Cytometric bead array to measure six cytokines in twenty-five microliters of serum.

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 70 ng/L (2 ) or 18 ng/L (1 ) for infection, and values 10 000 ng/L have been reported (1 ). IL-6 175 ng/L is predictive of sepsis, and values 747 ng/L are predictive for pneumonia (3 ). IL-6 is also believed to be predictive of necrotizing enterocolitis (3 ). IL-10 420 ng/L correlates with neonatal death (3 ). The ELISAs commonly used for cytokine detection require 50–100 L of serum ( 100–200 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 proand 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 2–3 logs. Recently, fluorescent bead array assays, such as the Cytometric Bead Array (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 antigens. We applied this system to pediatric samples and compared the results with those of standard ELISA analyses. The study was approved by the ethics committee of the Fig. 1. Calibration curves for the ELISAs and the CBA.