Tissue Microarray Analysis (TMA Analysis) on iGeneration LSC
iGeneration LSC instrumentation
provides researchers with unprecedented flexibility and quality
of quantitative automated analysis of fluorescently and
chromatically stained TMAs. The excellent
performance characteristics and
unique features of LSC technology make it the technology of
choice for tissue and TMA analysis.
Typical TMA analysis workflow
The experimental objective of this
example assay is to measure the expression of a target protein
(being evaluated as a potential biomarker) in non-necrotic
epithelium in human multi-tumor TMA. Two serially sectioned TMAs
were stained with antibodies to this protein and developed with
fluorescent and chromatic dyes as described in the table below.
Fluorescently Labeled TMA
Chromatically Labeled TMA
Analysis starts by setting up
protocols (or recalling existing ones from an extensive
parameters are easily adjusted:
End-points of a
particular assay are defined by the operator. In this
application the following primary end-points will be collected:
randomly sampled expression measurements of the target protein
as integral intensity values
percentage of cellular events that are positive for the target
protein on a per core basis
tissue area that is positive for the target protein on a per
technique of variable resolution scanning a fast but low-resolution
overview scan is followed by a higher resolution scan for
quantitative analysis. This rapid scan takes only a few minutes
and is performed to:
catalog the TMA core elements
integrity ( are there missing elements, tissue folding, etc.),
quality of staining and presence of artifacts that may affect
the validity of the results
Set the areas
for high resolution scanning
resolution scan areas can be placed automatically (or defined by
the operator) to obtain quantitative and image data.
As the high resolution images are
acquired, they are segmented to parse the image into events for
which various features are extracted.
The types of events that the
iGeneration system can produce break down into two broad
categories: Segmented events are events based upon the
signal levels of one or more of the dyes. Many times this is a
nuclear segmentation where a contour line is drawn around the
cell nuclei based on a DNA stain such as PI or Hematoxylin in
this example. This type of segmentation is shown below on the
left for this assay example. Note that this method is not
limited to nuclear segmentation. Any signal (or combination of
signals) can be the basis for a segmented event. Random
events are circular contours, of a user-defined size,
overlaid on images as shown below on the right.
Each segmented or random event,
regardless of type, is assigned features based on the morphology
as well as from the signal levels from each of the images.
Random event segmentation is a welcome alternative to event
segmentation in situations where cellular/event boundaries
cannot be established, i.e. tumorous mass, etc.
Event features are displayed in
scattergrams and histograms for population analysis. In
population analysis, the appropriate sub-populations of events
are identified and segregated in order to generate the desired
assay end points as well as ancillary parameters of interest.
Areas of distinct populations in the
scattergrams are identified as shown below.
between the event data and the image data can also be explored
in the iGeneration Cytometric Analysis Software.
For example, the color coding from the gated
regions is “carried back” to the images for visual confirmation
of the gating boundaries.
generated from the relevant scattergram and histogram gating
regions (Run Statistics) may be exported to CompuCyte's iBrowser
data integration software for additional analysis and viewing.
In this assay the intensity of the target protein expression
within each core element is compared to the lowest expressing
core using the
Kolmogorov-Smirnov test in the iBrowser
Statistics may be output to third party programs, such as Excel,
for further analysis.
The KS difference values are shown plotted in Excel, below.
Summary of key benefits of using
iGeneration of LSC technology for TMA analysis
Uniquely with LSC technology, chromatically and
fluorescently stained samples may be analyzed sequentially or
simultaneously utilizing a combination of fluorescent and
chromatic stains on the same slide.
Multiple fluorescent dyes can be used, allowing improved
quantification of immunohistochemistry (ISH) and fluorescence
in situ hybridization (FISH) TMAs.
The same chromatically stained slides which are optimal for
conventional manual interpretation can be utilized in LSC
Combination of highly quantitative and image data.
Rapid and unbiased automated analysis with the supporting
image data archived in an accessible format.
Automated Tissue Microarray Analysis – A New Paradigm
A Novel Approach to Automated Quantitative Analysis of HER2
LSC Analysis of Tissue Microarrays: Application to
Subcellular Localization of the Cell Cycle Inhibitor p27 and
Prostate Cancer Recurrence - Peter Gann, MD, ScD
Department of Pathology , University of Illinois at Chicago.
For more information, review the
CompuCyte Published Materials and