An introduction of an easy-operating and economical technique for tissue microarray preparation.

AIM: Tissue microarray (TMA) is a powerful and effective tool for in situ tissue analysis. However, manual TMA construction methods showed varied qualities. This study aimed to raise a standardised TMA preparation technique that can be easily operated and is economical. METHODS: A sampling needle was used to punch the tissue rods from the donor block and holes in the recipient block. To indicate the dots' positions and ensure vertical punching, a novel auxiliary device made using commercial three-dimensional printing technology was attached. The TMA block was made up of tissue rods and a recipient block. RESULTS: A 77-rod (7×11) TMA block was constructed. The rows and columns were fixed in straight lines. There was no specimen loss during the process of embedding. CONCLUSIONS: An alternative method for the construction of TMA blocks that met the basic requirement of many laboratories and can be effortlessly performed was presented.

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Validation of a low-cost modified technique for constructing tissue microarrays for canine mammary tumor analysis.

Compared with conventional histological paraffin blocks, tissue microarray (TMA) represents a "high-throughput tool" that provides rapid results, a time- and cost-effective approach and simultaneous investigation of several tissue samples under the same conditions. Given the large number of cases of dogs affected with mammary tumors, the complexity of these tumors and their similarity with breast cancer in women, this study aimed to validate a low-cost modified method to construct TMAs for canine mammary tumor analysis using immunomarkers. Carcinoma cases were selected from canine mammary carcinomas in mixed tumors (CMT) because this tumor type is the most heterogeneous among the histopathological types of mammary tumors observed in female dogs. Through a histopathological examination, tumor representativity was compared between conventional sections and histological sections obtained from the TMA block; both were stained with hematoxylin and eosin. An immunohistochemistry analysis was performed to compare the percentages of immunoreactive cells obtained in whole tissue sections versus those obtained from sections from the TMA block. Streptavidin-biotin peroxidase complex and anti-PCNA, anti-vimentin and anti-pancytokeratin antibodies were used. Statistical analysis consisted of the nonparametric Friedman's test (p≤0.05) and descriptive statistical analysis. Histopathological analysis showed tumor representativity in all TMA cores selected for the study. There was no difference between the immunohistochemical analysis of mammary tumors using conventional histological sections or sections obtained from a single 1-mm-diameter TMA core, regardless of the marker used: PCNA (p=0.279), pancytokeratin (p=0.243) and vimentin (p=0.967). The results did not change even when the means of any number of cores were compared among each other and with the conventional histological section: PCNA (p=0.413), pancytokeratin (p=0.177) and vimentin (p=1.0). Therefore, this study validates the use of a low-cost and modified TMA construction technique proposed by Pires et al. for canine mammary tumor analysis.

Tissue Microarrays.

Modern next-generation sequencing and microarray technologies allow for the simultaneous analysis of all human genes on the DNA, RNA, miRNA, and methylation RNA level. Studies using such techniques have lead to the identification of hundreds of genes with a potential role in cancer or other diseases. The validation of all of these candidate genes requires in situ analysis of high numbers of clinical tissues samples. The tissue microarray technology greatly facilitates such analysis. In this method minute tissue samples (typically 0.6 mm in diameter) from up to 1000 different tissues can be analyzed on one microscope glass slide. All in situ methods suitable for histological studies can be applied to TMAs without major changes of protocols, including immunohistochemistry, fluorescence in situ hybridization, or RNA in situ hybridization. Because all tissues are analyzed simultaneously with the same batch of reagents, TMA studies provide an unprecedented degree of standardization, speed, and cost efficiency.

Configurable low-cost plotter device for fabrication of multi-color sub-cellular scale microarrays.

The construction and operation of a low-cost plotter for fabrication of microarrays for multiplexed single-cell analyses is reported. The printing head consists of polymeric pyramidal pens mounted on a rotation stage installed on an aluminium frame. This construction enables printing of microarrays onto glass substrates mounted on a tilt stage, controlled by a Lab-View operated user interface. The plotter can be assembled by typical academic workshops from components of less than 15,000 Euro. The functionality of the instrument is demonstrated by printing DNA microarrays on the area of 0.5 cm2 using up to three different oligonucleotides. Typical feature sizes are 5 µm diameter with a pitch of 15 µm, leading to densities of up to 10(4)-10(5) spots/mm2. The fabricated DNA microarrays are used to produce sub-cellular scale arrays of bioactive epidermal growth factor peptides by means of DNA-directed immobilization. The suitability of these biochips for cell biological studies is demonstrated by specific recruitment, concentration, and activation of EGF receptors within the plasma membrane of adherent living cells. This work illustrates that the presented plotter gives access to bio-functionalized arrays usable for fundamental research in cell biology, such as the manipulation of signal pathways in living cells at subcellular resolution.

