Tissue Microarrays: Applications in Neuropathology Research, Diagnosis, and Education

Tissue microarrays (TMAs) are composite paraffin blocks constructed by extracting cylindrical tissue core "biopsies" from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates. Using this technique, up to 1000 or more tissue samples can be composited into a single paraffin block. Tissue microarrays permit high-volume simultaneous analysis of molecular targets at the DNA, mRNA, and protein levels under identical, standardized conditions on a single glass slide, and also provide maximal preservation and utilization of limited and irreplaceable archival tissue samples. This versatile technique facilitates retrospective and prospective human tissue studies, animal tissue studies, and cell line cytospin cell block studies. In this review, we present the technical aspects of TMA construction and sectioning, validation aspects of the technique, TMA advantages and limitations, and a sampling of the broad range of TMA uses in modern neuropathologic clinical diagnosis, research, and education. A specific illustration of the most widely employed and increasingly important TMA application is also presented: confirmation via TMA-based immunohistochemistry of the differential expression of a marker (IGFBP2) initially identified by gene expression profiling to be overexpressed in glioblastoma.     

Depositing archived paraffin tissue core biopsy specimens in paraffin tissue microarrays using a paraffin tissue punch modified with a countersink

Paraffin tissue microarrays (PTMAs) introduced by Kononen et al in 1998 have become a widely used technique in routine pathology and even more so in research. Kononen used a tissue puncher/arrayer (Beecher Instruments, Sun Prairie, WI, USA) to take paraffin tissue core biopsy specimens (PTCBs) of 0.6-2 mm in diameter from routine paraffin tissue blocks and transfer them to another paraffin block with up to 1000 holes. As pointed out by Mengel et al, however, it is not possible to use the Kononen/Beecher system to construct PTMAs out of archived PTCBs. To overcome this drawback in the extremely popular Beecher system, the paraffin tissue punch was modified by incorporating a conical 4 mm deep countersink. This countersink was milled with a conical precision cutter that can be bought in an ordinary hardware store (cost <5 US dollars). The countersink facilitates the insertion of an archived PTCB into the paraffin tissue punch and enables the construction of PTMAs with previously archived PTCBs using the widely distributed Beecher system. Moreover, this paraffin tissue punch can be used for other systems to create PTMAs, such as the low-budget systems designed by Vogel.     

The application of tissue microarrays to cancer research

Tissue microarrays are paraffin blocks containing multiple cyclindrical tissue biopsy cores taken from individual donor paraffin-embedded tissue blocks and placed into a recipient block with defined array coordinates. Tissue microarray technology facilitates rapid assessment of the clinical relevance of molecular markers by enabling the simultaneous analysis of hundreds of tissue specimens. One of the applications of this technology is to significantly accelerate advances in cancer research through more efficient assessment of novel markers of outcome and response and, as a result, a more rapid application of this knowledge to clinical practice.     

Radiographic determination of tissue thickness in paraffin blocks: application to the construction of tissue microarrays.

The determination of tissue thickness in paraffin blocks in the histology laboratory has been largely based on visual estimates. More accurate methods are required for the construction of tissue microarrays (TMAs) to assure a greater yield of cores in sections through the TMA block. We describe an accurate radiographic method to determine tissue thickness in donor paraffin blocks and have validated its application to TMA construction. Individual radiographic analysis was performed on paraffin donor blocks used for the construction of TMAs for determination of donor block tissue thickness. Consecutive numbered slide sections through the TMA block were then examined for the presence or loss of cores in the 150th TMA slide (from the final third of the TMA block) and correlated with the thickness of the individual donor blocks determined radiographically. At the 150th TMA slide, 202 of 1340 cores (15.1%) were depleted. Radiographic measurement showed a greater thickness of the donor paraffin block tissue (2.02 mm) corresponding to the retained cores as compared with the donor tissue (1.54 mm) of the depleted cores (P < 0.001). With progressive slide sections through a TMA block, the retention of tissue cores shows a significant correlation with donor block tissue thickness. Radiographic determination of tissue thickness in donor paraffin blocks can be used in TMA construction. Prior knowledge of tissue thickness in TMA construction can prompt compensatory steps that can enhance the yield of valuable samples and assure sufficient numbers of adequate cores for statistical analysis in biomarker evaluations.     

