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Measurement of lymphocyte aggregation by flow cytometry-physiological implications in chronic lymphocytic leukemia
Nili Dezorella1,2, Sigi Kay1, Shoshana Baron1
1The Department of Hematology, Tel Aviv Sourasky Medical Center, Tel-Aviv, Israel, 64239.
Insights
Flow cytometry accurately quantifies lymphocyte aggregation, a key response to stimuli. This method reveals disease-specific aggregation patterns in lymphoproliferative disorders, offering new diagnostic insights.
Area of Science:
- Immunology
- Cell Biology
- Clinical Diagnostics
Background:
- Lymphocyte aggregation is a crucial physiological response to extracellular stimuli.
- Current methods for assessing lymphocyte aggregation are qualitative or semi-quantitative.
- Quantitative assessment is needed for accurate analysis and disease correlation.
Purpose of the Study:
- To evaluate flow cytometry for quantitative, accurate, and reproducible measurement of lymphocyte aggregation.
- To investigate the significance of aggregation responses in various lymphoproliferative diseases.
- To correlate aggregation responses with immunophenotyping and IGHV mutational status in chronic lymphocytic leukemia.
Main Methods:
- Lymphoid cells aggregation induced by anti-CD19 antibodies or phorbol ester.
- Quantification of aggregation using flow cytometry (forward/side scatter) and ImageStreamX.
- Evaluation of methodology's accuracy, reproducibility, and limitations.
Main Results:
- Flow cytometry effectively quantifies lymphocyte aggregation induced by extracellular stimuli.
- Aggregation responses differ across various lymphoproliferative diseases.
- CD19-induced aggregation correlates with aberrant chronic lymphocytic leukemia characteristics, independent of IGHV mutational status.
Conclusions:
- Flow cytometry provides a robust method for measuring T and B cell aggregation.
- Quantitative aggregation analysis can elucidate mechanisms in lymphoproliferative diseases.
- This technique offers a valuable analytical tool for disease research.
Background:
Cellular aggregation is a physiological response of lymphocytes to various extracellular stimuli. Currently, lymphocytes aggregation is only evaluated qualitatively or by semiquantitative methods. In this study, we assessed the capacity of flow cytometry to measure lymphocytes aggregation in a quantitative, accurate, and reproducible manner, and examined the significance of aggregation responses in various lymphoproliferative diseases.
Methods:
Extracellular triggers such as anti-CD19 antibodies or phorbol ester were utilized to induce lymphoid cells aggregation in a concentration dependent manner. Aggregation was quantified by flow cytometry based on the forward or side scatter (SSC), or by dark-field SSC of aggregates measured by ImageStreamX. Accuracy, reproducibility, and limitations of the methodology were evaluated. Aggregation responses were measured in various types of lymphoproliferative diseases, and correlated with immunophenotyping and IGHV mutational status in chronic lymphocytic leukemia.
Results:
Lymphoid aggregates provoked by extracellular stimuli elevate the forward and SSC signals relatively to the number of cells in each event. Aggregation responses vary among different types of lymphoproliferative diseases. Moreover, elevated levels of CD19-induced aggregation are associated with aberrant chronic lymphocytic leukemia characteristics, but not with IGHV mutational status of the disease
Conclusions:
We have demonstrated that flow cytometry can provide accurate and reproducible measurement of both primary as well as T and B cell lines aggregation in response to extracellular stimuli. The use of quantitative evaluation of activation driven or other cellular aggregation may provide an analytical tool to elucidate biochemical and molecular mechanisms associated with lymphoproliferative diseases. © 2015 International Clinical Cytometry Society.
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