminobenzidine (K3468, DAKO) for 10 min.
Analysis of KIT and PDGFRA Sequences
First, five consecutive sections were prepared and the same chip on the consecutive sections were collected for genetic test. The sequence analysis of KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18) was performed according to the protocols reported previously[15–17]. Primer sequences are available upon request. In short, genomic DNA was isolated from paraffin-embedded tissue samples using a standard phenol/chloroform organic extraction protocol. Sequencing reactions were conducted in both forward and reverse directions. The results were compared with the sequences of human KIT (NM_001093772) and PDGFRA (NM_006206) genes in National Center for Biotechnology Information.
Section and H&E staining
IHC on the TMA section
Analysis of KIT and PDGFRA sequences
The sequences of the analyzed genes obtained from TMA were consistent with those obtained from their corresponding original slides. Figure6 shows TGGAAG deletion of the codons 557 and 558 in exon 11 of KIT gene.
Development of the high throughput TMA
After 20 years of development, TMA technology has been well established in terms of both methodology and instruments. In recent years, high-density TMA has become a standard laboratory tool for identifying and validating diagnostic and prognostic biomarkers for a variety of diseases, such as breast cancer, prostate cancer[9, 19] and lung cancer. Application of 2 mm punchers allows construction of tissue arrays with about 50–100 specimens, while application of 0.6 mm punchers allows construction of a typical array of 300–500 specimens with a spacing of 0.8 mm between sample centers per block in regular tissue cassettes. A recipient block could accommodate up to 1,000 samples since the core of diameter can be as small as 0.43 mm. Removing small diameter cores of tissue means that the tissue in the main block is preserved and does not significantly damage the structure of the tumor in the donor block, which can be used for further conventional sectioning should this become necessary. However, there are still several shortcomings in the conventional TMA block.
Some shortcomings of the high throughput TMA
Firstly, tissues are embedded horizontally on the surface of a paraffin wax block with the thickness of tissue often less than 2–3 mm after being sectioned for H&E staining. If TMA is sectioned carefully by an experienced technician, at least 200 sections would be obtainable. Prior use of the donor block for immunohistochemistry as well as gene analysis in special cases may reduce this number, as the cores obtained will be shorter. Therefore, the productivity from one conventional TMA block is limited. Thus, the sections are often obtained at one time, otherwise at least ten sections would be lost because of trimming.
Secondly, tumor within the donor block is a three-dimensional structure, and so is the core. This means that the composition of any individual core may change as the sample is sectioned. A core may contain tumor cells at one end but only stroma at the other. If arrays are not well constructed or some cores are shorter than others, some cores will “sectioned out” before others. Previous investigators have reported a 10–30% loss of tissue when constructing conventional TMA from a large number of cores using manual or automated methods[7, 21–23].
Invention of a new high output TMA technique
The current new method could improve the output of TMA. As demonstrated, it is possible to construct a TMA from strip-shaped tissue rods taken from tumor abundant area and produce up to 1000 sections using appropriate simple tools. This method involves repositioning the rods in a vertical orientation, which enables the production of approximately 1000 sections from each rod of tissue to be used for H&E and IHC staining. Such sections could also, in principle, be used for other molecular procedures such as FISH. The IHC staining and DNA sequencing results showed that the TMA tissue maintained its antigens and DNA after the rods of tissues being planted in the recipient block.
Jhavar has described a method for preparing TMA from core biopsy tissues by sectioning small part of the tissues out of the prostate needle biopsy donor block and re-embedding the sections vertically in a new block. Miettinen has also developed a simple method for generating multi-tissue blocks without special equipment. However, these procedures are unsuitable for long rods of tissues. The rods of tissues could easily become slanted and are very difficult to orientate when re-embedded. Although our method is similar to theirs, it is more feasible for long rod of tissue. It took our 2 years to improve the method and ensure the rods could stand vertically in a TMA.
The present method of re-embedding tissue vertically is rare in TMA construction. A similar “checkerboard method” has been proposed by Battifora[1, 25] to produce tissue arrays of a multi-tumor tissue block (MTB). In this checkerboard method, multiple (up to 100) chunks of formalin-fixed de-paraffinized or fresh normal or tumor tissues were reset first in agar and then in paraffin wax in a checkerboard pattern, and more than 1,000 sections with thickness of 5 μm could be obtained from one sausage type “MTB” block or checkerboard type “MTB” block. In our method, tissue rods were planted corresponding to the original samples rather than re-embedded samples in the chips.
