Measurement of lymphocyte aggregation by flow cytometry-physiological implications in chronic lymphocytic leukemia

Nili Dezorella, Sigi Kay, Shoshana Baron, Mika Shapiro, Ziv Porat, Varda Deutsch, Yair Herishanu, Ben Zion Katz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)257-266
Number of pages10
JournalCytometry Part B - Clinical Cytometry
Volume90
Issue number3
DOIs
StatePublished - 1 May 2016

Keywords

  • CD19
  • aggregation
  • chronic lymphocytic leukemia
  • flow cytometry
  • lymphocytes
  • side scatter

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