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Concept: Tissue microarray

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Tissue microarrays were originally developed to enable alignment of multiple tissue cores in a single paraffin block and to enable high-throughput laboratory analysis. However, a major drawback is the loss of tissue cores during slide preparation, especially when sectioning the tissue block. Tissue cylinders directly aligned in the metal box without preheating tend to detach from the surrounding paraffin, which results in incomplete or folded tissue sections. The proposed solution is preheating all tissue cylinders on a hot plate to facilitate fusion between the paraffin within the core and the paraffin surrounding the core. In this study, 6 tissue microarray blocks were constructed from 528 tissue cores extracted from various formalin-fixed, paraffin-embedded human tissue samples. The tissue cores in the arrays revealed good homogenization with the surrounding paraffin wax, and the tissue sections were obtained intact. Both hematoxylin-eosin and immunohistochemical staining confirmed satisfactory results. This simple and economical method is easily performed in the laboratory without expensive instrumentation.

Concepts: Histology, Immunohistochemistry, Paraffin, Tissue, Staining, Microarray, Array, Tissue microarray

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Over the last two decades, an innovative technology called Tissue Microarray (TMA), which combines multi-tissue and DNA microarray concepts, has been widely used in the field of histology. It consists of a collection of several (up to 1000 or more) tissue samples that are assembled onto a single support - typically a glass slide - according to a design grid (array) layout, in order to allow multiplex analysis by treating numerous samples under identical and standardized conditions. However, during the TMA manufacturing process, the sample positions can be highly distorted from the design grid due to the imprecision when assembling tissue samples and the deformation of the embedding waxes. Consequently, these distortions may lead to severe errors of (histological) assay results when the sample identities are mismatched between the design and its manufactured output. The development of a robust method for de-arraying TMA, which localizes and matches TMA samples with their design grid, is therefore crucial to overcome the bottleneck of this prominent technology.

Concepts: Histology, Control theory, Tissue, DNA microarray, Microarrays, Multiplex, Tissue microarray, Microscope slide

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The aim of this study was to define the concordance between tissue microarrays (TMAs) of different sizes and whole slide for 15 different antibodies in endometrial cancer and study the use of TMAs in preoperative endometrial samples. Cores of preoperative and hysterectomy specimens of 14 endometrial cancer and three atypical hyperplasia cases were collected in TMA blocks. Two 0.6-mm and two 2.0-mm cores were used from each sample. Different antibodies were tested in TMAs and compared with results of whole slides of hysterectomy. Tested antibodies were as follows: ER, PR, p53, Ki-67, MLH1, PMS2, MSH2, MSH6, ARID1A, stathmin, IMP3, L1CAM, PTEN, β-catenin, and p16. Seventeen cases with four cores per paraffin block (both 0.6 and 2.0 mm in duplicate) and 15 different antibodies resulted in a total of 1020 cores for both preoperative and hysterectomy specimen. Overall, 2.0-mm cores were more assessable for evaluation than 0.6-mm cores (96.0 versus 79.5%, p < 0.01). For most antibodies, a substantial to good agreement between hysterectomy TMA and whole slide was present, with lowest agreement for p16 and stathmin and perfect agreement for mismatch repair proteins. Preoperative TMAs showed for most antibodies moderate to perfect agreement with hysterectomy TMAs. In conclusion, 2.0-mm cores are the preferred size for immunohistochemical studies in endometrial cancer. For all tested antibodies, TMAs are a good alternative for whole slide analysis in scientific studies with large patient cohorts, even in preoperative endometrial samples. However, caution is required for interpretation of TMA results of p16 and stathmin.

Concepts: Sample, Menopause, Histology, Hereditary nonpolyposis colorectal cancer, P53, Endometrial cancer, Microarrays, Tissue microarray

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Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection and quantification of the expression of up to six markers, plus a nuclear counterstain, on a single tissue section. Recent studies have shown the potential for mIF to advance our understanding of complex disease processes, including cancer. It is important that the technique be standardised and validated to facilitate its transition into clinical use. Traditional approaches to mIF rely on manual processing of sections, which is time-consuming and a source of significant variation between samples/individuals. Here we determined if an automated diagnostic tissue stainer could be used for mIF incorporating tyramide signal amplification (TSA), and how the final image quality compared with sections stained semi-automatically or manually. Using tissue microarrays of fixed human breast tumour sections, we observed comparable antibody labelling between the diagnostic autostainer and manual technique. The diagnostic autostainer produced higher signal intensity with similar spectral unmixing efficiency. We also found that microwave treatment for antibody stripping during TSA labelling could be replaced by the heating option incorporated within the diagnostic-use autostainer. These data show that diagnostic autostainers used for traditional immunohistochemistry protocols can be readily adapted to achieve rapid preparation of high-quality sections using a TSA method for clinical mIF.

Concepts: Antibody, Cancer, Blood, Anatomical pathology, Histology, Immunohistochemistry, Staining, Tissue microarray

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We sought to test recent guidelines for preserving immunoreactivity of precut slides, to quantify loss of immunoreactivity, and to determine potential for preservation by altering storage conditions.

