|Year : 2017 | Volume
| Issue : 4 | Page : 203-209
Assorted archetypal tissue analysis: A breakthrough in oncopathology
Sushmita Swain1, Harish Kumar2, Sujatha Ramachandra2, Shyam S Behura2
1 Department of Oral Pathology and Microbiology, Kalinga Institute of Dental Sciences, Kalinga Institute of Industrial Technology, Deemed to be University, Bhubaneswar, Odisha, India
2 Department of Oral and Maxillofacial Pathology, Kalinga Institute of Dental Sciences, Kalinga Institute of Industrial Technology, Deemed to be University, Bhubaneswar, Odisha, India
|Date of Web Publication||26-Dec-2017|
Dr. Sujatha Ramachandra
Department of Oral and Maxillofacial Pathology, Kalinga Institute of Dental Sciences, Kalinga Institute of Industrial Technology, Deemed to be University, Bhubaneswar, Odisha
Source of Support: None, Conflict of Interest: None
Assorted Archetypal Tissue Analysis, commonly known as Tissue Microarray (TMA) technology is a highly throughput research tool that has greatly facilitated and accelerated tissue analysis by in-situ technologies. It involves core needle biopsies of multiple tissues constructed in the same block and is an innovative method where dozens of biomarkers can be applied to hundreds of samples simultaneously sparing costs, resources, and tissues.TMA provides a judicious use of precious tissue and gives experimental uniformity. Analyzing a large number of samples improves statistical precision in addition to enhanced speed and quality of analysis making it extremely useful in pathology research and practice. This article reviews the sequential development, modification. and applications of TMA and also comments on how TMA facilitates translation research at different levels.
Keywords: Assorted archetypal tissue analysis, tissue microarray, tissue core
|How to cite this article:|
Swain S, Kumar H, Ramachandra S, Behura SS. Assorted archetypal tissue analysis: A breakthrough in oncopathology. J NTR Univ Health Sci 2017;6:203-9
|How to cite this URL:|
Swain S, Kumar H, Ramachandra S, Behura SS. Assorted archetypal tissue analysis: A breakthrough in oncopathology. J NTR Univ Health Sci [serial online] 2017 [cited 2018 Jul 18];6:203-9. Available from: http://www.jdrntruhs.org/text.asp?2017/6/4/203/221525
| Introduction|| |
The current biomedical research is being fundamentally changed by the development of high-throughput screening techniques. To validate, prioritize, and select the best target from tens of thousands of candidate genes and proteins is a challenging task. Microarray involves analysis of molecular targets at the cellular level, assessment of their expression, and evaluation of their clinical significance, which would provide significant additional information to target selection.
Microarray is a gene expression profiling technology, which helps to analyze changes in the multi-gene patterns of expression and broader bioactivity functions of genes to produce either quantitative or qualitative data. Various types of microarrays exist such as chemical, DNA, protein, antibody, transfixation, and tissue microarrays (TMA).
TMA was first reported in 1987 from National Human Genome Research Institute, Bethesda, USA., It has been used for study of tumor biology, the assessment of novel molecular biomarkers and laboratory quality assurance. It is a simple mechanical method that employs a novel sampling approach to produce minute cylindrical tissue cores of regular size and shape subsequently arranged on a single paraffin block.
Comparisons of expression patterns between non-diseased and diseased tissues resulted in the identification of thousands of potentially disease-related gene. Although the initially used molecular genetic analysis methods such as Multi-tissue Northern/Western Blot analyses, protein arrays and Reactive Polymerase Chain Reaction (PCR) were ideal for throughput analysis, these had the disadvantage of tissue disintegration. Later, in-situ technologies such as RNA in situ Hybridization (RNA-ISH) or Fluorescence in situ Hybridization (FISH) were introduced but large scale analyses were inconvenient. Moreover, it was not possible to cut more than 200 regular sections from one tissue block. This also resulted in the exhaust of valuable tissue resources.
