No abstract

Infections and sepsis are among the most common reasons for neonatal morbidity and mortality. Early diagnosis is difficult because clinical presentation is highly variable and signs are often subtle and common to a variety of conditions. Among the proposed early indicators of infection and sepsis are serum concentrations of interleukin (IL)-6, IL-8, and IL-10. It is believed that IL-8 is a sensitive indicator of infection and that high concentrations of IL-6 and IL-10 are indicators of sepsis and predictors of mortality (1-3 ). The concentrations of each of these cytokines in serum vary by several orders of magnitude (1-3 ). Literature-reported cutoff values for IL-8 are Ͼ70 ng/L (2 ) or Ͼ18 ng/L (1 ) for infection, and values Ͼ10 000 ng/L have been reported (1 ). IL-6 Ͼ175 ng/L is predictive of sepsis, and values Ͼ747 ng/L are predictive for pneumonia (3 ). IL-6 is also believed to be predictive of necrotizing enterocolitis (3 ). IL-10 Ͼ420 ng/L correlates with neonatal death (3 ).The ELISAs commonly used for cytokine detection require 50 -100 L of serum (ϳ100 -200 L of peripheral blood in the neonate) per cytokine. To determine the stage of an infection, measurement of several cytokines at multiple time points can be of importance (3 ). Combining pro-and antiinflammatory cytokines in a single assay yields an overall view on the patient's inflammatory status; may allow differentiation among infection, sepsis, and enterocolitis; and thus may improve diagnostic accuracy. In neonates, however, particularly preterm neonates, such combined measurements are often hampered by lack of sufficient obtainable blood (1 ). Furthermore, although the ELISAs are adequate for measuring these cytokines, they often require multiple dilutions to cover a wide concentration range because their dynamic range is only ϳ2-3 logs.Recently, fluorescent bead array assays, such as the Cytometric Bead Array TM (CBA; BD Biosciences, San Jose, CA) (4 ), that involve measurement by flow cytometry have become available, and they have widened the assay dynamic range greatly (4,5 ). This in turn improved efficiency by decreasing the required number of dilutions. These assays are multiplexed such that numerous substances are measured simultaneously in a single well. The CBA assay consists of a mixture of six types of beads uniform in size but containing different fluorescence intensities of a red-emitting dye. A different capture antibody (Ab) against one of six cytokines is covalently coupled to each type of bead. Cytokines bound to these Abs are detected by use of Abs labeled with phycoerythrin. The fluorescence intensity measured with phycoerythrin is proportional to the cytokine concentration in the sample and is quantified from a calibration curve. The wider dynamic range is attributable to the use of florescence detection, which has a range of 4 -5 logs, and the highly efficient capturing capability of the Ab-linked particles (4 ). Particles in suspension are much more efficient than the static surface of ELISA well bottoms in capturing antig...

No abstract

The multiple cellular and soluble elements of the immune system respond in a coordinated way, orchestrated by cytokines, to preserve the integrity of the organism. In this study, we describe a new and unique whole blood method that, with minimal sample manipulation, allows an overall evaluation of immune responses by simultaneously measuring cell activation and cytokine secretion. The identification of cells actively secreting cytokines is based on the stabilization of tumor necrosis factor a (TNFa) at the cell surface through the use of a specific inhibitor of the TNFa-converting enzyme. This inhibitor does not affect the release of cytokines other than TNFa and makes it possible to assess, in the same measurement, the phenotype of TNFa þ -secreting cells and quantify multiple secreted cytokines by using a specific and highly sensitive flow cytometry-based bead immunoassay. Upon stimulation of normal peripheral blood samples with either phorbol 12-myristate 13 acetate (PMA) plus ionomycin or lipopolysaccharide (LPS), both the number of TNFa þ cells and the amount of secreted cytokines progressively increased, the former becoming detectable first. After stimulation for 3 h with PMA plus ionomycin, cellular responses were associated with surface TNFa expression on the majority of CD3 þ T cells and secretion of Th1-associated cytokines: interferon c, interleukin (IL)-2, and to a lesser extent IL4. In turn, stimulation with LPS induced a response mainly by inflammatory cells. After 4 h of LPS-stimulation, the majority of CD14 þ monocytes showed surface TNFa expression; in parallel, high amounts of soluble IL1b, IL6, and IL8 became detectable. Likewise, stimulation of blood samples with cytomegalovirus (CMV) lysates induced viralspecific immune responses detectable in seropositive but not seronegative volunteers; such responses were associated with the detection of increased numbers of TNFa þ monocytes, TNFa þ /CD8 þ T cells and TNFa þ /CD8 À T lymphocytes in association with an increased secretion of IFNc, IL6 and TNFa.

Analytical cytometry has significant potential beyond cellular analysis. The inherent capability of flow cytometers to efficiently discriminate between uniformly sized particles based on their intrinsic properties provides the foundation for multiplex bead assays. The technology can be exploited in designing immunoassays, Western blot-like antibody assays, and nucleic acid hybridization assays. This chapter focuses on immunoassay applications. The multiplex bead assays have recently evolved as a new and increasingly popular area for flow cytometry, becoming a good alternative to enzyme-linked immunosorbent assay for efficient evaluation of panels of analytes. This chapter provides detailed information about two bead platforms, the BD Cytometric Bead Array kits and the BD Cytometric Bead Array Flex Set Assays.