组织芯片的制备与应用

组织芯片(tissue chip)又称组织微阵列(tissue microarray)是一种将一系列标准小组织标本整齐有序地排放在石蜡块上,从而在一张切片上对数十个、数百个、乃至上千个组织样本同时进行分析的技术方法。组织芯片能够在分子水平对肿瘤和其它疾病进行快速、全面、高通量的原位分析,作为一种新型的组织学研究工具,不仅在肿瘤研究中具有价值而且还将在病理学的其他领域发挥重要作用。     

Constructing tissue microarrays without prefabricating recipient blocks: a novel approach

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.     

The construction of high-density paraffin tissue microarrays with 0.43-mm-diameter paraffin tissue core biopsies is technically feasible

Paraffin tissue microarrays (PTMAs) are blocks of paraffin containing up to 1,000 paraffin tissue core biopsies (PTCBs). The growing number of publications in recent years bears eloquent witness to the advantages of these PTMAs in high-throughput molecular profiling of tumor specimens. In order to conserve the often minute quantities of available tumor tissue with precisely recorded follow-up data and to store the greatest possible number of PTCBs in one block, researchers often try to reduce PTCBs to the smallest possible diameter. Until now, the smallest feasible diameter for PTCBs was 0.6 mm. Experiments with diameters below 0.6 mm have failed due to the instability of the paraffin tissue punch. The process described allows the construction of PTMAs with PTCBs only 0.43 mm in diameter utilizing simple, inexpensive, self-made paraffin tissue punches and predrilled recipient blocks.     

Agarose mold embedding of cultured cells for tissue microarrays.

There are several indications for the placement of samples of cultured cells in tissue microarrays (TMAs). To optimize this technique, three embedding procedures were compared: embedding of fixed cells pelleted by centrifugation, embedding of cells dispersed in an agarose matrix, and embedding of pelleted cells packed into the center of hollow agarose molds. TMAs were made from these preparations. The number of cells per tissue spot and the number of histologic sections that could be obtained from the preparations were determined. The agarose matrix and agarose mold techniques resulted in the longest core samples, while the cell pellet and agarose mold methods resulted in the greatest cell density. Thus, the use of cylindrical agarose molds optimizes both the number of cells present on a histologic section of a TMA, and the number of histologic sections that can be obtained from a TMA. This technique results in a paraffin-embedded cell preparation that yields a cell density of approximately 1000 cells per 0.6-mm diameter circular histologic section, and that produces uniform core samples the full thickness of the donor block. Histologic sections of TMAs prepared in this manner were validated in immunohistochemical and in situ hybridization assays.     

Multitissue array review: a chronological description of tissue array techniques, applications and procedures

We performed a systematic search for literature dealing with tissue microarray technology. During the last two decades, these procedures have developed into a powerful tool for the high-throughput analysis of tissue specimens. This technology offers the following advantages: amplification of a scarce resource, experimental uniformity (tissue of multiple patients are treated in an identical manner), decreased assay volume, preservation of original blocks, amenability to a wide range of techniques and evaluation of tissue from multiple patients on the same slide. Depending on the shape of the tissue sample and the method used to obtain it, multitissue array techniques may be classified into two different groups: rod-shaped tissue techniques and core tissue techniques. These techniques have been used for quality control, diagnosis, and teaching and screening purposes. Some technical aspects should be considered when deciding which technique should be used: the number, size and origin of tissue samples; the quality of paraffin wax, the distance between samples and the depth in the receptor block; antigenicity preservation and block sectioning. This review shows different techniques, the use of which is dependent on the requirements of the arrays and the technical possibilities.     

A simple and economical method for the manual construction of frozen tissue arrays

Tsao S‐C, Wu C‐C, Wen C‐H, Huang Y‐C, Chai C‐Y. A simple and economical method for the manual construction of frozen tissue arrays. APMIS 2010; 118: 739–43. Tissue microarray has been developed to enable multiple cores of tissue in one or more new paraffin blocks. Currently, almost all tissue microarrays are made by coring cylindrical tissues from formalin‐fixed and paraffin‐embedded tissues. The disadvantages of formalin‐fixed and paraffin‐embedded tissues include the poor preservation of antigenicity of certain proteins and mRNA degradation induced by the fixation and embedding process. However, frozen tissue array construction presents technical difficulties, and tissue array devices are expensive, particularly for small‐ and medium‐sized laboratories. We describe a simple manual method for producing well‐aligned tissue arrays by a capsule freeze method that allows us to successfully perform hematoxylin–eosin and immunohistochemical stain. All 120 tissue samples were collected and constructed into blocks by this capsule freeze method. The capsules were not affected during the sectioning process, and the capsule material always disappeared during the aqueous steps of the stain processing. The frozen tissue arrays were smoothly sectioned without the use of a tape transfer system and immunohistochemical study was performed with satisfactory results. This alternative method can be applied in any laboratory, and is both simple and economical.     