No-cost manual method for preparation of tissue microarrays having high quality comparable to semiautomated methods.

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.

A novel tissue microarray instrumentation: the HT-1 tissue microarrayer.

BACKGROUND: Tissue microarray (TMA) is a novel and useful tool to efficiently analyze gene expression in histological tissues. AIM: Cost-efficient and easy to use automated tissue arrayers will provide a better instrumentation to generate TMAs. Thus, we designed and produced our tissue microarrayer to meet these needs. MATERIALS AND METHODS: The HT-1 tissue microarrayer we designed and manufactured consists primarily of four parts, including an instrument to make array pores for the recipient paraffin blocks, a punch needle, an instrument for negative-pressure embedding, and a special manipulator. By using the HT-1, 14 different TMAs were made to accommodate 312 cases of tissues and TMA sections were tested by hematoxylin-eosin (H&E) staining, in situ hybridization, and immunohistochemistry. RESULTS: Expand: Hematoxylin and eosin staining showed that the tissue cylinders were similar, even, and in order on the slides. Most importantly, the HT-1 microarrayer can make array pores in the recipient paraffin block with a single application in seconds. The HT-1 also contains a unique negative pressure system for embedding TMA blocks. In addition, HT-1 can make tissue cylinders with the same levels and depth for equally embedded and sectioning. CONCLUSIONS: The HT-1 tissue microarrayer is a device that is simple, economical and easy to use.

Quality control by tissue microarray in immunohistochemistry.

AIMS: An external positive control section is included in each immunohistochemical analysis as a well recognised and validated technique for standardising results. The method is time-consuming and expensive. On the contrary, internal controls are warranted and inexpensive, but their use is only feasible in selected diagnoses. The aim of this work is to show how the method of the authors allows improving the interpretation and cuts costs in the immunohistochemical analysis of bone marrow specimens. METHODS: A paraffin-embedded tonsil tissue cylinder was sampled from a donor block using an automated sampler and included as an 'internal control' together with a bone marrow biopsy in a recipient block, avoiding the use of external tonsil tissue control. To validate this technique, the authors compared the quality of immunohistochemistry, the workload and costs with routine external control in 50 consecutive bone marrow biopsies. RESULTS: Processing simultaneously the sample and the tissue control in the same block, 60 external positive control tests were spared. Only a few minutes were taken for the preparation of the recipient blocks, and no particular technical skill was required. Considering that the volume of antibodies used for the analysis of each sample was not increased, a considerable amount of the disposable material was saved. The workload of technicians was decreased and some potential technical bias was avoided. The time required for pathologists to interpret the slides was also reduced. CONCLUSIONS: In conclusion, this seems to be a feasible, cost-cutting and quality-improving technique, not limited to haematopathology but potentially extensible to other fields of pathology.

Trapping cells on a stretchable microwell array for single-cell analysis.

There is a need for a technology that can be incorporated into routine laboratory procedures to obtain a continuous, quantitative, fluorescence-based measurement of the dynamic behaviors of numerous individual living cells in parallel, while allowing other manipulations, such as staining, rinsing, and even retrieval of targeted cells. Here, we report a simple, low-cost microarray platform that can trap cells for dynamic single-cell analysis of mammalian cells. The elasticity of polydimethylsiloxane (PDMS) was utilized to trap tens of thousands of cells on an array. The PDMS microwell array was stretched by a tube through which cells were loaded on the array. Cells were trapped on the array by removal of the tube and relaxation of the PDMS. Once that was accomplished, the cells remained trapped on the array without continuous application of an external force and permitted subsequent manipulations, such as staining, rinsing, imaging, and even isolation of targeted cells. We demonstrate the utility of this platform by multicolor analysis of trapped cells and monitoring in individual cells real-time calcium flux after exposure to the calcium ionophore ionomycin. Additionally, a proof of concept for target cell isolation was demonstrated by using a microneedle to locally deform the PDMS membrane in order to retrieve a particular cell from the array.

An inexpensive method of small paraffin tissue microarrays using mechanical pencil tips.

