Circulating Human Immunodeficiency Virus (HIV) p24 Antigen-Positive Lymphocytes: A Flow Cytometric Measure of HIV Infection

Bm, Ohlsson-Wilhelm; Cory, JM; Ha, Kessler; Me, Eyster; Rapp, Fred; Landay, Alan

doi:10.1093/infdis/162.5.1018

Cited by 23 publications

(28 citation statements)

References 20 publications

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“…This method proved to be more sensitive and accurate for quantitative studies and faster than other methods of HIV-1 detection, such as the reverse transcriptase (RT) assay or determination of syncytium formation. Detection of HIV antigens on peripheral blood mononuclear cells by FCM is a useful method for monitoring HIV replication in vivo by monitoring the number of circulating CD4 ϩ cells positive for p24 (63,131,235), p24 and nef (213), or p18 and p24 (107). In all these works, the percentage of cells expressing these antigens was statistically correlated with the clinical status of the patient.…”

Section: Cd38mentioning

confidence: 99%

Applications of Flow Cytometry to Clinical Microbiology

Alvarez-Barrientos¹,

Arroyo²,

Cantón³

et al. 2000

Clinical Microbiology Reviews

162

112

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Section: Cd38mentioning

confidence: 99%

Applications of Flow Cytometry to Clinical Microbiology

Alvarez-Barrientos¹,

Arroyo²,

Cantón³

et al. 2000

Clinical Microbiology Reviews

162

112

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“…In order to confirm the previously published observations, PBMCs from 7 HIV seropositive individuals with varying stage of infection and 4 HIV seronegative individuals were labeled according to the procedure of OhlssonWilhelm et al (1). In addition, anti-CD4-PE was used to differentiate the CD4ϩ lymphocytes from other lymphocyte subsets.…”

Section: Intracellular P24 Quantification Using the Standard Methodsmentioning

confidence: 88%

“…Flow cytometry has been used by others to detect HIV-1-infected cells in patient blood (1,(8)(9)(10)(11)(12), usually by intracellular labelling for the HIV-1 p24 core antigen with an HIV-1 p24 mAb (1,9-11). Of note, these original studies did not include isotype-matched control mAbs.…”

Section: Discussionmentioning

confidence: 99%

“…The difficulties of using isotype controls led to the use of HIV-1 seronegative control subjects to establish cutoff points for selection of positive cells (1,9,11). Different FIG.…”

Section: Discussionmentioning

confidence: 99%

“…By PCR-driven fluorescent in situ hybridization (FISH) and flow cytometry, the number of infected PBMCs ranges from 4% to 15% when measuring HIV-1 proviral DNA, and from less than 1% to 8% for HIV-1 tat mRNA (8). The proportion of lymphocytes with demonstrable intracellular p24 varies from 0-25% (1,(9)(10)(11)(12) when measured by flow cytometry with an inverse correlation between the number of p24-antigen positive cells and CD4ϩ T cell numbers.…”

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confidence: 99%

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Specificity of binding of HIV-1 anti-p24 antibodies to CD4+ lymphocytes from HIV-infected subjects

et al. 1998

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The clinical utility of a flow cytometric assay (FCA) for intracellular HIV p24 antigen was evaluated in a group of HIV‐1‐infected subjects. A previously described method, p24‐FCA (1), was modified to minimize nonspecific staining and to include irrelevant isotype‐matched control antibodies. Blood mononuclear cells from 32 HIV‐1 seropositive subjects and 14 HIV‐1 seronegative controls were examined. In HIV‐1 seropositive individuals, the proportion of CD4+ lymphocytes that bound the p24 monoclonal and the isotype control antibodies were not different. The frequency of cells binding p24 antibodies increased with declining CD4 counts and was highest in patients with low CD4+ lymphocyte counts. Although results of p24‐FCA do reflect disease progression, the high nonspecific binding of monoclonal antibodies in infected subjects obscures specific p24 binding and precludes its use as an accurate measure of infection within single cells. Cytometry 33:83–88, 1998. © 1998 Wiley‐Liss, Inc.

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Rapid single-step method for flow cytometric detection of surface and intracellular antigens using whole blood

Francis¹,

Connelly²

1996

Cytometry

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Fixation/permeabilization methods used for the detection of intracellular antigens by flow cytometry often result in the destruction of cellular morphology and surface immunoreactivity, properties useful in flow cytometry for the characterization of cells in heterogeneous populations. In addition, a majority of these methods are incompatible with whole blood and require that peripheral blood leukocytes (PBLs) be purified prior to fixation. This article describes a new technique for the rapid detection of both intracellular and cell surface antigens, while preserving cell morphology, through the use of a single‐step fixation/permeabilization reagent, ORTHO PermeaFix (OPF). OPF is compatible with whole blood, allowing for the direct preparation of PBLs without prior cell separation. An additional red blood cell lysing reagent was not required because RBC lysis occurred upon resuspension of OPF‐treated whole blood samples in isotonic solution. Discrimination of leukocyte populations by light scatter after OPF treatment was comparable to matched unfixed live cells. In addition, absolute lymphocyte and white blood cell (WBC) counts were not significantly affected when OPF‐treated cells were compared with unfixed cells. Treatment of whole blood from 7 normal donors showed no significant difference in percentage of cells positive for CD2, CD3, CD4, CD8, CD16, or CD19 between fixed and unfixed samples when cells were stained before fixation, and no difference in CD3, CD4, CD8, CD16, or CD19 percentages when cells were stained following fixation. Monoclonal antibodies specific for intracellular antigens located at various sites within the cell were tested on fixed samples. OPF‐treated peripheral blood lymphocytes showed greater than 95% reactivity for the inner mitochondrial membrane protein bcl‐2, and the cytoskeletal cytoplasmic protein vimentin. TIA‐1, a cytolytic granule‐associated protein, showed differential reactivity within lymphocyte subsets, from a low of 8 ± 2% in CD4+ cells to 89 ± 6% in CD16+ cells, when whole blood from five normal donors was fixed and stained. Reh cells treated with OPF showed greater than 95% reactivity for the internuclear protein TdT. A comparison of OPF with two other fixation/permeabilization procedures, 1% paraformaldehyde followed by 45% ethanol and 0.25% paraformaldehyde followed by 0.2% Tween 20, showed that only OPF could be used both prior to or following cell surface staining with no effect on antigen detection while allowing optimal detection of all of the intracellular antigens tested. © 1996 Wiley‐Liss, Inc.

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Circulating Human Immunodeficiency Virus (HIV) p24 Antigen-Positive Lymphocytes: A Flow Cytometric Measure of HIV Infection

Cited by 23 publications

References 20 publications

Applications of Flow Cytometry to Clinical Microbiology

Applications of Flow Cytometry to Clinical Microbiology

Specificity of binding of HIV-1 anti-p24 antibodies to CD4+ lymphocytes from HIV-infected subjects

Rapid single-step method for flow cytometric detection of surface and intracellular antigens using whole blood

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