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A novel multiparameter flow cytometric assay for inosine triphosphatase expression analysis in leukocytes
Wim H M Vroemen1, Imke C A Munnix, Jaap A Bakker
1Department of Clinical Chemistry & Hematology, Atrium Medical Center Parkstad, Heerlen, The Netherlands.
Insights
This study developed a flow cytometry assay to measure inosine triphosphate (ITPase) expression in leukocytes. Activated monocytes and phagocytes showed higher ITPase levels than lymphocytes.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Inosine triphosphate (ITPase) is an enzyme involved in purine metabolism.
- Understanding ITPase expression in leukocytes is crucial for immune function research.
- Previous studies lacked methods to quantify ITPase in specific leukocyte populations.
Purpose of the Study:
- To develop and validate a flow cytometry assay for assessing ITPase expression in human leukocytes.
- To determine the qualitative and quantitative expression of ITPase across different leukocyte subsets.
- To investigate the subcellular localization of ITPase within leukocytes.
Main Methods:
- Developed a multiparameter flow cytometry assay using a Fix & Perm strategy.
- Analyzed ITPase expression (percentage of positive cells and median fluorescent intensity - MFI) in peripheral blood leukocytes from healthy volunteers (n=20).
- Utilized immunocytochemistry to determine ITPase subcellular localization.
Main Results:
- ITPase is expressed intracellularly in all leukocyte populations.
- ITPase expression levels remained stable for at least 48 hours post-sampling.
- Activated monocytes exhibited significantly higher ITPase MFI than total monocytes, followed by granulocytes, then lymphocytes.
- Within lymphocytes, T-helper cells showed higher ITPase levels compared to cytotoxic T cells, B cells, and NK cells.
Conclusions:
- The developed flow cytometry assay reliably quantifies ITPase expression in leukocyte subpopulations.
- Phagocytes, particularly activated monocytes, demonstrate higher intracellular ITPase levels compared to lymphocytes.
- This study provides novel insights into ITPase distribution within the human immune system.
Abstract:
The aim of this study was to assess inosine triphosphate (ITPase) expression in the different leukocyte populations present in peripheral blood samples of a nonimmune compromised control group. For this purpose, a multiparameter flow cytometric assay was developed and performed to study ITPase expression in peripheral leukocyte subpopulations of healthy volunteers (n = 20). Qualitative ITPase expression was assessed by determining the percentage of ITPase-positive cells. Quantitative data were obtained by measuring the median fluorescent intensity (MFI). Subcellular localization of ITPase was analyzed using immunocytochemistry. Immunocytochemistry showed that ITPase is present in all leukocytes and localized intracellular. Based on this finding, a multiparameter flow cytometric assay was developed using a Fix & Perm strategy. Qualitative and quantitative ITPase expression remained stable (variation, <10%) for at least 48 h after blood sampling. MFI values showed that activated monocytes contained significantly more ITPase when compared to the total monocyte fraction (P < 0.0001), which subsequently had a higher amount of expression than granulocytes (P < 0.0001). In addition, the phagocyte subpopulations ([activated] monocytes and granulocytes) contained significantly higher levels of ITPase when compared to lymphocytes (P < 0.0001). Within the lymphocyte fraction, it appeared that T-helper cells contained significantly higher ITPase levels when compared to cytotoxic T cells, B lymphocytes, and natural killer cells (P < 0.0001). Our study is the first which describes a flow cytometry assay to analyze ITPase expression in leukocytes qualitatively as well as quantitatively and visualizes the intracellular localization of ITPase in leukocytes. © 2012 International Society for Advancement of Cytometry.