To overcome the above limitations, TMA technique was developed where hundreds of different minute tissue samples are placed on one microscope glass slide and subjected to joint analysis by in situ methods.
In the year 1965, Lillie  came forward with a technique of special blocking and trimming procedure for cross section of multiple small tubular structures , which was then in 1986 modified by Battifora by introducing multi-tumor (sausage) tissue block (MTTB) that were wrapped as fixed tissue rods., In 1988, Kraaz  came up with a technique of multiblock control for immunohistochemistry in which 4 mm of skin punch biopsy was modified with a mandrill and cores were placed into a warm cast., Similarly, Battifora  in 1990 introduced checkerboard tissue block in which fixed tissue rods were stacked with agar plates., Kraaz punching method was brought up in 1994 by Rose  for teaching purpose in which the multi-block slides were presented in grid pattern by careful hand positioning., In the same year, Sundblad  was credited for the simplified multi-tissue block technique in which he removed wedge shaped tissue rods from the surface of paraffin donor block and processed further as Multitumor Sausage Blocks (MTSB) as controls (stack of paraffin plates of tissue rods)., In 1998, Kononen  introduced TMA, which consisted of punching paraffin tissue cores, arrangement in a Cartesian Coordinate system  and development of manual tissue arrayer.
In 2000, modification of specified multiple tissue core array was done by Gillet  using a 11-gauge core cut needle. He punched tissue cores and installed into a recipient block with preformed holes, punched with a 13-gauge needle., In 2003, Vogel  constructed TMA with pre-drilled ordinary steel embedding molds, while Hidalgo  constructed manually using a bone marrow aspiration needle. In the same year, Matysiak  constructed it with semi-automated Kononen tissue arrayer., In 2005, TMA from suspension cells were constructed by Montgomery  using paraffin-embedded cell pellets in Eppendorf tubes and punching the cells out of the tube., Chen , on the other hand, constructed TMA using double-sided adhesive tape technique with X-ray film backbone, without prefabricating recipient blocks. Wang  in 2006 constructed TMA using a handmade paper mold, while stabilization body such as agar was used by Zhou  (2007) for TMA technology in frozen pathological tissues. Vogel  experimented with evenly long core biopsies created with a cutting board and a cutting board arrays in 2008 and in 2010, he combined the drilling technique with the adhesive tape technique for constructing TMA from needle biopsy specimen.,,
Improvement of the checker technique by punching the prostrate biopsies out of the checkers with the kononen technique (beecher tissue arrayer) was done by McCarthy  in 2011., Pilla  in 2012 improved the TMA technique by implementation of a barcode driven error control of design and execution of a TMA by using the laboratory information system. In 2013, Deng  came up with the idea of 'Patch TMA' where TMA was made on a slide using cores of retrieved already stained sections., Shi, in the same year, introduced tissue rods punched parallel to the donor block surface to ensure equal length of the rods and the tissue of interest in every section to be cut., Inexpensive self-made tissue punches were introduced by Gracia-Gracia  useful in paraffin TMAs.
Tissue microarray: Construction and its design
For maximizing the benefits of TMA, extensive planning and attention to details are required prior to construction.
Tissue microarray construction
Relevant clinical data and tissue is collected. The archival formalin-fixed paraffin-embedded tissues are stained with regular stains before starting as eventually the quality of TMA will be represented by the quality of each tissue selected.,
Mapping of slides
Mapping of the area of interest with ink on the freshly stained slide on a scanning power is the optimal method in selecting a tissue for arraying from a donor block. The depth of tissue should be importantly considered as the width of the same.
