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.

Protocol Set-up

Analysis starts by setting up protocols (or recalling existing ones from an extensive library.)

Hardware parameters are easily adjusted:

• Laser, PMT and photodiode settings

• Scan resolution

• Scan magnification

End-points of a particular assay are defined by the operator. In this application the following primary end-points will be collected:

• Cellular and randomly sampled expression measurements of the target protein as integral intensity values
• The percentage of cellular events that are positive for the target protein on a per core basis
• The percent tissue area that is positive for the target protein on a per core basis

Using the 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:

• Locate and catalog the TMA core elements

• Review tissue 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

High resolution scan areas can be placed automatically (or defined by the operator) to obtain quantitative and image data.

Data Extraction

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.

Relationships 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.

Results Reporting

Numerical data 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 

Run 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 analysis.

• Combination of highly quantitative and image data.

• Rapid and unbiased automated analysis with the supporting image data archived in an accessible format.

Relevant Reading

1. Automated Tissue Microarray Analysis – A New Paradigm

2. A Novel Approach to Automated Quantitative Analysis of HER2 Expression

3. 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.

More Information

For more information, review the CompuCyte Published Materials and CompuCyte Bibliography.