Alissa Minkovsky, Limor Cohen, Stacy E F Melanson, David R Walt
American Journal of Clinical Pathology
Background: Changes in local cytokine levels accompany immune-mediated anti-tumor responses. However, cytokine responses are challenging to measure systemically, outside the tumor microenvironment. The lack of sensitivity in the sub-pg/mL range with existing assays is an obstacle in developing peripheral blood-based biomarkers for the field of immuno-oncology. The objective of this study was to clinically validate a new ultra-sensitive immunoassay platform with EDTA plasma patient specimens for eventual translation as a blood-based biomarker in immuno-oncology.
Methods: Here, we describe the validation of ultra-sensitive single molecule array (Simoa) assays for IL-2, IL-6, IL-10, and IFNγ. Simoa cytokine assays have been carried out using serum but no studies have been carried out using plasma, which is more readily processed and prevents platelet activation, which can alter cytokine levels ex vivo. Ninety patient EDTA plasma specimens were obtained from excess CBC material. A subset of patient samples used was expected to be enriched for high endogenous cytokine levels by selecting for patients with high CRP levels. Up to 2.5 mL of whole blood was centrifuged at 2,000 x g for 15 minutes, and the supernatant plasma was then aliquoted and stored at –80°C. Either 29 uL (IL-6, IL-10) or 42 µl (IL-2, IFNγ) of thawed plasma was used for each Simoa assay. Simoa assays were performed using antibody pairs to form enzyme-labeled immunocomplexes on beads and were detected using single molecule counting with the HD-2 Analyzer (Quanterix, Lexington, MA). We evaluated various analytical parameters (linearity, precision, accuracy, lower limit of quantification, cross-reactivity, interferences) of the assay and assessed analyte stability to optimize for eventual clinical implementation.
Results: The Simoa assays for IL-2, IL-6, IL-10, and IFNγ were both linear and precise. The lower limit of quantitation was 0.01 pg/mL for IL-6 and IL-10, and 0.04 pg/mL for IL-2 and IFNγ. None of the four cytokine assays cross-reacted with up to 1,000 pg/mL recombinant protein. All analytes were stable in EDTA plasma at room temperature for up to six hours with the exception of IL-6; levels decreased by approximately 10% at six hours. There was no interference from hemolysis, icterus, and lipemia. Vigorous handling of whole blood specimens and transport by pneumatic tube did not significantly affect analyte levels.
Conclusions: IL-2, IL-6, IL-10, and IFNγ are largely stable in EDTA plasma at room temperature for up to four hours after blood draw. Lower limits of quantitation by Simoa surpass those reported for ELISAs by an average of 722-fold for the four analytes measured. Simoa immunoassays are a robust technique to measure ultra-low concentrations of cytokines with high precision. Simoa cytokine immunoassays represent a promising approach for monitoring anti-tumor immune responses in peripheral blood.