Immunophenotyping

Introduction

Immunophenotyping is the analysis of heterogeneous populations of cells for the purpose of characterizing the presence and proportions of the various populations of interest. Typical samples are leukocytes from peripheral blood obtained in fine needle aspirates, such as those taken form lymph nodes. LSC-based immunophenotyping is a well-developed application in clinical research settings, well-suited to paucicellular specimens such as FNAs and pediatric blood.

For more information, see the LSC Application Notes.

Highlights of Analysis with iGeneration Imaging Cytometers

The flexibility of the iGeneration platform allows the use of customized “Clatch” slides, as shown in the diagram below, or multi-well plates.

A full antibody panel for immunophenotyping includes up to 24 different immunophenotypic markers in the Clatch slide. A multi-well plate can expand the number of antibodies in the panel or the number of samples being examined in a single analysis.

Immunophenotyping on the iGeneration allows samples to be analyzed with higher throughput in a multi-well plate. Advanced processing tools can aid in identifying cells. Cells are identified by either their fluorescence or their ability to scatter laser light. Scatter of laser light by cells can be detected by a forward scatter detector.

Each well of a 96-well plate may be used to characterize the population for two or more immunophenotypic markers. The populations can be resolved using scattergrams.

In the example shown above:

Cells that are negative for CD3 or CD4 are seen as white dots.
Cells that are positive for CD3 only are seen as red dots.
Cells that are positive for both CD3 and CD4 are seen as yellow dots.

The relative percentages of these three populations can be easily ascertained from the scattergram. With this approach using a full antibody panel, the immunophenotypic characteristics of the population may be determined as shown below.

Adapted from: Clatch, RJ, Foreman, JR, Walloch, JL. 1998. Simplified Immunophenotypic Analysis by Laser Scanning Cytometry. Cytometry. 34:3-16.

Benefits of Using iGeneration Technology

Using advanced data processing, cells may be identified for analysis using either fluorescence characteristics or the cells’ ability to absorb or refract light. Using carrier types such as 96-well microtiter plates can facilitate both sample processing and throughput. Multiple probes can be analyzed and widefield images obtained in a single sample; and the results correlated to the quantitative data in field images. The iGeneration platform combines the advantages of flow cytometry and image analysis in a single system. The iGeneration inverted format supports a diversity of carrier types, allowing flexibility in matching the needs of your application with the appropriate carrier type.

More Information

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