Concepts: Food preservation, Tissue microarray

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To investigate the relationship between YY1AP1 and various clinicopathological features of colon adenocarcinoma (COAD), we conducted immunohistochemical (IHC) analyses of human tissue microarrays. We found that YY1AP1 protein expression was significantly higher in tumor tissue of the colon and liver, and was significantly lower in tumor tissue of the kidney. An analysis that employed the SurvExpress database indicated that increased expression of YY1AP1 mRNA was significantly associated with the overall survival of COAD patients. To clarify the validity of YY1AP1 or PCNA as determined by the IHC analysis was performed on 59 paired samples from COAD and adjacent normal tissue. Statistically significant differences of immunoreactivity for YY1AP1 or PCNA protein expression was observed between COAD tissue and adjacent normal tissue. High protein expression levels of YY1AP1 and PCNA were also found to be significantly correlated with M-class and distant metastasis. We also determined that YY1AP1 was correlated with PCNA expression in COAD samples, and Kaplan-Meier survival curves indicated that YY1AP1 protein expression was significantly associated with poor survival. Finally, a univariate analysis demonstrated that YY1AP1 protein expression was related to YY1AP1 score, and multivariate analysis revealed that the YY1AP1 protein expression level was an independent risk factor of overall COAD survival. Taken together, our findings indicate that YY1AP1 expression plays an important role in the tumorigenesis and progression of COAD and could serve as a clinical prognostic indicator for COAD.

Concepts: Cancer, Statistics, Blood, Statistical significance, Messenger RNA, Multivariate statistics, Adenocarcinoma, Tissue microarray

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The complement pathway has potential contributions to both white (WM) and grey matter (GM) pathology in Multiple Sclerosis (MS). A quantitative assessment of complement involvement is lacking.

Concepts: Scientific method, Evaluation methods, Multiple sclerosis, Tissue, Complement system, Quantitative research, Classical complement pathway, Tissue microarray

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For identification of clinically relevant masses to predict status, grade, relapse and prognosis of colorectal cancer we applied MALDI imaging mass spectrometry (IMS) to a tissue micro array (TMA) containing formalin-fixed and paraffin-embedded tissue samples from 349 patients. Analysis of our MALDI-IMS data revealed 27 different m/z signals associated with epithelial structures. Comparison of these signals showed significant association with status, grade and Ki-67 labeling index. 15 out of 27 IMS signals revealed a significant association with survival. For 7 signals (m/z 654, 776, 788, 904, 944, 975, and 1013) the absence, and for 8 signals (m/z 643, 678, 836, 886, 898, 1095, 1459, and 1477) the presence was associated with decreased life expectancy, including 5 masses (m/z 788, 836, 904, 944, and 1013) that provided prognostic information independently from the established prognosticators pT and pN. Combination of these 5 masses resulted in a 3-step classifier that provided prognostic information superior to univariate analysis. In addition, a total of 19 masses were associated with tumor stage, grade, metastasis, and cell proliferation. Our data demonstrate the suitability of combining IMS and large-scale TMAs to simultaneously identify and validate clinically useful molecular marker.

Concepts: Cancer, Mass spectrometry, Lung cancer, Microarray, Array, MALDI imaging, Mass spectrometry imaging, Tissue microarray

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The construction of tissue microarrays (TMAs) with cores from a large number of paraffin-embedded tissues (donors) into a single paraffin block (recipient) is an effective method of analyzing samples from many patient specimens simultaneously. For the TMA to be successful, the cores within it must capture the correct histologic areas from the donor blocks (technical accuracy) and maintain concordance with the tissue of origin (analytical accuracy). This can be particularly challenging for tissues with small histological features such as small islands of carcinoma in situ (CIS), thin layers of normal urothelial lining of the bladder, or cancers that exhibit intratumor heterogeneity. In an effort to create a comprehensive TMA of a bladder cancer patient cohort that accurately represents the tumor heterogeneity and captures the small features of normal and CIS, we determined how core size (0.6 vs 1.0 mm) impacted the technical and analytical accuracy of the TMA. The larger 1.0 mm core exhibited better technical accuracy for all tissue types at 80.9% (normal), 94.2% (tumor), and 71.4% (CIS) compared with 58.6%, 85.9%, and 63.8% for 0.6 mm cores. Although the 1.0 mm core provided better tissue capture, increasing the number of replicates from two to three allowed with the 0.6 mm core compensated for this reduced technical accuracy. However, quantitative image analysis of proliferation using both Ki67+ immunofluorescence counts and manual mitotic counts demonstrated that the 1.0 mm core size also exhibited significantly greater analytical accuracy (P=0.004 and 0.035, respectively, r(2)=0.979 and 0.669, respectively). Ultimately, our findings demonstrate that capturing two or more 1.0 mm cores for TMA construction provides superior technical and analytical accuracy over the smaller 0.6 mm cores, especially for tissues harboring small histological features or substantial heterogeneity.Laboratory Investigation advance online publication, 23 January 2017; doi:10.1038/labinvest.2016.151.

Concepts: Cancer, Carcinoma in situ, Anatomical pathology, Histology, Urinary bladder, Tissue, Microarrays, Tissue microarray

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Tissue microarrays were first constructed in the 1980s but were used by only a limited number of researchers for a considerable period of time. In the last 10 years there has been a dramatic increase in the number of publications describing the successful use of tissue microarrays in studies aimed at discovering and validating biomarkers. This, along with the increased availability of both manual and automated microarray builders on the market, has encouraged even greater use of this novel and powerful tool. This chapter describes the basic techniques required to build a tissue microarray using a manual method in order that the theory behind the practical steps can be fully explained. Guidance is given to ensure potential disadvantages of the technique are fully considered.

Concepts: Time, Scientific method, Tissue, Theory, Microarray, Microarrays, The Technique, Tissue microarray