Cylindrical cores of paraffin-embedded tissue are removed from pre-existing donor tissue blocks for construction of TMA, which may be of surgical pathology autopsy or research material. A blank recipient block made from low melting point paraffin (52–56° C) is taken and these tissue cylinders are then inserted into the blocks. Care should be taken to prevent bubble formation within the block as it may cause problems during the arraying process., Ideal measure of at least 45 × 20 mm in dimension with practical array field of 22 × 18 mm is considered which would easily hold 500 or more 0.6mm cores. Sub-array should ideally be large enough to be entirely seen at 4× magnification.,
In the manual method [Figure 1], visual section during punching by use of magnifying glass or stereomicroscope as a guide is performed, while in the automated method selection, marking, editing as well as punch coordinates are saved using an on-screen display and software tools. Punching speed becomes faster as well as the block capacity is seven times more in automated method [Figure 2]. In automated device (Beecher's Instruments, USA), pre-marked slide images of tissue blocks are displayed by the video-merge unit side by side to the donor block image while regions of interest are marked by pathologists by hand manually before arraying. [Figure 3] shows a recipient block containing 18 cores.
Array design and sampling
The main purpose of arraying is to match the core size to the number of cores and transfer the tissue cylinders into recipient block. The diameter of needles varies and comes in 0.6, 1.0., 1.5, 2.0 mm, which corresponds to 500, 200, 100, 50 cores, respectively, in the recipient block. Before construction of a tissue array, making a template grid with selected area size and individual sub-arrays proves to be very useful. The size of the needle should be selected based on quantity and quality of available tissue. As the needle diameter increases, the spacing between the cores must increase. The core diameter should be inversely proportional to the size of the sample. There should be no air bubbles, the needles should be clean, and the stylet should move freely. Recipient hole should be made prior to the coring of donor tissue to avoid deformity of holes due to elasticity of paraffin. A 'pointer' core on the top left of the array area of the recipient block can be made for providing orientation. After arraying of recipient block with desired tissue cores, tempering is done by keeping it in an incubator at 37°C overnight. Cooling is important to make the block hold all the cores firmly and helps to cut 100–150 sections of 3–5 microns with ease by help of microtome as shown in [Figure 4].,, [Figure 5] shows a prepared TMA slide after the routine staining procedure. [Figure 6] and [Figure 7] shows tissue cores under various magnifications.
Applications of tissue microarray
TMA technology has facilitated high throughput analyses on a large series of tissues in a single experiment as in Immunohistochemistry (IHC) and ISH.
- Validation of diagnostic biomarkers
- Validating the genomic and proteomic data across multi-tumor types in a limited time frame in annotated clinical samples is a very important use of TMA. It also serves as surrogate endpoints for clinical studies, as screening tools., In order to improve diagnostic accuracy, conventional histology can be incorporated with molecular biomarkers. Hence, with improvised TMA technology, the practical diagnostic value of histologically similar neoplasms can be assessed by new biomarkers identified by gene expression profiling 
- Validation of prognostic biomarkers:
- Correlations of biomarker expression with clinical end points like disease-free survival or overall survival are now becoming possible as tumor banks and clinical sample cohorts are maturing with regard to clinical follow up data. Changes in protein expression is identified in tissue samples and is co-related with clinico-pathological parameters
- TMA can also be constructed on basis of patients receiving specific treatments and its response to therapy leading to validating its predictive value ,
- TMA technology is being not only used in cancer research but it is also widening its horizon in non-neoplastic pathology research such as neurodegenerative, dermatological, cardiac, and placental diseases 
- Testing of new antibodies and probes as well as determination of optimal staining conditions can be done using TMAs in order to define the functional status of the tumor prior to therapy clinically., Clinical application also includes xenograft tumor assays, which help to measure susceptibility or response to a drug ,
- IHC staining procedures and interpretation of internal and external quality control assurances against a validated benchmark can be made using TMAs ,
- Microbial detection and identification along with microbial typing and gene expression profiling to determine host genomic polymorphism are also useful applications of TMAs 
- Tissue immunoblotting proposed by Chung et al. to overcome inherent limitation of IHC proteomic profiling on TMA for quantitative protein analysis, resulted in better functional protein array construction by tissue trans-blotting approach in which the proteins from a paraffin-embedded TMA section was transferred from glass slide to a nitrocellulose membrane
- Tissue repository and education.
