Inactivation of HIV-infected H9 cells in whole blood preparations by lysing/fixing reagents used in flow cytometry

Nicholson, Janet K. A.; Browning, Sandra W.; Orloff, Sherry L.; McDougal, J. Steven

doi:10.1016/0022-1759(93)90180-f

Cited by 21 publications

(15 citation statements)

References 12 publications

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“…These specimens must be handled with appropriate biohazard precautions as described previously (12). Several publications have also addressed specific issues relating to FC testing of HIVpositive specimens (13)(14)(15).…”

Section: Sample Requirementsmentioning

confidence: 99%

Validation of cell‐based fluorescence assays: practice guidelines from the ICSH and ICCS – part II – preanalytical issues

Davis¹,

Dasgupta

Kussick

et al. 2013

Cytometry Part B Clinical

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Section: Sample Requirementsmentioning

confidence: 99%

Validation of cell‐based fluorescence assays: practice guidelines from the ICSH and ICCS – part II – preanalytical issues

Davis¹,

Dasgupta

Kussick

et al. 2013

Cytometry Part B Clinical

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“…Immunofluorescence staining was performed by using a standardized lysed whole blood staining technique [17][18][19]. The monoclonal antibodies (MAbs) were directly conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (RD).…”

Section: Direct Immunofluorescence Stainingmentioning

confidence: 99%

Changes in Antigen Expression on B Lymphocytesduring HIV Inf ection

Ginaldi¹,

Martinis²,

D'Ostilio³

et al. 1998

Pathobiology

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Involvement of the B cell compartment during HIV infection plays an important role in the development of immune deficiency. The aim of this study was the identification of specific antigen expression changes on B lymphocytes in HIV infection as surrogate markers in this cell population of certain functional aspects that could be easily measured. We investigated the level of expression of a series of constitutive surface markers in B lymphocytes (HLA-DR, CD19, CD20, CD21, CD22) from 30 HIV-seropositive adult patients and 20 normal controls. By means of quantitative flow cytometry, we assessed the number of antigen molecules per cell using standard beads to convert fluorescence intensity into antibody-binding capacity (ABC). We correlated these results with disease stage and cellular markers of immune activation. The expression of CD20 was significantly increased when B cells from HIV-infected individuals were compared with those from uninfected subjects. No differences were found in the density expression of HLA-DR on activated CD3+ T cells between HIV+ and HIV– subjects. In contrast, B cells from HIV+ patients showed a significantly lower number of HLA-DR molecules per cell compared to normal controls. A significantly lower number of CD21 molecules per cell was also found on B lymphocytes from HIV+ patients compared to normal controls. No differences in CD19 and CD22 expression levels on B cells between HIV-infected patients and controls were detected. No differences between HIV disease stages were detected for CD19, HLA-DR, CD21 and CD22. In contrast, differences between stages were found for CD20 expression, which showed significant changes in individuals with less than 200 CD4 T cells/µl. The data presented here demonstrate that B lymphocytes of HIV-infected individuals exhibit specific changes in receptor density expression during HIV infection and that these changes are often correlated with progression of disease, as measured by CD4 counts. No correlations were found between the percentages of HLA-DR+ T cells and the ABC values of the B cell markers studied. These antigen expression modulations may contribute to the humoral abnormalities during HIV infection and to the development of severe, recurrent or multiple bacterial infections. Therefore, quantitative flow cytometry may be of value in HIV infection both for clinical and biological studies. The study of antigen density changes on B cells in HIV infection may allow a better understanding of the humoral immune defects observed in these patients and provide insights into the functional defects of B cell compartment in HIV-infected individuals.

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“…Peripheral blood lymphocyte subpopulations were determined by two-color immunofluorescence using a standardized lysed whole blood staining technique [13,14], Briefly, the samples were stained with matching combinations of monoclonal antibodies (MoAbs), directly conjugated with fluorescein isothiocyanate (FITC) or phvcoerythrin (PE): 10 pi of each appropriately labeled MoAb were add ed to 100 pi of well-mixed anticoagulant whole blood. The tubes were vortexed and then incubated at room temperature for 20 min in the dark.…”

Section: Monoclonal Antibody and Flow Cytometrymentioning

confidence: 99%

Activated Naive and Memory CD4+ and CD8+ Subsets in Different Stages of HIV Infection

Ginaldi

Martinis²,

D'Ostilio³

et al. 1997

Pathobiology

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The aim of this study was to evaluate the changes and the correlations between the main lymphoid phenotypes indicative of activation and/or functional states during the course of HIV infection. Immunophenotype studies by flow cytometry were performed on blood samples from 59 HIV-1-positive patients, divided into four stages, and 18 seronegative healthy controls, to determine the expression of HLA-DR, CD29 and CD45RA on CD4+ and CD8+ lymphocytes. HLA-DR expression was elevated on the total lymphocyte population and in both the main T subsets. Its presence on CD4+ lymphocytes probably has a different significance in the first phase of infection when it is indicative of reactive activation, in contrast to the more advanced stages of disease when it favors the spread of HIV infection among this cellular subset. The increasing state of immune activation is also confirmed by a proportional decrease in the expression of CD45RA, substantial stability of CD29 and an increase in double-negative CD4+ cells as the infection proceeds. Also CD8+HLA-DR+ lymphocytes increase during the course of disease. The parallel increase of the CD8+CD45RA+ subset in asymptomatic patients suggests the presence in this phase of infection of peripheral blood immature and activated CD8+ cells. Similarly to CD4+, the CD29 subset of CD8+ lymphocytes remains unchanged compared to controls during disease progression. In both CD4+ and CD8+ subsets we observed the increase of a double-negative sub-population of uncertain significance. HLA-DR, the memory marker CD29 and the naive marker CD45RA seem to be the more promising and helpful indicators for a better staging of diseae and may provide information that accurately correlates with progression of infection. The peculiar trend of the described phenotypic alterations could represent changes in the immune response to HIV during disease progression and facilitate the definition of specific immune patterns in different stages of HIV infection.

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Inactivation of HIV-infected H9 cells in whole blood preparations by lysing/fixing reagents used in flow cytometry

Cited by 21 publications

References 12 publications

Validation of cell‐based fluorescence assays: practice guidelines from the ICSH and ICCS – part II – preanalytical issues

Validation of cell‐based fluorescence assays: practice guidelines from the ICSH and ICCS – part II – preanalytical issues

Changes in Antigen Expression on B Lymphocytesduring HIV Inf ection

Activated Naive and Memory CD4+ and CD8+ Subsets in Different Stages of HIV Infection

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