Ingrid Schmid

A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry

A sensitive method for quantification of cells undergoing apoptosis that permits the simultaneous measurement of dual-color cell surface immunofluorescence is presented. Unfixed cells are stained with 7-amino-actinomycin D (7-AAD) for discrimination of live from early apoptotic cells and from cells which have lost membrane integrity (late apoptotic or necrotic, dead cells). Owing to its spectral characteristics 7-AAD can be combined with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) cell surface staining. After staining, the samples can be treated with paraformaldehyde (PF) solution to eliminate the risk for exposure of laboratory personnel to biohazardous agents and to preserve the cells through fixation for later analysis on the flow cytometer. The value of the method is shown on the measurement of apoptosis in human thymocytes and in human peripheral blood mononuclear cells (PBMC) exposed to various inducers of active cell death. The method is validated by fluorescent activated cell sorting in combination with morphologic examination of the sorted cells. The technique we are presenting is particularly valuable in a clinical setting because it allows rapid multiparameter analysis of apoptosis in combination with cell surface phenotype on biohazardous samples with single laser instrumentation.

Quality control in the flow cytometric measurement of T-lymphocyte subsets: The Multicenter AIDS Cohort Study experience

Since 1984, the Multicenter AIDS Cohort Study (MACS) has utilized four flow cytometry laboratories to measure T-lymphocyte subset levels semiannually in a large cohort of homosexual men. This report summarizes the steps taken in the MACS laboratories to attain comparability of lymphocyte subset determinations across the centers and over time. Identical flow cytometers, monoclonal antibodies, and analytic procedures have been used, and over a period of time, the procedure for sample preparation was also standardized. Interlaboratory proficiency testing utilizing identical specimens analyzed in the four laboratories was performed to evaluate the comparability of the data among the laboratories. Our results verify that such testing can identify technical bias in flow cytometric evaluations performed at different laboratories. Temporal laboratory consistency in flow cytometric measurements was evaluated using data from each sites HIV-seronegative homosexual reference group. Both sequential 95% confidence intervals (mean +/- 2 x SEM) and the within-person standard deviations of the immune measurements were considered. Significant variation in CD3, CD4, and CD8 lymphocyte subset percentages over time in the seronegative reference population was observed. Our observations indicate that the lymphocyte subset values of this seronegative group should be used to adjust those obtained on the seropositive study participants during a particular time period, thereby allowing improved discrimination of the effects of HIV on T cells in infected individuals. The data presented are of use for designing epidemiologic and intervention studies in HIV-1-infected individuals, especially for calculating sample sizes. The methods we have used to assess the quality of data in the MACS have general application to quality control programs in flow cytometry laboratories. In particular, comparison of sequential confidence intervals and within-person standard deviations for lymphocyte subset determinations on control populations are essential to a comprehensive proficiency testing program because they permit assessment of consistency within a laboratory over time.Since 1984, the Multicenter AIDS Cohort Study (MACS) has utilized four flow cytometry laboratories to measure T-lymphocyte subset levels semiannually in a large cohort of homosexual men. This report summarizes the steps taken in the MACS laboratories to attain comparability of lymphocyte subset determinations across the centers and over time. Identical flow cytometers, monoclonal antibodies, and analytic procedures have been used, and over a period of time, the procedure for sample preparation was also standardized. Interlaboratory proficiency testing utilizing identical specimens analyzed in the four laboratories was performed to evaluate the comparability of the data among the laboratories. Our results verify that such testing can identify technical bias in flow cytometric evaluations performed at different laboratories. Temporal laboratory consistency in flow cytometric measurements was evaluated using data from each sites HIV-seronegative homosexual reference group. Both sequential 95% confidence intervals (mean ± 2 × SEM) and the within-person standard deviations of the immune measurements were considered. Significant variation in CD3, CD4, and CD8 lymphocyte subset percentages over time in the seronegative reference population was observed. Our observations indicate that the lymphocyte subset values of this seronegative group should be used to adjust those obtained on the seropositive study participants during a particular time period, thereby allowing improved discrimination of the effects of HIV on particular time period, thereby allowing improved discrimination of the effects of HIV on T cells in infected individuals. The data presented are of use for designing epidemiologic and intervention studies in HIV-1-infected individuals, especially for calculating sample sizes. The methods we have used to assess the quality of data in the MACS have general application to quality control programs in flow cytometry laboratories. In particular, comparison of sequential confidence intervals and within-person standard deviations for lymphocyte subset determinations on control populations are essential to a comprehensive proficiency testing program because they permit assessment of consistency within a laboratory over time.

