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Laboratories worldwide find it challenging to identify enough tissues and cases for verification and validation studies of low-incidence, rare antigens. These antigens have a low frequency of occurrence in the population, or have little or no expression in normal tissues. Validation studies are essential to assure testing standardization before introducing a new instrument, product, or test into the clinical laboratory. The College of American Pathologists has published comprehensive guidelines for the verification and validation of new immunohistochemical tests introduced into the laboratory menu. Within the guidelines, varied numbers of cases are required for nonpredictive versus predictive markers. However, regarding low-incidence antigens, the laboratory medical director determines the extent of validation required. Recommended practical solutions available to clinical laboratories for low-incidence validation include developing internal resources using the laboratory information system with retrospective and prospective search(s) of archival material and purchase of tissue microarray blocks, slides, or cell lines from external resources. Utilization of homemade multitissue blocks has proved to be extremely valuable in biomarker research and demonstrated great utility in clinical immunohistochemistry laboratories. Participation in External Quality Assessment program(s) may provide insufficient numbers or the ability to calculate concordance rates. However, supplementation with in-house tissues can allow a laboratory to reach the optimal number of cases needed for verification and/or validation schemes. An alternative approach is conducting a thorough literature search and correlating staining patterns of the new test to the expected results. These solutions may be used uniquely or together to assure consistent standardized testing., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.)

Tissue microarrays (TMAs) are important tools to conserve precious tissue resources from increasingly smaller biopsies and to control experimental costs and variation across sample sets. The quality assurance assessment of TMA materials created at centralized biobanks has not been standardized. Herein, we outline 2 processes for the construction of TMAs ("recipient block" and "tape" methods) and the associated preconstruction quality control measures (pathology review, protein and RNA assessment, map creation, and storage conditions) developed by the AIDS Cancer Specimen Resource (ACSR) Network's Science and Technology Core. These steps provide a suggested framework for quality assessment that allows end-users, receiving materials from tissue banks, confidence in their experimental results.

The use of controls is a hallmark for quality control in anatomic pathology. However, standardization of controls between laboratories has been a significant issue. Differential processing techniques between institutions and a multitude of preanalytical difficulties can result in different immunostain intensities. So called histoid controls, xenografts or culture cell lines, have been discussed in the past but with no recent followup. Herein is presented a histoid termed a 3D tissue microarray control (3D TMAC) control to help alleviate the burgeoning need for control standardization. A breast and cervix 3D TMAC control were tested for staining quality for 11 different antibodies commonly tested in either breast or cervical cancer work ups. We additionally looked at a small run of 5 days of CK5 and HER2 for reproducibility of the 3DRSTMA. Staining quality of 9 of the antibodies stained appropriately and 2 stained inappropriately, mammoglobin and GCDFP. Two of the antibodies were not reported to have any staining properties in the 3D TMAC, p16 and mammoglobin. Of these, p16 had appropriate staining and mammoglobin did not. In the 5 runs of CK5 and HER2, there was good reproducibility between stains assessed by both visual and computer-assisted methods, with membrane intensity coefficients of variation of 3.58% and 3.18%, respectively. The 3D TMAC has the potential to markedly improve intralaboratory and interlaboratory standardization practices.

