Flow cytometric microsphere-based immunoassay: analysis of secreted cytokines in whole-blood samples from asthmatics.

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-gamma (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-gamma than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.

Assessment of the Th1/Th2 paradigm in whole blood in atopy and asthma. Increased IFN-gamma-producing CD8(+) T cells in asthma.

Atopy is characterized by an immune system that is biased to T helper cell, type 2 (Th2) activation. This condition predisposes to asthma, a disease in which a Th2 activation was found in blood and lungs. However, most blood studies have considered purified cells, which might give an incomplete view of immune reactions. In this study, we assessed in whole blood cultures the Th1/Th2 paradigm in atopy and asthma. Sixty-nine subjects (31 atopic asthmatics, six nonatopic asthmatics, 13 atopic nonasthmatics, and 19 control subjects) were included in this study. Interleukin-4 (IL-4), interferon gamma (IFN-gamma), and IL-12 were assayed in stimulated whole blood culture supernatants by using a flow cytometer microsphere-based assay. Intracellular IL-4 and IFN-gamma were detected in T cells and CD8(+) T cells by flow cytometry. Atopy was characterized by a higher production of IL-4, which was correlated to total IgE levels, and by an impairment of the T-cell capacity to produce IFN-gamma. This impairment was correlated to the number of positive skin tests. In asthma, the overproduction of IL-4 was still found if atopy was present. Unexpectedly, an overproduction of IFN-gamma was found, which was related to an increased capacity of CD8(+) T cells to produce IFN-gamma. The number of IFN-gamma-producing CD8(+) T cells was related to asthma severity, to bronchial hyperresponsiveness, and to blood eosinophilia. In addition, this number was correlated to IL-12 production. These results show that in addition to the well-known Th2 inflammation in asthma, there are IFN-gamma-producing CD8(+) T cells in the blood, possibly controlled by IL-12.

Simultaneous detection of multiplex-amplified human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA using a flow cytometer microsphere-based hybridization assay.

The feasibility of performing a multiplex assay for the detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNAs and hepatitis B virus (HBV) DNA is demonstrated. This assay is based (i) on the coamplification of a 142-bp fragment from the gag region of the HIV-1 genome and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment from the 5' noncoding region of the HCV genome, and a 104-bp fragment from the pre-C and C gene regions of the HBV genome, using three sets of specific primers; (ii) on the capacity of these four biotinylated PCR products to hybridize to their specific oligonucleotide probe-coated microspheres; and (iii) on the ability of the flow cytometer to discriminate between distinct fluorescent-microsphere categories. Absence of cross-hybridization between the unrelated oligonucleotide probes and PCR products generated by the multiplex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infectious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that multiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quantitation and detection of the major viral agents of infectious diseases in a single plasma sample.

Relationships between natural T cells, atopy, IgE levels, and IL-4 production.

BACKGROUND: Th2 cells govern allergic disorders. Mechanisms leading to the Th2 commitment are dominated by the requirement of IL-4. A potential source of this triggering IL-4 could be the CD4 + subset of a small population of T cells, natural T (NT) cells. Indeed, this subset is involved in IgE responses in mice and produces promptly high amounts of IL-4 in both mice and man. METHODS: NT cells were identified in peripheral blood by flow cytometry with antibodies against Valpha24 and Vbeta11, recognizing the T-cell receptor specific for NT cells. Simultaneous staining with anti-CD3, anti-CD4, or anti-CD8 antibodies was performed. The frequency of NT cells in man was studied according to the presence of atopy defined by the positivity of skin tests, according to total IgE levels in serum, and according to IL-4 concentration of whole-blood culture supernatants determined by a flow cytometer microsphere-based assay. RESULTS: Seventy subjects were included, of whom 30 were atopic. The number of CD4+ NT cells was higher in atopics than in nonatopics (P=0.009). This number was correlated to the total IgE levels (r = 0.34, P = 0.03). In addition, the number of CD4 + NT cells, but also of CD8 + NT cells, was correlated to the levels of IL-4 (r=0.71, P=0.01, and r=0.6, P=0.03, respectively). CONCLUSIONS: These results show that the number of NT cells, particularly the CD4+ subset, is related to atopy, IL-4 production, and IgE levels. Therefore, this population of T cells is likely to play a role in the Th2 commitment initiating atopic diseases.