BACKGROUND: Tissue microarray technology has provided a high throughput means of evaluating potential biomarkers in archival pathological specimens. This study was carried out in order to produce tissue microarray blocks using mechanical pencil tips without high cost. METHOD: Conventional mechanical pencil tips (Rotring Tikky II Mechanical Pencil 1.0 mm) were used to cut out 1 mm wax cylinders from the recipient block, creating from 36 to 72 holes. Three cores of tumor areas were punched out manually by using the mechanical pencil tips from donor paraffin embedded tissue blocks and transferred to the holes of the paraffin tissue microarrays. RESULTS: This technique was easy and caused little damage to the donor blocks. We successfully performed H&E slides and immunodetection without substantial tissue cylinder loss. CONCLUSION: Our mechanical pencil tip technique is the most inexpensive easy technique among the literature. It also takes a reasonable amount of time and reduces antibody consumption during immunohistochemistry.

Practical considerations of image analysis and quantification of signal transduction IHC staining.

The dramatic increase in computer processing power in combination with the availability of high-quality digital cameras during the last 10 years has fertilized the grounds for quantitative microscopy based on digital image analysis. With the present introduction of robust scanners for whole slide imaging in both research and routine, the benefits of automation and objectivity in the analysis of tissue sections will be even more obvious. For in situ studies of signal transduction, the combination of tissue microarrays, immunohistochemistry, digital imaging, and quantitative image analysis will be central operations. However, immunohistochemistry is a multistep procedure including a lot of technical pitfalls leading to intra- and interlaboratory variability of its outcome. The resulting variations in staining intensity and disruption of original morphology are an extra challenge for the image analysis software, which therefore preferably should be dedicated to the detection and quantification of histomorphometrical end points.

Tissue microarray for routine clinical breast biomarker analysis. The British Columbia Cancer Agency 2008 experience.

Clinical use of tissue microarrays for immunohistochemical analysis of breast biomarkers, namely estrogen receptor, progesterone receptor, and HER2, was instituted in our laboratory in 2008. The method has proved reliable and cost-effective. We report the results of the initial year of testing with this method.

Use of tissue microarray to facilitate oncology research.

HPV-positive oropharyngeal squamous cell carcinomas (OSCC) represent a distinct disease entity from traditional OSCC. We hypothesized that for HPV DNA-positive cases, p16 expression status differentiates the biologically relevant ones.We determined HPV16DNA viral load in a cohort of 79 oropharyngeal squamous cell cancers by real-time polymerase chain reaction (PCR). We used cervical cancer as a disease model for HPV-initiated epithelial cancer. In cervical cancer, p53 and Rb expression is reduced, while p16 expression is increased. We used TMA technology to facilitate interrogation of this cohort for p53, Rb, and p16 protein expression using a quantitative, in situ method of protein analysis (AQUA analysis). Our results indeed delineate three biologically and clinically distinct types of oropharyngeal squamous cell cancers based on HPV-DNA determination and p16 expression status: one class of HPV-negative/p16-nonexpressing (HPV-negative), one class of HPV-positive/p16-nonexpressing (HPV-inactive), and one class of HPV positive/p16-expressing (HPV-active) oropharyngeal tumors. We demonstrated that only the HPV-active tumors share a similar molecular phenotype to cervical cancers, and are the ones associated with favorable prognosis.

Making and using inexpensive manually constructed tissue micro-array: experience of a tertiary care hospital in India.

BACKGROUND: Tissue micro-array enables the analysis of a large number of tissues simultaneously. Widespread use of this technology is hampered by the high cost of commercial array instruments. We describe our experience of constructing tissue micro-array in a simple method using easily available and inexpensive instruments. MATERIALS AND METHODS: We used an 11-19 gauge (G) bone marrow trephine biopsy needle/ small sized slotted screwdriver to punch holes in the wax blocks. Cores were taken from donor tissue blocks using a bone marrow trephine biopsy needle and arrayed into host paraffin wax blocks. A detailed database was constructed for each array constructed. RESULTS: The array blocks were used over a period of one year as internal control for immunohistochemistry (IHC), quality control and research. It took about 10 minutes to construct a nine-dot array and about one hour for a 56-dot array. During IHC, the average loss of control dots was less than one per cent. We did not see any loss of antigenicity in the control sections even after four weeks storage. DISCUSSION: Tissue array construction by the technique described here is inexpensive and reliable alternative to automated instruments. Because it is easy to modify the arrays by varying the core size, it is easy to adapt this to individual labs and requirements. We recommend using blocks with cores in 3 x 3 to 5 x 4 grids as controls in IHC and for standardizing antibodies and array blocks with a larger number of cores for research.