Advantages and disadvantages of our high output TMA technique
The advantages of our current approach are summarized as follows: a) the procedure and facility are simple, omitting hole-punching step, and no special, expensive instrument is required. With only a little practice, an operator could successfully select the needed rods from a “library” of tissues and plant them into a recipient paraffin block, b) the rods are almost immobile because they are long enough to stand stably, and c) the most improvement of the method is very high TMA output.
There are several practical limitations in vertically TMA construction. For example, pre-malignant lesions such as carcinoma in situ are not big enough for making high output TMA. On the other hand, carcinomas with large stromal component (such as pancreatic ductal adenocarcinoma) or large mucinous component (such as colorectal adenocarcinoma) contain a small proportion of viable cells on surface area; therefore they are not suitable for making high output TMA.
Compared with punching cylindrical core-based TMA method, the high output TMA technology developed in this study appears to be relatively easy, time-saving and cost-efficient. Therefore, this method is more feasible for general research groups to prepare high output TMA slides from appropriate tissue samples.
We are grateful to Zhichen Wang for his initial help in making the knife and cutting the rods of tissues, Dr Rongkui Luo for his assistance in the initial selection of suitable cases and reviewing of the archive slides, Dominic Yuan for his English proofreading. This study was funded by (i) Youth Science Fund of the National Natural Science Foundation of China, grant number 81101809, (ii) Shanghai Pujiang Program, grant number 09PJD009, and (iii) Shanghai Municipal Health Bureau Program, grant number2009020.
- Battifora H: The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest. 1986, 55 (2): 244-248.PubMedGoogle Scholar
- Wan WH, Fortuna MB, Furmanski P: A rapid and efficient method for testing immunohistochemical reactivity of monoclonal antibodies against multiple tissue samples simultaneously. J Immunol Methods. 1987, 103 (1): 121-129. 10.1016/0022-1759(87)90249-3.View ArticlePubMedGoogle Scholar
- Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP: Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998, 4 (7): 844-847. 10.1038/nm0798-844.View ArticlePubMedGoogle Scholar
- Jawhar NM: Tissue microarray: a rapidly evolving diagnostic and research tool. Ann Saudi Med. 2009, 29 (2): 123-127. 10.4103/0256-4947.51806.PubMed CentralView ArticlePubMedGoogle Scholar
- Avninder S, Ylaya K, Hewitt SM: Tissue microarray: a simple technology that has revolutionized research in pathology. J Postgrad Med. 2008, 54 (2): 158-162. 10.4103/0022-3859.40790.View ArticlePubMedGoogle Scholar
- Faratian D, Graham A, Rae F, Thomas J: Rapid screening of tissue microarrays for Her-2 fluorescence in situ hybridization testing is an accurate, efficient and economic method of providing an entirely in situ hybridization-based Her-2 testing service. Histopathology. 2009, 54 (4): 428-432. 10.1111/j.1365-2559.2009.03257.x.View ArticlePubMedGoogle Scholar
- Schraml P, Kononen J, Bubendorf L, Moch H, Bissig H, Nocito A, Mihatsch MJ, Kallioniemi OP, Sauter G: Tissue microarrays for gene amplification surveys in many different tumor types. Clin Cancer Res. 1999, 5 (8): 1966-1975.PubMedGoogle Scholar
- Veeck J, Dahl E: RNA expression analysis on formalin-fixed paraffin-embedded tissues in TMA format by RNA in situ hybridization. Methods Mol Biol. 2010, 664: 135-150. 10.1007/978-1-60761-806-5_14.View ArticlePubMedGoogle Scholar
- Schlomm T, Chun F, Erbersdobler A: From gene to clinic: TMA-based clinical validation of molecular markers in prostate cancer. Methods Mol Biol. 2010, 664: 177-189. 10.1007/978-1-60761-806-5_17.View ArticlePubMedGoogle Scholar
