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Published on: September 24, 2010
Detection of intracellular HIV in lymphocytes by flow cytometry
N Gadol1, G J Crutcher, M P Busch
1Irwin Memorial Blood Centers, San Francisco, California 94118-0318.
Insights
This study optimized flow cytometry for detecting intracellular Human Immunodeficiency Virus type 1 (HIV-1) antigens. The assay effectively monitors disease progression and antiviral therapy effects in patients with advanced HIV-1.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- Flow cytometry is crucial for immunological assays.
- Optimizing intracellular antigen detection in HIV-1 (Human Immunodeficiency Virus type 1) is vital for clinical monitoring.
Purpose of the Study:
- To optimize flow cytometric assays for direct detection of intracellular HIV-1 antigens in clinical blood samples.
- To develop a specific assay for monitoring HIV-1 disease progression and treatment efficacy.
Main Methods:
- Tested various permeabilization and gating techniques for flow cytometry.
- Developed an optimal procedure involving blood lysis, fixation with formaldehyde, and permeabilization with Tween 20.
- Used FITC-labeled anti-p18 or anti-p24 monoclonal antibodies and PE-anti-Leu M9 (CD33) for cell labeling and exclusion of monocytes/granulocytes.
Main Results:
- The optimized assay showed increased mean percentages of HIV p24 antigen-positive cells in patients with advanced disease.
- The assay successfully identified changes in HIV antigen expression correlating with disease advancement.
Conclusions:
- The developed flow cytometry assay is valuable for monitoring HIV-1 disease progression and antiviral therapy response in patients with advanced disease.
- The assay's sensitivity is currently insufficient for consistently detecting low levels of HIV-infected cells in asymptomatic individuals.
Abstract:
Various permeabilization procedures and defined gating techniques were tested in order to optimize existing flow cytometric assays and devise a specific assay for the direct detection of intracellular HIV-1 antigens in clinical blood specimens. In our optimal procedure, blood lysed with Orthomune Lysing Reagent was fixed with 3.7% formaldehyde for 10 min at room temperature and then permeabilized with 0.2% Tween 20 for 15 min at room temperature. Cells from whole blood were labeled with either FITC-anti-p18 or FITC-anti-p24 monoclonal antibodies and PE-anti-Leu M9 (CD33) in order to exclude monocytes and granulocytes from the lymphocyte gate. The assay demonstrated that mean percentages of HIV p24 antigen positive cells were increased in patients with advanced disease. The assay in its present form is useful for monitoring disease progression and for monitoring the effects of antiviral therapy in individuals, but it is not currently sensitive enough to detect consistently the low levels of HIV infected peripheral blood cells in asymptomatic individuals.
