TMA Grand Master - recommended reading

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1.    The tissue microarray technique is widely used in biomarker studies. This paper describes a modern, next generation TMA protocol for biomarker studies using our TMA Grand Master tissue microarrayer in combination with 3DHISTECH’s Pannoramic 250 Flash II (digital slide scanner) and Case Center (server application for storing digital slides). The article is illustrated with a high quality video which gives step by step guidance for a successful TMA project.

Zlobec, I., Suter, G., Perren, A., and Lugli, A. (2014).
A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies.
J. Vis. Exp. 91.

Abstract

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a ‘donor’ block into a ‘recipient’ block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 ‘recipient’ blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.
You can find out more details about this research here.


 

2.    Molecular pathology is an emerging discipline within pathology is commonly used in diagnosis of cancer. Our TMA Grand Master tissue microarreyer has the potential to assist the molecular pathology workflow, by extracting tissue cores from the donor block and inserting them into clean tubes. The DNA extracted from these tissue cores could be used later for molecular analysis such as PCR or DNA sequencing. In this article the authors studied the possibility of cross contamination within samples punched with the same device. They used a homemade semi-automated tissue microarreyer and our fully-automated TMA GM tissue microarryer for this task. There was no cross-contamination between samples punched with the same device. The authors concluded that TMA instrumentation is appropriate for use as an accessory to molecular applications.

Vassella, E., Galván, J.A., and Zlobec, I. (2015).
Tissue Microarray Technology for Molecular Applications: Investigation of Cross-Contamination between Tissue Samples Obtained from the Same Punching Device.
Microarrays 4, 188–195.

Abstract

Background: Tissue microarray (TMA) technology allows rapid visualization of molecular markers by immunohistochemistry and in situ hybridization. In addition, TMA instrumentation has the potential to assist in other applications: punches taken from donor blocks can be placed directly into tubes and used for nucleic acid analysis by PCR approaches. However, the question of possible cross-contamination between samples punched with the same device has frequently been raised but never addressed. Methods: Two experiments were performed. (1) A block from mycobacterium tuberculosis (TB) positivetissue and a second from an uninfected patient were aligned side-by-side in an automated tissue microarrayer. Four 0.6 mm punches were cored from each sample and placed inside their corresponding tube. Between coring of each donor block, a mechanical cleaning step was performed by insertion of the puncher into a paraffin block. This sequence of coring and cleaning was repeated three times, alternating between positive and negative blocks. A fragment from the 6110 insertion sequence specific for mycobacterium tuberculosis was analyzed; (2) Four 0.6 mm punches were cored from three KRAS mutated colorectal cancer blocks, alternating with three different wild-type tissues using the same TMA instrument (sequence of coring: G12D, WT, G12V, WT, G13D and WT). Mechanical cleaning of the device between each donor block was made. Mutation analysis by pyrosequencing was carried out. This sequence of coring was repeated manually without any cleaning step between blocks. Results/Discussion: In both analyses, all alternating samples showed the expected result (samples 1, 3 and 5: positive or mutated, samples 2, 4 and 6: negative or wild-type). Similar results were obtained without cleaning step. These findings suggest that no cross-contamination of tissue samples occurs when donor blocks are punched using the same device, however a cleaning step is nonetheless recommended. Our result supports the use of TMA technology as an accessory to PCR applications.
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3.    This methodology article describes a study about the possibility to construct new TMA blocks using tissues from older arrays available and supplementary donor blocks in order to get a TMA with rare disease entities. This task requires high precision, the authors chose our TMA Grand Master to execute this job. Today, the TMA-GM is the most automated and sophisticated device for the construction of TMAs, allowing the precise selection of specific regions of interest (ROI) on the donor blocks. In this paper, the authors described the successful transfer of tissue cores from existing TMAs to new ones, relocating tissue of interest together with others obtained from “normal” FFPE donor blocks.

Lacombe, A., Carafa, V., Schneider, S., Sticker-Jantscheff, M., Tornillo, L., and Eppenberger-Castori, S. (2015).
Re-Punching Tissue Microarrays Is Possible: Why Can This Be Useful and How to Do It.
Microarrays 4, 245–254.

Abstract

Tissue microarray (TMA) methodology allows the concomitant analysis of hundreds of tissue specimens arrayed in the same manner on a recipient block. Subsequently, all samples can be processed under identical conditions, such as antigen retrieval procedure, reagent concentrations, incubation times with antibodies/probes, and escaping the inter-assays variability. Therefore, the use of TMA has revolutionized histopathology translational research projects and has become a tool very often used for putative biomarker investigations. TMAs are particularly relevant for large scale analysis of a defined disease entity. In the course of these exploratory studies, rare subpopulations can be discovered or identified. This can refer to subsets of patients with more particular phenotypic or genotypic disease with low incidence or to patients receiving a particular treatment. Such rare cohorts should be collected for more specific investigations at a later time, when, possibly, more samples of a rare identity will be available as well as more knowledge derived from concomitant, e.g., genetic, investigations will have been acquired. In this article we analyze for the first time the limits and opportunities to construct new TMA blocks using tissues from older available arrays and supplementary donor blocks. In summary, we describe the reasons and technical details for the construction of rare disease entities arrays.
You can find out more details about this research here.
 

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