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Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry.

M R Loken¹, J M Brosnan, B A Bach

¹Becton Dickinson Immunocytometry Systems, San Jose, California 95131.

|January 1, 1990

Summary

Accurate lymphocyte identification in peripheral blood is achieved using CD45/CD14 fluorescence and light scatter. This method ensures maximal lymphocyte recovery and enables precise quantification for reliable flow cytometry analysis.

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Area of Science:

Immunology
Hematology
Flow Cytometry

Background:

Distinguishing lymphocyte populations from other leukocytes in peripheral blood is crucial for accurate immunological assessments.
Traditional methods may face challenges in achieving precise cell population isolation and quantification.

Purpose of the Study:

To establish a reliable method for accurately distinguishing and quantifying lymphocytes in peripheral blood samples.
To optimize flow cytometry gating strategies for maximal lymphocyte recovery and purity.

Main Methods:

Utilizing a combination of CD45/CD14 immunofluorescence and forward/orthogonal light scatter for cell identification.
Establishing a light scattering window to encompass at least 98% of the target lymphocyte population.
Employing immunofluorescence to define the purity of the established gate and identify non-lymphocytes.

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Main Results:

Accurate discrimination between lymphocytes and other leukocyte subsets was achieved.
Maximal lymphocyte recovery (>= 98%) was consistently obtained through optimized gating.
The method allowed for the accurate identification of non-lymphocytes within the gate, enabling precise percentage calculations.

Conclusions:

The combined use of CD45/CD14 fluorescence and light scatter provides a robust approach for lymphocyte identification and quantification.
This technique enhances the accuracy of flow cytometry analysis by accounting for non-lymphocyte interference.
The established gating strategy ensures reliable and reproducible results in immunological studies.