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Detection and quantitation of human immunodeficiency virus-infected peripheral blood mononuclear cells by flow
J J McSharry1, R Costantino, E Robbiano
1Department of Microbiology, Albany Medical College, New York 12208.
Insights
A new flow cytometry assay can detect and quantify human immunodeficiency virus (HIV)-infected cells in patients. This method shows promise for monitoring HIV disease progression, correlating with clinical status and T4 cell counts.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- Human immunodeficiency virus (HIV) infection requires sensitive methods for monitoring disease progression.
- Current methods for detecting HIV antigens in patients may be limited by factors like immune complexes.
- Accurate quantitation of infected cells is crucial for assessing patient prognosis.
Purpose of the Study:
- To develop and validate a flow cytometric assay for detecting and quantifying HIV-infected peripheral blood mononuclear cells (PBMCs).
- To assess the correlation between the percentage of HIV-infected cells and clinical status in HIV-seropositive patients.
- To compare the efficacy of flow cytometry for antigen detection with standard enzyme-linked immunosorbent assays (ELISAs).
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) were isolated from HIV-seropositive patients.
- Intracellular HIV antigens (p24, nef) were detected using indirect immunofluorescence with monoclonal antibodies.
- Cell DNA content was measured via propidium iodide staining after RNase treatment.
- Two-color fluorescence analysis was performed using flow cytometry.
Main Results:
- Flow cytometry successfully detected and quantified HIV-infected cells using antibodies against p24 or nef antigens.
- A significant correlation (P=0.0001) was observed between the percentage of p24-positive cells and poorer patient prognosis.
- An inverse correlation was found between p24-positive cells and T4 cell counts in patients in CDC groups III and IV.
- Cell-associated antigen detection by flow cytometry did not correlate with serum antigen detection by ELISA, likely due to serum immune complexes.
Conclusions:
- Flow cytometry provides a rapid, sensitive, and quantitative method for determining HIV antigen status in patients.
- This assay may serve as a more effective indicator of HIV disease progression compared to current antigen detection methods.
- The assay's ability to correlate with clinical status highlights its potential clinical utility in managing HIV infection.
Abstract:
A flow cytometric assay has been developed to detect and quantitate human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells obtained from HIV-seropositive patients. Peripheral blood was obtained from patients attending an acquired immune deficiency syndrome clinic, and mononuclear cells were separated by centrifugation onto Ficoll-Hypaque. The cell layer at the interface was removed, washed in phosphate-buffered saline without Ca2+ and Mg2+, and fixed with 90% methanol, and intracellular HIV antigens were detected by indirect immunofluorescence with monoclonal antibodies to HIV antigens as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G F(ab')2 antibody as the secondary antibody. DNA content was determined by propidium diiodide staining after RNase treatment. These fluorochrome-treated cells were analyzed for two-color fluorescence by flow cytometry. The results showed that HIV-infected cells in peripheral blood that have been treated with monoclonal antibodies to the p24 or nef antigens of HIV can be detected and quantitated by flow cytometry. The percentage of p24 antigen-positive mononuclear cells had a significant correlation (P = 0.0001) with the clinical status of the patient, i.e., those with a high percentage of p24 antigen-positive cells had a poorer prognosis than those with a lower percentage of p24 antigen-positive mononuclear cells. In addition, for those in Centers for Disease Control groups III and IV, there was an inverse correlation between the percentage of p24 antigen-positive mononuclear cells and the number of T4 cells. However, cell-associated antigen detection by flow cytometry did not correlate with detection of antigen in sera of HIV-seropositive patients by the standard antigen capture enzyme-linked immunosorbent assay. This lack of correlation was probably due to the presence of immune complexes in the sera of HIV-seropositive patients. These results suggest that flow cytometry can be used as a rapid, sensitive, and quantitative assay system for the determination of the antigen status of HIV-seropositive patients and that it may be more useful as an indicator of disease progression than the currently used antigen detection methods.