Quantitation of Biomarkers Using Flow Cytometry in Clinical Pathology: Comparative Sensitivity and Reliability of Multi-Analytes Platform and Multiplex Cytometry in the Quantitation of Circulating Cytokines
Stricker-Krongrad, A., Shoemake, C., Zhong, M., Liu, J., Bouchard, G.F.
Background: Circulating Th1/Th2/Th17 cytokines IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, and
IL-17a become elevated in response to inflammation and are key regulators of immune
responses. Measuring the expression profiles of cytokines is important in monitoring
polarization of the immune response; results should be independent of the quantitation
methods if they are to be accepted as validated clinical pathology biomarkers.
Objective: The aim of this study was to evaluate the effect of quantitation methods on
the detection of biomarkers of inflammation.
Methods: Female C57BL6 mice were treated orally with vehicle or dexamethasone,
then challenged with lipopolysaccharide (LPS) IV 1 – 1.5 hours later. At 0.5, 1, 2, 4, and
6 hours after LPS challenge, blood samples were collected and plasma was analyzed
with the Rodent MAP V3.0 Antigens assay or the Mouse Cytokine Panels A & B assay
(Myriad-RBM; Luminex platform), and the Mouse Th1/Th2/Th17 cytokines (Becton-
Dickinson: BD CBA) assay on a BD Accuri C6 flow cytometer platform. Results were
compared between assays.
Results: Reproducible quantitation of circulating TNF-α and IL-6 levels were obtained
with assays from both Myriad-RBM and BD Biosciences. The BD CBA cytokine assay was
not as sensitive as the Myriad-RBM assays in detecting and quantitating circulating IL-2
and IL-4 and IL-17A levels, but was more sensitive and reliable in measuring circulating
IFN-γ levels. Reliable circulating IL-4 measurements were not achieved by either assay.
Conclusions: Quantitation of circulating biomarkers of inflammation can be achieved
using multiplexed flow cytometry, but careful considerations have to be made for the
validation of assays.