Nitric oxide induces necrotic but not apoptotic cell death in oligodendrocytes

We have investigated the mechanism of nitric oxide-induced damage in glial cells. Genomic DNA isolated from astrocytes and microglia, treated for 18 h with varying concentrations of a nitric oxide donor, was analysed by electrophoresis. No DNA damage was evident. Oligodendrocytes, treated with 2 mM nitric oxide for 3-48 h, showed single stranded breaks at 48 h but no laddering of nucleosomic fragments of DNA. When analysed by electron microscopy, ultrastructural changes in oligodendrocytes treated with 1 mM nitric oxide for 24 h showed intact nuclei but alterations in membranes and organelles characteristic of necrosis, including disrupted mitochondria with dissolution of their christae. Astrocytes, a glial cell type that we have previously shown to be much less sensitive to nitric oxide-induced damage, did not show ultrastructural changes. DNA analysis by flow cytometry of glial cells treated with nitric oxide supported the apparent necrotic-type death in oligodendrocytes. Double staining of oligodendrocytes, using Hoechst 33342 and propidium iodide for the simultaneous assessment of both apoptotic and necrotic cells, demonstrated that, while the proportion of dead cells increased with time and increasing concentrations of nitric oxide, the death was due to necrosis and not apoptosis. In this study, we demonstrate that direct exposure to soluble nitric oxide, produced in vitro from a nitric oxide donor chemical, ultimately kills oligodendrocytes by necrosis. Microglia and astrocytes maintain DNA and organelle integrity when exposed to exogenous nitric oxide.

Elevated relative fluorescence intensity of CD38 antigen expression on CD8+ T cells is a marker of poor prognosis in HIV infection: Results of 6 years of follow‐up

Relative fluorescence intensity measurements from a flow cytometer were used to evaluate expression of CD38 and HLA-DR antigens. These molecules are associated with cellular activation and are present at increased levels on the CD8+ lymphocytes of HIV-infected subjects. In the current study, the prognostic value of mean fluorescence intensity measurements of CD38 and HLA-DR on CD8+ cells was compared to results from our previous study in which we reported prognostic value for an elevated percentage of CD8+ cells that were positive for expression of the CD38 antigen (Giorgi et al.: JAIDS 6:904-912, 1993). Using the proportional hazards model, elevated mean fluorescence intensity of CD38 expression on CD8+ cells had prognostic value for development of AIDS that was almost identical to the prognostic value of the percentage of CD8+ cells that were positive for expression of CD38. This prognostic value was in addition to that provided by the patients CD4+ cell measurement. To our knowledge, this is the first report that a measurement of fluorescence intensity can be used as a prognostic marker in an immunodeficiency disease. Efforts are needed to establish methods that will allow widespread application of this observation in the clinical management of HIV-infected subjects.

Preparation of Cells and Reagents for Flow Cytometry

Flow cytometry is widely used for analyzing the expression of cell surface and intracellular molecules (on a per cell basis), characterizing and defining different cell types in heterogeneous populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. This technique is predominantly used to measure fluorescence intensity produced by fluorescent‐labeled antibodies or ligands that bind to specific cell‐associated molecules. A procedure for direct and indirect staining of single‐cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single‐cell suspensions is also included. Alternate protocols describe intracellular staining of unfixed cells in the presence of a detergent and staining of nonviable cells to facilitate discrimination of dead cells in fixed or permeabilized cell preparations.

Selective alterations in immunoregulatory lymphocyte subsets in early HIV (human T-lymphotropic virus type III/lymphadenopathy-associated virus) infection.

In order to characterize the effects of HIV (human T-lymphotropic virus type III/lymphadenopathy-associated virus) on the immune system, Leu8− and Leu8+ subsets of CD4 and CD8 cells were studied in seropositive homosexually active men without acquired immune deficiency syndrome (AIDS). Controls included both heterosexual men and HIV-seronegative homosexually active men. The decrease in CD4 levels, observed in HIV-seropositive men who were asymptomatic, as well as in those who had persistent generalized lymphadenopathy or constitutional symptoms of HIV infection, occurred proportionally in both the Leu8− and the Leu8+ CD4 subsets. This observation, that HIV infection does not selectively diminish either subset of CD4 cells, indicates that the selective loss of T cell-mediated functions which accompanies the development of AIDS is not related to preferential loss of the Leu8+ CD4 subset. Among CD8 cells, however, HIV infection resulted in a threefold elevation in the number of Leu8− CD8 cells, while the number of Leu8+ CD8 cells remained constant. The increase in Leu8− CD8 cells was present in recent seroconverters, persistently seropositive men, and patients with AIDS. We propose that the increase in Leu8− CD8 cells represents an HIV-specific cytotoxic T-cell response. These cells may operate by killing infected CD4 cells, thereby partially controlling viral infection while simultaneously contributing to the destruction of the immune system.