Tissue microarrays, or the advent of chips in pathology

Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The method consists to core tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. Despite the low amount of tissue analyzed by tissue microarray, different studies have demonstrated a high concordance of protein expression between this technique and the conventional tissue sections.     

Combination of nucleic acid and protein isolation with tissue array construction: using defined histologic regions in single frozen tissue blocks for multiple research purposes

Precise dissection of defined histological regions for nucleic acid and protein isolation is a precedent step in finding out cancer-related alterations, and high quality tissue microarrays are demanded in the validation of screened genetic alterations by multiple in situ approaches. In this study, a combined technique was developed by which sample isolation and tissue array construction could be performed on the defined morphological region(s) in single tissue block. The RNA and protein samples generated from the selected portions were of good quality and sufficient for multiple experimental purposes. The frozen tissue arrays constructed on a novel recipient are suitable for multiple in situ evaluations including immunohistochemical staining and mRNA hybridisation. In most cases, the data obtained from in situ assays coincided well with the ones revealed by RT-PCR and Western blot hybridisation. The potential experimental bias caused by cell contamination can be amended by tissue array-based retrospective examination. The combination of tissue-selective sample preparations with tissue array construction thus provide a tool by which comprehensive cancer research can be performed on defined histological regions in a series of single frozen tissue blocks.     

An easy method for manual construction of high-density tissue arrays.

Tissue array technique is a powerful tool for high-throughput in situ analysis on a large cohort of cases. This report describes an easy and effective method for manual construction of high-density tissue array containing 88 (11 x 8) samples, each of which measures 2 mm in diameter. No extraneous device is needed except a conventional 16-gauge bone marrow biopsy trephine apparatus to puncture the paraffin blocks. The authors constructed 563 cases of epithelial neoplasm into 7 blocks. The sectioning is smooth and does not require adhesive tape. They performed immunohistochemical staining for cytokeratins 7 and 20 on these tissue array slides. The samples rarely fell off during the antigen retrieval and staining procedure. The results were generally in agreement with those in previous reports. The authors offer a satisfactory alternative method for building custom arrays for any laboratory that is unable to afford a tissue array apparatus.     

Frozen Tumor Tissue Microarray Technology for Analysis of Tumor RNA, DNA, and Proteins

Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. One difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. We have modified this technology by using frozen tissues embedded in OCT compound as donor samples and arraying the specimens into a recipient OCT block. Tumor tissue is not fixed before embedding, and sections from the array are evaluated without fixation or postfixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. While paraffin tissue arrays can be problematic for immunohistochemistry and for RNA in situ hybridization analyses, this method allows optimal evaluation by each technique and uniform fixation across the array panel. We show OCT arrays work well for DNA, RNA, and protein analyses, and may have significant advantages over the original technology for the assessment of some genes and proteins by improving both qualitative and quantitative results.     

Simple, inexpensive, and precise paraffin tissue microarrays constructed with a conventional microcompound table and a drill grinder

In 1998, paraffin tissue microarrays (PTMAs) as paraffin blocks containing up to 1,000 cylindrical paraffin tissue core biopsy specimens (PTCBs) for high-throughput molecular profiling of tumor specimens were introduced. PTCBs can be constructed using a manual tissue puncher/arrayer (Beecher Instruments, Sun Prairie, WI; cost, at least $7,000). Furthermore, custom-built PTMAs such as the MaxArray are created by companies such as Zymed Laboratories (South San Francisco, CA; PTMA with 96 holes, about $900). In our search for a less expensive alternative, we constructed PTMAs with up to 558 PTCBs by using a drill grinder, a drill stand, and a microcompound table (Proxxon, Niersdorf, Germany; cost, <$300).     

A rapid and efficient method for testing immunohistochemical reactivity of monoclonal antibodies against multiple tissue samples simultaneously

A flexible, efficient and rapid method was developed whereby a small volume of monoclonal antibody could be used to immunohistochemically stain many different tissues, simultaneously, on one standard glass slide. This method is based on the preparation of 'cores' of paraffin-embedded tissue from standard histology blocks. The paraffin cores are straightened, inserted into a casing cut from an ordinary drinking straw, mounted in a paraffin block and sectioned. Over 120 individual tissue samples can be organized on a slide and stained for screening or characterization with 0.25 ml of diluted primary antibody. Advantages of this paraffin core method include: great economies in time, reagents, tissue specimens and antibodies; ease of producing multiple, regular, stable, easily handled tissue core samples; direct identification of intratissue regions of interest for inclusion; efficient use of rare tissue samples; versatility for rapid construction of multiple tissue slides containing any combination of relevant tissues from a 'library' of tissue cores; and, no need for deparaffinization and reembedding of tissues.     

