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
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 (BectonDickinson: 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.
A. Stricker-Krongrad, PhD; C. Shoemake, DVM; M. Zhong, PhD; J. Liu, PhD; G. Bouchard, DVM
Sinclair Research Center, LLC, Auxvasse, MO
LPS administration induces an acute inflammation that is associated with increases of a number of inflammatory cytokines in the peripheral blood of treated animals. Dexamethasone inhibits the effect of LPS on cytokine synthesis. IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, and IL-17A become elevated in response to inflammation; these cytokines are also key regulators of immune responses1 . IL-2, IFN-γ, and TNF-α are associated with the Th1 response. IL-4, IL-6, and IL-10 are associated with the Th2 response. IL-6 and IL-17A are associated with the Th17 response. Measuring the expression profiles of these cytokines is important in monitoring the polarization of the immune response and the results should therefore be independent of the quantitation methods. To evaluate the effect of quantitation methods, the 7 circulating Th1/Th2/Th17 cytokines were quantified in plasma of LPS-treated mice with two different multiplex platforms (Myriad-RBM and BD Biosciences).
Materials & Methods
Figure 1 Legend:
- Female C57BL6 mice, 6 – 9 months old, were dosed orally with either vehicle (0.5% methylcellulose in water) or dexamethasone (5 mg/kg) LPS challenge was administered IV 1 – 1.5 hours later
- LPS challenge was administered IV 1 – 1.5 hours later
- Blood was drawn at 0.5, 1, 2, 4, and 6 hours post-LPS challenge
- Samples were processed to collect plasma
- Samples were 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 assay (Becton-Dickson: BD CBA) on a BD Accuri C6 flow cytometer platform
The plasma samples analyzed with the Myriad-RBM Luminex platform were analyzed with the Mouse Cytokine Panel A&B at the 4-hour time point, while samples of the other time points were analyzed with the Rodent MAP V3.0 Antigen assay. Plasma samples from the 2 and 4-hour time points only were analyzed with the BD CBA cytokine assay. Numbers in red indicate levels below the lower limit of quantification.
Download the poster to view Figure 1 and Tables 1-4
- None of the 7 Th1/Th2/Th17 cytokines were detected in plasma of untreated mice with BD CBA assay (data not shown).
- Variations in concentrations of cytokines were consistent and acceptable when determined in diluted plasma samples with BD CBA (Table 4).
- IL-6 was the only cytokine that was quantified comparably with the BD CBA assay and the Myriad-RBM assays, and was also the only cytokine that should be quantitated in diluted plasma with the BD CBA assay due to levels exceeding the upper limit of quantification of the assay.
- In the BD CBA assay, the time course effect of LPS on plasma TNF-α was consistent with previous reports2,3, and the dexamethasone inhibitions were comparable between the 2 and 4-hour plasma samples. In the Myriad-RBM platform, while LPS was shown to increase plasma TNF-α to similar levels in both 2 and 4-hour samples, dexamethasone had much weaker inhibition in 4-hour samples than in 2-hour samples. (Tables 2 and 3)
- Plasma IFN-γ was increased in the 4-hour samples as previously reported4 following LPS challenge in the BD CBA assay, whereas an opposite trend was observed for the IFN-γ secretion in the Myriad-RBM assays (Table 2).
- IL-10 was quantified at lower plasma levels with a less potent inhibition of dexamethasone in BD CBA assay than in Myriad-RBM assay. No other differences were found for IL-10 quantification between the two assay platforms. (Tables 2 and 3)
- IL-4, the signature Th2 cytokine1 , was supposed to not be induced by LPS treatment. The lack of signal in IL-4 quantification with BD CBA assay reflected the specificity of this kit in IL-4 measurement
- Raphael I, Nalawade S, Eagar TN and Forsthuber TG. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine. 2015; 74:5 – 17.
- Dinges MM and Schlievert PM. Role of T cells and gamma interferon during induction of hypersensitivity to lipopolysaccharide by toxic shock syndrome toxin 1 in mice. Infect Immun. 2001; 69: 1256 – 64.
- Baumgarten G, Knuefermann P, Nozaki N, et al. In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of Toll-Like Receptor-4. J Infect Dis. 2001; 183: 1617 – 24.
- Pini M, Castellanos KJ, Rhodes DH and Fantuzzi G. Obesity and IL-6 interact in modulating the response to endotoxemia in mice. Cytokine. 2013; 61: 71 – 7.