Differential tropism of HIV-1 isolates for distinct thymocyte subsets in vitro.

ObjectiveUnderstanding the interaction between HIV and developing thymocytes is crucial in determining how HIV infection perturbs the immune system. We determined which thymocyte subsets can harbor and express HIV. DesignHIV expression in mature and immature thymocytes obtained from surgical specimens from non-infected children was determined after in vitro infection with the syncytium-inducing, cytopathic NL4–3 and the non-syncytium-inducing, relatively noncytopathic JR-CSF isolates. MethodsIntracellular staining for the HIV p24gag antigen was combined with cell surface phenotyping to determine thymocyte subsets expressing HIV. Infection was quantitated by polymerase chain reaction on sorted subsets. ResultsNL4–3 replicated faster and to higher titers and caused a more severe decrease of all CD4-bearing thymocytes than did JR-CSF. In addition, both immature CD1+ and mature CD1− thymocytes expressed NL4–3, whereas only mature CD1 -cells expressed JR-CSF. The tropism of NL4–3 for these immature cells suggests a mechanism for a more profound impact on T-cell maturation than that seen with JR-CSF. We also found that thymocytes lacking cell surface CD4 (CD4-CD8− and CD4-CD8+ subsets) expressed virus with either isolate late in infection, when viral levels were high. The CD4-CD8− cells expressing HIV were mature CD3bright T-cell receptor (TCR)α/βbright cells. ConclusionsThese results show that NL4–3 can be expressed by thymocytes at immature and mature stages of differentiation and cause severe loss of CD4+ cells. Thus, tropism of a virus for immature cells can affect the capability of the thymus to produce new T lymphocytes leading to a greater impact on development and functions of the immune system. It is proposed that this in vitro model can be used to study pathogenic mechanisms in the thymus.

Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D

This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.

Major Expansions of Select CD8+ Subsets in Acute Epstein-Barr Virus Infection: Comparison with Chronic Human Immunodeficiency Virus Disease

CD8+ lymphocyte phenotypes were characterized during acute Epstein-Barr virus (EBV) infection, and a comparison was made to previous studies of human immunodeficiency virus (HIV). This was of interest because CD8+ cells contribute to immunologic control of both infections, but the usual outcome of EBV infection is benign, whereas untreated HIV infection is fatal. During acute EBV infection, CD8+ cells expressed elevated levels of the activation antigens CD38 and HLA-DR, similar to that during chronic HIV infection. Within 16 weeks, when EBV latency is established, CD8+ cell activation had resolved. In contrast, activation persists in HIV infection. Expression of CD38 and HLA-DR on CD8+ cells could be a marker for ongoing viral replication in both infections. Other CD8+ cell alterations observed in this study of acute EBV infection included increases in both CD62L- and CD62L+ CD8+ cells and unique kinetics in the expansion of the CD57+CD8+ cell subset.

Measurement of lymphocyte subset proliferation by three-color immunofluorescence and DNA flow cytometry.

We developed a method for simultaneous flow cytometric analysis of three-color immunofluorescence and DNA content. We show here that staining with 7-amino-actinomycin D (7-AAD) at 10 microg/ml using a phosphate-citrate buffer at low pH containing saponin for cell membrane permeabilization yields good resolution DNA histograms with low coefficients of variation. Furthermore, light scatter properties of cells are preserved after permeabilization; this permits gating on cell populations that differ in scatter signals on the flow cytometer. Because of the low pH of the phosphate-citrate staining buffer, Alexa488, a pH-independent green-fluorescent fluorochrome is used instead of fluorescein-isothiocyanate (FITC) for cell surface staining in combination with phycoerythrin (PE) and with allophycocyanin (APC) which are both pH insensitive. Removal of 7-AAD after staining and replacing it with non-fluorescent actinomycin D (AD) retains DNA staining and allows detection of Alexa488, PE and APC cell surface immunofluorescence without interference from fluorescent 7-AAD in solution for clear identification of cell subpopulations even after prolonged stimulation in culture. Thus, using a four-color benchtop flow cytometer, measurement of Alexa488, PE and APC three-color immunofluorescence can be combined with 7-AAD DNA content analysis. Furthermore, we demonstrate that sample storage overnight without fixation for later analysis on the flow cytometer is possible without compromising results. Application of the method to the assessment of the differential proliferative responses of lymphocyte subsets of human peripheral blood mononuclear cells (PBMC) that were costimulated with CD3 and with CD28.2 is presented.