Manual assessment of human epidermal growth factor receptor 2 (HER2) protein expression by immunohistochemistry (IHC) in gastric and gastroesophageal junction (GGEJ) adenocarcinomas is prone to interobserver variability and hampered by tumor heterogeneity and different scoring criteria. Equivocal cases are frequent, requiring additional in situ hybridization analysis. This study aimed to evaluate the accuracy of digital image analysis for the assessment of HER2 protein expression. In total, 110 GGEJ adenocarcinomas were included in tissue microarrays with 3 tissue cores per case. Two immunoassays, PATHWAY and HercepTest, and fluorescent in situ hybridization analysis were performed. The Visiopharm HER2-CONNECT Analysis Protocol Package was applied through the ONCOtopix digital image analysis software platform. HER2 membrane connectivity, calculated by the Analysis Protocol Package, was converted to standard IHC scores applying predetermined cutoff values for breast carcinoma as well as novel cutoff values. Cases with excessive cytoplasmic staining as well as HER2 amplified IHC negative cases were excluded from analysis. Applying HER2-CONNECT with connectivity cutoff values established for breast carcinoma resulted in 72.7% sensitivity and 100% specificity for the identification of HER2 positive gene amplified cases. By application of new cutoff values, the sensitivity increased to 100% without decreased specificity. With the new cutoff values, a 36% to 50% reduction of IHC equivocal cases was obtained. In conclusion, HER2-CONNECT with adjusted cutoff values seem to be an effective tool for standardized assessment of HER2 protein expression in GGEJ adenocarcinomas, decreasing the need for in situ hybridization analyzes.

Manual tissue microarray (TMA) construction had been introduced to avoid the high cost of automated and semiautomated techniques. The cheapest and simplest technique for constructing manual TMA was that of using mechanical pencil tips. This study was carried out to modify this method, aiming to raise its quality to reach that of expensive ones. Some modifications were introduced to Shebl's technique. Two conventional mechanical pencil tips of different diameters were used to construct the recipient blocks. A source of mild heat was used, and blocks were incubated at 38°C overnight. With our modifications, 3 high-density TMA blocks were constructed. We successfully performed immunostaining without substantial tissue loss. Our modifications increased the number of cores per block and improved the stability of the cores within the paraffin block. This new, modified technique is a good alternative for expensive machines in many laboratories.

Background: Cancer biomarker studies using the combination of tissue microarray and automated quantitative assessment of immunofluorescence (TMA-AQUA) have been successfully performed for various types of human carcinoma, but its performance characteristics have yet to be evaluated in human lymphoma., Methods: A pilot TMA was constructed containing duplicate 1.5 mm cores from 15 cases of mantle cell lymphoma (MCL), 3 cases of low-grade B-cell lymphoma, and 3 cases of benign lymphoid tissue. Protein expression of c-Myc, Cdc2, Cyclin D1, Ki-67, Mcm2, and p27 by immunofluorescence and chromagenic staining were evaluated by AQUA and visual scoring, respectively. Gene expression of cMYC, CDC2, and CCND1 was determined by quantitative nuclease protection assay., Results: Protein expression between duplicate cores determined by AQUA showed excellent correlation for all markers [correlation coefficient (R)=0.79 to 0.94] and Cyclin D1 expression was significantly higher in MCL cases compared with non-MCL cases (P=0.00019). Overall correlation of AQUA with scoring of chromagenic staining by 2 pathologists was good for all markers (R=0.56 to 0.90), except Cdc2 (R=0.25). Localization of expression to cytoplasmic and/or nuclear compartments was comparable with chromagenic staining patterns for all markers except Ki-67 and Mcm2, where a significant difference between nuclear and cytoplasmic expression could not be appreciated by AQUA, despite clear nuclear localization by chromagenic staining. Correlation of gene expression with protein expression was variable for CDC2, cMYC, and CCND1 (R=0.32, 0.35, and 0.69)., Conclusions: TMA-AQUA has the potential to be successfully used as a high-throughput protein biomarker screening platform for MCL; however, appropriate target protein selection and antibody performance validation are factors that need to be considered.

