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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.
You can find out more details about this research here.
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.