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.

AIMS: Fluorescence in situ hybridization (FISH) testing is the 'gold standard' method for Her-2 status assessment in breast cancer patients, yet is only employed in about 30% of tests carried out because of cost and labour considerations. We have previously described tissue microarray (TMA)-based testing to eliminate cost constraints, and now describe a rapid screening approach to reduce time spent testing. METHODS AND RESULTS: We examined 88 cases of invasive breast cancer on TMAs comparing formal FISH scoring with a rapid screening technique. Each core was screened by two observers and results recorded as positive, equivocal or negative. Each approach was timed. Data were analysed by comparing the rapid screening results with formal counts. Using rapid screening, two-thirds of negative and half the positive cases could be eliminated with 100% accuracy. It took 2 min per observer per case to rapid screen six TMA cores at x100 magnification. The remaining cases required formal counting, which took no longer than with whole-section techniques. CONCLUSION: Rapid screening of TMAs for routine Her-2 FISH testing is safe, economical and time efficient. The technique ensures that all patients receive 'gold standard' testing.

Tissue microarray technology in the routine assessment of HER-2 status in invasive breast cancer: a prospective study of the use of immunohistochemistry and fluorescence in situ hybridization.

AIMS: To compare tissue microarray (TMA) and whole-section (WS) techniques in the routine assessment of HER-2 status in invasive breast cancer by immunohistochemistry and fluorescence in situ hybridization (FISH). METHODS AND RESULTS: HER-2 status was assessed prospectively in 106 consecutive cases of excised high-grade and/or node-positive breast carcinoma using both WS- and TMA-based techniques. Whole sections were assessed by immunohistochemistry with FISH being performed on equivocal cases (scoring 2+ on HercepTest) and randomly selected 3+ cases included for quality assurance. Five 0.6-mm cores from each tumour allowed accurate immunohistochemical and FISH testing in >95% of cases. Ninety-seven per cent concordance was achieved between WS and TMA approaches to FISH analysis, the only discrepancies being in cases that were borderline or near borderline by both techniques. TMA and WS approaches were comparable in terms of time for preparation and scoring. CONCLUSIONS: TMA technology is a robust method of assessing HER-2 status in invasive breast cancer. This is directly comparable to the current standard methodology using whole sections. The use of TMA technology offers several advantages over existing full-section methods in terms of cost, quality control, facilitation of future research and the facility to provide a high-throughput testing methodology.

Quantitative PCR and HER2 testing in breast cancer: a technical and cost-effectiveness analysis.

We performed a technical and cost-effectiveness analysis of quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) for the assessment of HER2 in breast cancer. We evaluated 44 frozen and 55 formalin-fixed paraffin-embedded (FFPE) breast carcinoma specimens by Q-RT-PCR, immunohistochemical analysis, and fluorescent in situ hybridization (FISH). Immunohistochemical and FISH analyses were performed on individual slides and on tissue microarray. Costs of techniques were calculated to study 1 case and 10 or 40 cases. Q-RT-PCR provided reliable data in frozen and FFPE specimens, which were significantly correlated. HER2 messenger RNA levels were significantly stratified in agreement with immunohistochemical data (P < .05). There was complete concordance between Q-RT-PCR and immunohistochemical results for negative and strongly positive (3+) cases. The intermediate immunohistochemical group (2+), including FISH+ and FISH- cancers, could also be stratified by Q-RT-PCR. Cost analysis documented the advantage of Q-RT-PCR in all US Food and Drug Administration-approved assays. Our data support the use of Q-RT-PCR for testing breast cancer specimens to select patients for HER2 inhibitory therapy.

ImmunoCell-Array: a novel technology for pathway discovery and cell profiling.

A major challenge in drug discovery is the definition of the activity of therapeutic compounds before they enter clinical trials. Currently, many tools are available to profile drug response at a multiparametric level. Furthermore, the novel discipline of systems biology is offering interpretative cues to the amount of data generated by the so-called omics technologies. Nonetheless, novel approaches are needed to comprehensively evaluate drug response. This review will describe a recent technology for protein profiling, the ImmunoCell-Array, which, in to the authors' opinion, can enter the scenario of profiling technologies with the goal of offering a comprehensive description of proteome dynamics in a cellular context.