- Kallioniemi A: DNA copy number analysis on tissue microarrays. Methods Mol Biol. 2010, 664: 127-134. 10.1007/978-1-60761-806-5_13.View ArticlePubMedGoogle Scholar
- Gulmann C, Loring P, O'Grady A, Kay E: Miniature tissue microarrays for HercepTest standardisation and analysis. J Clin Pathol. 2004, 57 (11): 1229-1231. 10.1136/jcp.2004.018689.PubMed CentralView ArticlePubMedGoogle Scholar
- Gately K, Kerr K, O'Byrne K: Design, construction, and analysis of cell line arrays and tissue microarrays for gene expression analysis. Methods Mol Biol. 2011, 784: 139-153. 10.1007/978-1-61779-289-2_10.View ArticlePubMedGoogle Scholar
- Shebl AM, Zalata KR, Amin MM, El-Hawary AK: An inexpensive method of small paraffin tissue microarrays using mechanical pencil tips. Diagn Pathol. 2011, 6: 117-10.1186/1746-1596-6-117.PubMed CentralView ArticlePubMedGoogle Scholar
- Fowler CB, Man YG, Zhang S, O'Leary TJ, Mason JT, Cunningham RE: Tissue microarrays: construction and uses. Methods Mol Biol. 2011, 724: 23-35. 10.1007/978-1-61779-055-3_2.View ArticlePubMedGoogle Scholar
- Rubin BP, Singer S, Tsao C, Duensing A, Lux ML, Ruiz R, Hibbard MK, Chen CJ, Xiao S, Tuveson DA, and others: KIT activation is a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res. 2001, 61 (22): 8118-8121.PubMedGoogle Scholar
- Grabellus F, Ebeling P, Worm K, Sheu SY, Antoch G, Frilling A, Schmid KW: Double resistance to imatinib and AMG 706 caused by multiple acquired KIT exon 17 mutations in a gastrointestinal stromal tumour. Gut. 2007, 56 (7): 1025-1026. 10.1136/gut.2006.115923.PubMed CentralView ArticlePubMedGoogle Scholar
- Hou YY, Tan YS, Xu JF, Wang XN, Lu SH, Ji Y, Wang J, Zhu XZ: Schwannoma of the gastrointestinal tract: a clinicopathological, immunohistochemical and ultrastructural study of 33 cases. Histopathology. 2006, 48 (5): 536-545. 10.1111/j.1365-2559.2006.02370.x.View ArticlePubMedGoogle Scholar
- Pires AR, Andreiuolo Fda M, De Souza SR: TMA for all: a new method for the construction of tissue microarrays without recipient paraffin block using custom-built needles. Diagn Pathol. 2006, 1: 14-10.1186/1746-1596-1-14.PubMed CentralView ArticlePubMedGoogle Scholar
- Jhavar S, Corbishley CM, Dearnaley D, Fisher C, Falconer A, Parker C, Eeles R, Cooper CS: Construction of tissue microarrays from prostate needle biopsy specimens. Br J Cancer. 2005, 93 (4): 478-482. 10.1038/sj.bjc.6602726.PubMed CentralView ArticlePubMedGoogle Scholar
- Vogel UF: The construction of high-density paraffin tissue microarrays with 0.43-mm-diameter paraffin tissue core biopsies is technically feasible. Virchows Arch. 2008, 453 (1): 43-46. 10.1007/s00428-008-0622-9.View ArticlePubMedGoogle Scholar
- Mucci NR, Akdas G, Manely S, Rubin MA: Neuroendocrine expression in metastatic prostate cancer: evaluation of high throughput tissue microarrays to detect heterogeneous protein expression. Hum Pathol. 2000, 31 (4): 406-414. 10.1053/hp.2000.7295.View ArticlePubMedGoogle Scholar
- Hoos A, Cordon-Cardo C: Tissue microarray profiling of cancer specimens and cell lines: opportunities and limitations. Lab Invest. 2001, 81 (10): 1331-1338. 10.1038/labinvest.3780347.View ArticlePubMedGoogle Scholar
- Mengel M, Kreipe H, von Wasielewski R: Rapid and large-scale transition of new tumor biomarkers to clinical biopsy material by innovative tissue microarray systems. Appl Immunohistochem Mol Morphol. 2003, 11 (3): 261-268.View ArticlePubMedGoogle Scholar
- Miettinen M: A simple method for generating multitissue blocks without special equipment. Appl Immunohistochem Mol Morphol. 2012, 20 (4): 410-412. 10.1097/PAI.0b013e318245c82f.View ArticlePubMedGoogle Scholar
- Battifora H, Mehta P: The checkerboard tissue block. An improved multitissue control block. Lab Invest. 1990, 63 (5): 722-724.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.