Monitoring Cytokine Storms in Real Time

Pathogens, autoimmune and malignant diseases, genetic disorders, and some therapeutic interventions can lead to life-threatening systemic inflammatory syndromes. The common feature of cytokine release syndrome (CRS), or, in particularly severe cases, cytokine storm (CS), is a massive release of cytokines due to excessive activation of immune cells.¹

CS has been shown to play an important role in severe COVID-19 cases and reported as a major cause of death.² Pathogenesis heightens with immune dysregulation that leads to uncontrolled multisystem inflammatory response, caused by overproduction of pro-inflammatory cytokines.³ The increase in inflammatory mediators is correlated with a reduction of innate and adaptive cytotoxic antiviral function. Monitoring and accurate knowledge regarding clinical worsening is crucial to choosing appropriate interventions aimed to reduce mortality.⁴

Real-time cytokine measurement

An approach to monitoring cytokines is Ella, a fully-automated, microfluidic platform that supports multiplex sandwich immunoassays. “Simply add your samples and wash buffer and then let it run. Ella automates all aspects of the assay run and provides fully analyzed results in 90 minutes,” said Steve Crouse, senior vp & GM ASD at Bio-Techne. “Ella provides triplicate results for each sample and minimizes pipetting by offering a factory-generated standard curve.”

All Bio-Techne CS assays have coefficients of variation (CVs) below 10%, which leads to consistency when Ella is used as a distributed platform. “We have demonstrated that the performance of the sample on any of our lots of CS plates used on any of our instruments will be the same,” said Crouse. “Consistent data are important for monitoring a patient over time as well as comparing results from different locations.”

Bio-Techne collaborated with Mount Sinai’s Human Immune Monitoring Center, led by Miriam Merad, MD, PhD, Mount Sinai endowed professor in cancer immunology, and Sacha Gnajtic, PhD, associate professor of immunology and hematology/oncology, to identify cytokines that produced the most adverse response in a COVID-19 infection and to develop the CS test.⁵

The 4-plex CS panel designed for COVID-19 quantifies IL-lβ, IL-6, IL-8, and TNFα. “Along with fixed panels like our CS panel, we offer numerous cytokine and chemokine assays that can be combined in different ways up to an 8-plex to support different immune monitoring strategies,” said Crouse.

The COVID-19 pandemic produced the most research on the role of CS and the importance of monitoring cytokines to adjust treatment in severely ill patients. “This is starting to transition,” said Crouse. “Cell therapy is probably the largest driver, but other disease states or therapeutic approaches also require monitoring. However, to use monitoring broadly as a standard of care, it needs to clearly show that results can lead to a specific clinical intervention or outcome. This is not yet well defined in most instances but current research efforts point to immune monitoring becoming more prevalent over the next couple of years.”

Ella is a broad-based immunoassay platform with over 200 available markers and is used in a range of applications such as clinical trials for new drugs since it can be distributed to multiple clinical research organizations (CROs) for global trials. In cell therapy, it can also be used to look at interferon-γ as an indicator of T cell activation, for QC release of the cell therapy, in addition to patient monitoring. “From every instrument and every lot of assays you can trust that data are consistent, reproducible, and comparable over long periods and across multiple sites,” said Crouse.

Other approaches to monitoring

Immunoassays or ELISAs are typically used to measure cytokines. Other sandwich immunoassays also allow for cytokine quantification. “It is hard to achieve the same level of data precision as the 90-minute Ella CS assay while still maintaining sensitivity. A very sensitive platform is imperative for the detection of endogenous cytokine levels,” said Crouse.

A University of Michigan team also wanted to address the gap in monitoring cytokines in severely ill COVID-19 patients and reported on a microfluidic digital immunoassay platform under development that enables rapid 4-plex measurement of cytokines in COVID-19 patient serum. The assay employs single-molecule counting for an antibody sandwich immune-complex formation quenched at an early pre-equilibrium state resulting in a detection limit < 0.4 pg/mL and a linear dynamic range of 10³ while requiring an assay incubation time as short as 9 min.⁶

References

1. Jarczak D, Nierhaus A. Cytokine Storm—Definition, Causes, and Implications. J. Mol. Sci. 2022, 23, 11740. doi: 10.3390/ijms231911740
2. Mandel M, Harari G, Gurevich M, Achiron A. Cytokine Prediction of Mortality in COVID-19 Patients, Cytokine. 2020 Oct;134:155190. doi: 10.1016/j.cyto.2020.155190. Epub 2020 Jul 10.
3. Shcherbak SG, Anisenkova AY, Mosenko SV, Oleg S GlotovOS, Chernov AN, et al. Basic Predictive Risk Factors for Cytokine Storms in COVID-19 Patients. Front Immunol. 2021 Nov 10;12:745515.  doi: 10.3389/fimmu.2021.745515. eCollection 2021.
4. Bordoni V, Sacchi A, Cimini E,  Notari S,  Grassi G, et al. An Inflammatory Profile Correlates with Decreased Frequency of Cytotoxic Cells in Coronavirus Disease 2019. Clin Infect Dis. 2020 Nov 19;71(16):2272-2275.  doi: 10.1093/cid/ciaa577
5. Del Valle DM, Kim-Schulze S, Huang HH, et al. An Inflammatory Cytokine Signature Predicts COVID-19 Severity and Survival. Nat Med 26, 1636–1643 (2020). doi: 10.1038/s41591-020-1051-9
6. Song Y, Ye Y, Su SH, Stephens A, Cai T, et al. Digital Protein Microarray for COVID-19 Cytokine Storm Monitoring. Lab Chip. 2021 Jan 21; 21(2): 331–343. Published online 2020 Nov 19. doi: 10.39/d0lc00678e.