Mammographic and gross pathologic analysis of breast needle localization specimens. Use of a tissue analysis device

Histologic diagnosis of a nonpalpable breast lesion requires the following: (1) mammographically directed excision of the suspect lesion, (2) recovery of the target lesion from the tissue specimen, and (3) demonstration of the lesion in histologic section. Recovery of lesional tissue is a function of localization within the specimen, whereas histologic demonstration is a function of localization within the paraffin block. Both localizations are pitfalls enroute to accurate diagnosis of the small mammographic lesion. A technique of specimen analysis is presented that uses a specimen holder to assure precise localization of the lesion within the biopsy tissue. The excised specimen is immobilized within the holder, which incorporates a reference grid visible in the specimen radiograph and on the holder surface. The target lesion can be localized in the reference grid of the specimen radiograph and then excised from the corresponding area of the specimen, which remains in the holder until presented to the gross pathologist. Localization of the lesion within a specific paraffin block(s) will permit additional histologic levels for optimal examination while minimizing histologic work load. The advantages of this approach are discussed and contrasted with variations of standard gross technique.     

A simple method for the construction of small format tissue arrays

Tissue arrays can evaluate molecular targets in high numbers of samples in parallel. Array construction presents technical difficulties and tissue arrayers are expensive, particularly for small and medium sized laboratories. This report describes a method for the construction of 36 sample arrays using widely available materials. A blunted 16 gauge needle for bone marrow aspiration was used to extract paraffin wax cylinders and manually define a 6 x 6 matrix on a blank paraffin wax block. Tissue cores from 36 paraffin wax embedded premalignant lesions and invasive cervical carcinomas were injected into the matrix using a 14 gauge needle. This tissue array was sectioned using a standard microtome and used for the immunodetection of CD44 variant 9 and interleukin 18 with satisfactory results. This method can be applied in any laboratory, without the need of specialised equipment, offering a good alternative for the wider application of tissue arrays.     

Identification of tumor specimens by DNA analysis in a case of histocytological paraffin tissue block swapping

We report on a patient who was diagnosed with high-grade breast carcinoma by all the pre-surgery clinical evidence of malignancy, but histopathological reports did not reveal any such tumor residue in the post-surgical tissue block. This raised a suspicion that either exchange of block, labeling error, or a technical error took place during gross examination of the tissue. The mastectomy residue was unprocurable to sort out the problem. So, two doubtful paraffin blocks were sent for DNA fingerprinting analysis. The partial DNA profiles (8-9/15 loci) were obtained from histocytological blocks. The random matching probability for both the paraffin blocks and the patient's blood were found to be 1 in 4.43E4, 1.89E6, and 8.83E13, respectively for Asian population. Multiplex short tandem repeat analysis applied in this case determined that the cause of tumor absence was an error in gross examination of the post-surgical tissue. Moreover, the analysis helped in justifying the therapy given to the patient. Thus, with DNA fingerprinting technique, it was concluded that there was no exchange of the blocks between the two patients operated on the same day and the treatment given to the concerned patient was in the right direction.     

Increasing the efficiency of paraffin tissue microarrays by packing the paraffin tissue core biopsies in a honeycomb pattern.

Paraffin tissue microarrays (PTMAs) are blocks of paraffin holding up to 1000 paraffin tissue core biopsies (PTCBs) for high throughput molecular analysis. The number of PTCBs in a PTMA depends on the surface area of the PTMA, the diameter of and the distance between the PTCBs and on their arrangement inside the assembled PTMA. The PTCBs are usually arranged in a rectangular x-y pattern of rows and columns. This design facilitates the construction of a PTMA because the operator simply turns the wheels of an x-y-table for a set, unchanging distance. The evaluation of the stained sections is also relatively easy. However, this rectangular arrangement means wasted space in the PTMA. To reclaim this space, the PTCBs could be arranged in a honeycomblike pattern. For every 8 rows in the conventional rectangular arrangement, 1 additional row of PTCBs can be packed. However, the researcher has to become accustomed to this uncommon arrangement when filling and evaluating the PTMA. Automatic slide readers and specially designed computer programs for the digital evaluation of the PTMAs can be helpful. In summary, the arrangement of PTCBs in a honeycomblike pattern increases the density and number of specimens stored in a PTMA, thereby enhancing its efficiency.