Advantages of tissue microarray
TMAs allow the performance of tissue-based arrays on a large number of patient samples in an efficient and cost effective manner with respect to the use of laboratory reagents and technician time., Several hundred representative cores from several hundred patients may be included on a single glass slide for array. Thus, significant amount of tissue can be conserved. Source material for TMAs is readily available and is often linked to long-term outcome data. Studying morphological and molecular changes by construction of ex-vivo tumor progression model through different tumor progression stages is easily performed by constructing a TMA., In addition, methods for generating TMAs from fresh frozen tissue using blocks made from either Optimal Cutting Temperature (OCT) compound or from a mix of gelatin-sucrose have been described. TMAs can be generated from all tissue types including decalcified bone to core biopsies. The common types of TMA used in research field has been described in [Figure 8] and [Table 1].
Limitations of tissue microarray
Tissue heterogeneity, i.e., each core representing a fraction of lesion has been the major disadvantage of TMAs as they may not represent the entire tumor. In order to avenge out the problem associated with the tumor heterogenicity and consequent false positive and false negative results, two main strategies have been employed., The first is increasing the number of cores from each case, and second is, increasing the sample size in the study. Smaller cores permit sampling of different tumor areas and are therefore more likely to be representative of the entire tumor and they tend to inflict lesser degree of damage on the original tissue blocks.
There is marked heterogeneity within certain tumors such as glioblastoma, which cannot be captured in TMA studies. Study of rare or focal events such as number of immune cells in tumors is also cumbersome. Study of facets of tumor biology such as interactions between the tumor and its stromal components is difficult.
| Conclusion|| |
TMA has immensely revolutionized the histopathological analysis in research in the current world of high-throughput screening technology. It is amenable to a wide range of molecular techniques, provides judicious use of tissue specimen, gives experimental uniformity, and allows large samples to be arrayed on a single slide in an efficient and cost-effective manner.
TMA studies are likely to fasten the decision of molecular biomarkers acceptance in clinical setting and facilitate even much better efficient data analysis by its further automation and sophistication tool development.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Simon R, Mirlacher M, Sauter G. Tissue microarrays. Biotechniques 2004;36:98-105.
Miller MB, Tang YW. Basic concepts of microarray and potential applications in clinical microbiology. Clin Microbiol Rev 2009;22:611-33.
Aktas S. Tissue microarray. Current perpectives in pathology. Aegean Pathol J 2004;1:27-32.
Barrette K, Joost J, Oord V D, Garmyn M. Tissue microarray. J Invest Dermatol 2014;134:e24.
Solomon MC, Carnelio S, Gudattu V. Molecular analysis of oral squamous cell carcinoma – a tissue microarray study. Int J Cancer 2010; 47:166-72.
Lilie RD. Histopathologic Technic and Practical Histochemistry. 3rd
ed. McGraw-Hill Book Co.: New York, USA; 1965.
Vogel U. Overview on techniques to contract tissue arrays with special emphasis on tissue microarrays. Microarrays 2014;3:103-36.
Battifora H. The multitumor (sausage) tissue block: Novel method for immunohistochemical antibody testing. Lab Invest 1986;55:244-8.
Kraaz W, Risberg B, Hussein A. Multiblock: An aid in diagnostic immunohistochemistry. J Clin Pathol 1988;41:1337.
Battifora H, Mehta P. The checkerboard tissue block. An improved multitissue control block. Lab Invest 1990;63:722-4.
Rose DS, Maddox PH, Brown DC. Multiblock slides: A useful technique for teaching. J Clin Pathol 1994;47:88-9.
Sundblad AS. A simplified multitissue block. Am J Clin Pathol 1994;102:192-3.
Kononen J, Bubendorf L, Kallioniemi A, Brlund M, Schraml P, Leighton S, et al
. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998;4:844-7.
Leighton SB. Instrument for Constructing Tissue Arrays. U.S. Patent 6,103,518, 2000.
Gilett C, Springall R, Barnes D, Hanby AM. Multiple tissue core arrays in histopathology research: A validation study. J Pathol 2000;192:549-53.
