Unifying image analysis and flow cytometry software to analyse and present multi-parameter image data for quantitative pathology

Nguyen, T., Winterford, C., Stanley, A., Chang, C., Masel, A., Chojnowski, G. and Waterhouse, N.

QIMR Berghofer Medical Research Institute

Abstract:

Approaches to sample analysis in multi-parameter flow cytometry and imaging remain strikingly distinct despite their use of remarkably similar technologies for data acquisition (e.g. fluorescent dyes, lasers, filters, and PMTs/detectors etc). This may be largely due to the different size of data sets (100s vs 1000s of events) or different parameters being measured (counting overall fluorescence of cells in a sample vs analyzing location of a fluorophore in the cell or tissue). Further, multi-parameter analysis for quantitation of immune cells is commonly used in flow cytometry but tissue pathology remains largely restricted to measuring histological parameters (size of pathology) or single colours (e.g Dab staining).
A recent surge in immunotherapy has raised interest in multi-parameter imaging to quantitate immune cells, tumour cells and other cells in distinct areas of tissues (e.g. inside and outside the tumour margins). We have therefore been evaluating the use of InForm, which is proprietary software for analysis of images obtained using the Perkin Elmer Vectra Quantitative Imaging System. The data generated is then migrated to FCS Express for visualization and analysis using standard flow cytometry strategies. The benefits of this strategy is the ability to use spectral unmixing to accurately generate and analyse multi-parameter data in different tissue areas in a way that is easily understood by immunology researchers and clinicians who are familiar with results from flow cytometry software.

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High Content Analysis of a Robust 384-Well Cell Migration Assay

Sean Burke¹, Tim Baranowski², Jennifer A. Fronczak³, Jayne Hesley², Keren Hulkower³, and David Novo¹

¹De Novo Software, Inc. Los Angeles, CA, ²Molecular Devices, Inc. Sunnyvale, CA, ³Platypus Technologies, Madison, Wisconsin

Presented at CYTO 2011

Abstract:

Quantifying cell migration in an objective manner is of interest to researchers studying cancer metastasis and wound healing. The Oris™ Pro 384 Cell Migration Assay (Platypus Technologies) is a first-in-class, automatable, 384- well high throughput screening assay. By using a novel approach to image analysis, combining the ImageXpress® Velos laser scanning cytometer (Molecular Devices) and FCS Express 4 Image Cytometry™ (De Novo Software), we were able to rapidly analyze the effects of cell seeding density and inhibitors on the motility of three different cell lines.

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Homogeneous Multiplexed Assay for Hematopoietic Stem Cell Toxicity

Oksana Sirenko¹, Pierre Turpin¹, George Klarmann³,Jayne Hesley¹, Sean Burke², Michael Sjaastad², David Novo², H. Roger Tang¹, and Evan F. Cromwell¹

¹Molecular Devices, Inc. Sunnyvale, CA, ²De Novo Software, Inc. Los Angeles, CA, ³Lonza, Inc. Walkersville, MD

Presented at Lab Automation 2011

Abstract:

Many potential pharmaceuticals fail in clinical trials due to unacceptable toxicity, thus early discovery and elimination of toxic compounds is important to the drug discovery process. For example, in chemotherapy toxicity to hematopoietic stem cells (HSC) or bone marrow often limits dosing and duration and can cause anemia, neutropenia, or other side effects. One way to improve information on toxicity is to introduce more biologically relevant assays early in development. Hematopoietic progenitors are extremely sensitive to the toxic side effects of many compounds and can provide valuable information about both general and lineage-specific toxicity making them useful for screening of therapeutics for cancers and other diseases. Studies have shown that the inhibitory activity of several drugs, as measured in in vitro assays of hematopoietic progenitor differentiation, correlate with their in vivo activities in various animal models (2, 3). Toxic effects of drugs can also be lineage-specific (4, 5). However, primary cells, especially non-adherent hematopoietic progenitors, are more challenging to use in automated high-throughput assays compared to adherent cell lines. Current HSC assays using flow cytometry are labor intensive and less amenable to high-throughput. Here we report on the use of IsoCyte cytometer (Molecular Devices, Inc.) and FCS Express® 4 Image Cytometry software (De Novo Software) to automate the study of cell differentiation, cytotoxicity, and carcinogenesis in 96 or 384 multi-well plate formats at throughputs of 3-5 minutes per plate.

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