Background: Differentiating oncocytoma from its renal cell carcinoma (RCC) mimics, particularly chromophobe RCC, can be difficult, especially when limited tissue is available for evaluation. This study presents a panel of markers that are readily available, easy to use, and useful for differential diagnoses of renal tumors., Design: A renal cell neoplasm tissue microarray was constructed including oncocytoma (n=30), chromophobe RCC (n=18), conventional RCC (n=64), papillary RCC (n=50), and benign renal tissues (n=31). CK7, CD10, epithelial membrane antigen, renal cell carcinoma marker (RCCma), vimentin, and endogenous avidin-binding activity (EABA) were studied. An Automated Cellular Imaging System, was used to quantify the staining intensity., Result: EABA was positive in 97% of oncocytoma, 26% of conventional RCC and 35% of papillary RCC with granular/eosinophilic (G/E) features and 6% of chromophobe RCC. EABA was negative in RCC without G/E features. Vimentin and RCCma were positive in most RCC with G/E features (conventional, 78% and 71%; and papillary, 85% and 76%, respectively), and negative in oncocytoma. Vimentin was also negative in chromophobe RCC. CK7 was positive in up to 81% of papillary RCC and 63% of chromophobe RCC, and essentially negative in conventional RCC and oncocytoma., Conclusions: EABA is an excellent marker for oncocytoma, which can be useful in differentiating oncocytoma from chromophobe RCC. A panel of EABA, vimentin, and RCCma markers can be useful in discerning oncocytoma from RCC with G/E features. Vimentin can be useful in discriminating chromophobe RCC from papillary or conventional RCCs.

Generation of monospecific antibodies (msAbs) (multiepitope) through affinity purification of polyclonal antisera is a plausible strategy for high-throughput production of affinity reagents toward large sets of proteins. These antibodies are generated using readily accessible gene sequence information from publicly available databases. The resulting antibodies have the potential to be used in a variety of assays, probing differentially presented and altered proteins with high sensitivity and specificity. In the present study, 48 msAbs were compared with corresponding commercial analogs. Immunohistochemical staining properties were evaluated on tissue microarrays, representing various normal human tissues from 144 different individuals. MsAbs showed similar immunostaining patterns as compared with corresponding commercial analogs in 44 out of totally 48 (92%) antibody pairs analyzed. Although only few antibody pairs showed major discrepancies, minor dissimilarities were frequently seen. Our results suggest that msAbs are reliable and valuable tools in antibody-based proteomics, enabling analysis of protein expression patterns in cells and tissues. High-throughput strategies employing such antibodies provide a consistent approach in the exploration of the human proteome.

The tissue array is an economical and efficient tool for translational research. An important factor in constructing tissue arrays is the ability to reliably transfer the pathologic feature of interest from the donor block to the array block. The precision of this transfer is undermined by the distortion of dotted routine sections, which serve as the primary feature-finding device for manual array construction, and by certain tissues such as breast, colon, and skin that prove resistant to being punched. To evaluate and improve this transfer process, with a focus on breast tissue, we investigated a series of waterbath temperatures best suited to minimize microtomy-induced section distortion of breast tissue, and a novel donor block pretreatment before array construction. Our results show that routine sections deviate least from their blocks when transferred to slides in 46 degrees C waterbaths, but also that attention be paid to the crushing effect of the microtome. Core transfer rates are highest when donor blocks are stored at 4 degrees C before punching and when 0.6-mm diameter cores are taken from donor blocks.

The authors constructed tissue microarray (TMA) blocks from human postmortem brain including numerous core samples measuring 2 mm in diameter from various anatomic regions. These TMA sections were then processed using various stainings and pretreatment techniques to evaluate their properties. The loss of core samples ranged from 2% to 100% and was significantly influenced by the type of glass slide used; it was lowest (2-8%) with SuperFrost Plus slides. The losses were not significantly altered when applying the most demanding pretreatment procedures or using human brain tissue with a long postmortem delay. A slight influence on the quality and the repeatability of some of the IHC stainings was seen by the postmortem delay, by the brain region, or by the glass slide used. One special feature of the constructed brain TMA block including many anatomic brain regions is that persons who lack skills in neuroanatomy can identify various brain structures simply by following the x-y coordinates. Thus, the applications of this brain TMA block in neurologic research by scientists with different skill bases remain to be determined.