Vogel U, Bltmann B. A simple and cheap method to produce home-made high density multi-tissue arrays. Lab Invest 2003;83:328-34.
Hidalgo A, Pina 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.
Matysiak BE, Brodzeller T, Buck S, French A, Counts C, Boorsma B, et al
. Simple, inexpensive method for automating tissue microarray production provides enhanced microarray reproducibility. Appl Immunohistochem Mol Morph 2003;11:269-73.
Montgomery K, Zhao S, Van de Rijn M, Natkunam Y. A novel method for making “tissue” microarrays from small numbers of suspension cells. Appl Immunohistochem Mol Morphol 2005;13:80-4.
Chen N, Zhou Q. Constructing tissue microarrays without prefabricating recipient blocks. A novel approach. Am J Clin Pathol 2005;124:103-7.
Wang SL, Yang CH, Chen HH, Chai CY. A simple and economical method for the manual construction of well-aligned tissue arrays. Pathol Res Pract 2006;202:485-6.
Zhou L, Hodeib M, Abad JD, Mendoza L, Kore AR, Hu Z. New tissue microarray technology for analyses of gene expression in frozen pathological samples. BioTechniques 2007;43:101-5.
Vogel UF. Depositing archived paraffin tissue core biopsy specimens in paraffin tissue microarray using a paraffin tissue punch modified with a countersink. J Clin Pathol 2007;60:206-07.
Vogel U, Bultmann B. Application of a novel and low cost technique to construct paraffin tissue microarrays out of paraffinized needle biopsy specimens from patients with breast cancer. J Clin Pathol 2010;63:640-3.
McCarthy F, Dennis N, Flohr P, Jhavar S, Parker C, Cooper CS. High-density tissue microarrays from prostate needle biopsies. J Clin Pathol 2011;64:88-90.
Pilla D, Bosisio FM, Marotta R, Faggi S, Forlani P, Falavigna M, et al
. Tissue microarray design and construction for scientific, industrial and diagnostic use. J Pathol Inform 2012;3:42.
] [Full text]
Deng FM, Zhao Y, Kong X, Lee P, Melamed J. Construction of tissue microarrays using pre-existing slides as source of tissue when paraffin blocks are unavailable. J Clin Pathol 2013;66:627-9.
Shi Y, He D, Hou Y, Hu Q, Xu C, Liu Y, et al
. An alternative high output tissue microarray technique. Diagn Pathol 2013;8:9.
Waterwowrth A, Horgan K, Speirs V, Hanby AM. Tissue microarrays – big potential from small samples. Int J Oncology 2004;25:167-71.
Avninder S, Ylaya K, Hewitt SM. Tissue microarray: A simple technology that has revolutionized research in pathology. J Postgrad Med 2008;54:158-62.
] [Full text]
Fedor HL, De Marzo AM. Practical methods for tissue microarray construction. Met Mol Med 2005;103:89-101.
Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schrawl P, Leighton S, et al
. Tissue microarrays for highthroughput molecular profiling of tumor specimens. Nat Med 1998;4:844-7.
Olli P, Kallioniemi, Wagner U, Kononen J, Sauter G. Tissue microarray technology for high-throughput molecular profiling of cancer. Hum Mol Genet 2001;10:657-62.
Hewitt SM. Design, construction and use of tissue microarrays. Met Mol Biol 2004;261-72.
Sexena R, Badve S. Tissue microarray- construction and quality assurance. Dako handbook, immunohistochemical staining methods 5th
Voduc D, Kenney C, Nielsen TO. Tissue Microarrays in clinical oncology. Semin Radiat Oncol 2008;18:89-97.
Chung JY, Braunschweig T, Tuttle K, Hewitt SM. Tissue microarrays as a platform for proteomic investigation. J Mol Histo 2007;38:123-8.
Russo G, Zegas C, Giordono A. Advantage and limitations of microarray technology in human cancer. Oncogene 2003;22:6497-507.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]