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Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry
Published on: February 27, 2019
Immunologic differentiation of absolute lymphocyte count with an integrated flow cytometric system: a new concept for
T J Mercolino1, M C Connelly, E J Meyer
1Ortho Diagnostic Systems, Raritan, New Jersey 08869-0606, USA.
Insights
A new automated flow cytometry method simplifies immune status monitoring. This approach uses a three-test panel and software for accurate lymphocyte subset counts, reducing costs and complexity.
Area of Science:
- Immunology
- Biotechnology
- Medical Diagnostics
Background:
- Accurate immune status monitoring relies on precise lymphocyte subset counts.
- Traditional methods for measuring absolute lymphocyte counts can be complex and prone to errors.
Purpose of the Study:
- To present a novel, automated method for immune status monitoring using flow cytometry.
- To evaluate the accuracy and efficiency of this new approach compared to traditional methods.
Main Methods:
- Utilized a three-test panel (isotype control, CD4/CD8/CD3, CD16/CD19/CD3) with flow cytometry.
- Employed automated software for lineage-specific gating and absolute lymphocyte and subset counts.
- Validated automated gating against manual analysis and assessed quality control features.
Main Results:
- Automated software provided equivalent results to expert manual analysis, even with low cell counts.
- The sum of T, B, and NK cells correlated well with light scatter differential lymphocyte counts.
- The new method demonstrated reduced error rates and minimized technician-dependent variations.
Conclusions:
- The described automated flow cytometry method offers a viable alternative for immune status monitoring.
- This approach can decrease testing costs and complexity through objective analysis without compromising result quality.
Abstract:
We describe a method to obtain results for immune status monitoring that uses a three-test panel, comprised of isotype control and 2 specific Mab tests (CD4/CD8/CD3 and CD16/CD19/CD3), in conjunction with a flow cytometer that directly measures absolute counts. Automated software is used for lineage-specific gating of three-color immunofluorescence to determine lymphocyte and lymphocyte subset counts. The autogating function of this software is shown to yield equivalent results to manual analysis by an expert user, and to be effective when as few as 25 target cells are present. The software is also shown to perform automatic quality control checks of the sample preparation, reagent, and automated analysis. We demonstrate that the sum of T (CD3+), B (CD19+), and natural killer (NK, CD16 + CD3-) cells, as a determination of all lymphocytes, correlates well with lymphocytes measured using a light scatter differential. Moreover, T + B + NK lymphocyte count is shown to be less error-prone than lymphocyte count from light scatter differential, and to minimize errors that arise from between-technician variation in sample preparation. Our data suggest that the new approach that we describe could offer an alternative to the traditional two-stage methods for measuring absolute counts of lymphocyte subsets for immune status monitoring. As such this method could reduce, through objective automated analysis, testing cost and complexity, without sacrificing the quality of results.
