Cytokine Quantitation: Challenges and Opportunities

July 26, 2023

Cytokines are cell signaling proteins that are produced by immune, inflammatory, and other cells. They are pertinent biomarkers in bodily fluids for many serious diseases, and thus are important in medical diagnostics (Li et al., 2023). For example, the multiple organ failure in severe cases of Covid-19 and other conditions corresponds to excessive cytokine production (Cron et al., 2022). The prominence of cytokine research might be best illustrated by its global market size: $83.3 billion in 2022, predicted to reach $130.5 billion by 2028 (IMARC Group, 2022).

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To better understand the biology of cytokines and to improve the safety of corresponding biomedical applications, it’s critical to have a reliable means of cytokine quantitation (Deckers et al., 2023). Here, we provide an overview of pertinent issues and industry solutions to cytokine quantitation, and current research that required reliable measurements of cytokine levels.

An unstable needle in a haystack

Cytokine quantitation has many challenges (Liu et al., 2021). For example, in vivo cytokine concentration is typically quite low: in the picomolar range. Furthermore, this concentration is highly irregular because corresponding secretion processes are transient and dynamic. Cytokine half-life is brief: minutes to hours. Additionally, as discussed next, sample collection and handling can affect cytokine stability.

For reliable cytokine quantitation, it’s good practice to minimize the time between collecting a blood sample and processing it in the laboratory. Leukocytes, for example, can secrete substantial quantities of cytokines prior to separation of plasma from whole blood. Furthermore, the chosen method for separating serum from plasma—such as using heparin versus EDTA anticoagulants—can alter cytokine levels. Although heparin can induce release of cytokines from monocytes, EDTA does not. Additionally, the chosen method for storing a blood sample can affect its cytokine concentration. For example, storing whole blood in a refrigerator for three days generally stabilizes it against the comparatively rapid cytokine degradation that’s observed at room temperature. Cytokine levels in a sample are generally stable for no more than three freeze–thaw cycles.

Industry solutions to cytokine quantitation

What tools are available for cytokine quantitation? The targeted protein biomarker panels from Olink Proteomics (Olink, n.d.) require only 1 µL of biological sample and test against pertinent biomarkers, as evaluated by standard bioinformatics databases. The biochemical basis of the panel is proximity extension assay technology. Here, two complimentary DNA-labeled antibodies bind to the cytokine (antigen) of interest in the sample. The close proximity of the DNA labels upon antigen binding enables hybridization, which in turn enables amplification and then detection by quantitative PCR or next-generation sequencing. The sensitivity of the assay is comparable with or better than that of ELISA.

“The Olink technology platform scales from the high-plex product Olink Explore, to mid-plex Olink Target 96 and Target 48, down to customized low-plex Olink Focus and Olink Flex,” reports Olink’s Strategic Portfolio Director Dr. Dominik Muggenhumer. “For example, the Olink Target 48 cytokine panel simultaneously detects and quantifies 45 protein biomarkers involved in key pathways associated with cytokine signaling. Olink Flex allows researchers to build their own custom biomarker panel with up to 21 protein biomarkers that can be freely selected from a broad library of key cytokines, chemokines, and many other inflammatory markers.”

Representative cytokine research

Cytokine quantitation is essential to many lines of work. For example, Sparks et al. (2023) tested the effects of acute viral infections on immune system homeostasis and future viral challenges. Their study was on the response of 33 recovered mild, non-hospitalized Covid-19 patients (who did not have confounding comorbidities) to influenza vaccination, on average 151 days after diagnosis. There were sex-specific differences, such as males exhibiting some stronger immune responses (compared with females) for 28 days after vaccination, in part because of males’ enhanced ability to produce interferon cytokines. This sexually dimorphic response qualitatively differs from the immune response of the 40 control patients who had not contracted Covid-19. Sparks et al.’s work also reveals that even mild viral infections can alter the immune response for months afterward.

Vlachogiannis et al. (2023) tested the role of chemokines (small cytokines) in subarachnoid hemorrhage: inflammation of the space surrounding the brain. In a study of 29 patients, they found that elevated levels of certain chemokines in the cerebrospinal fluid 10 days after injury corresponded to more severe clinical outcomes 1 year post-injury. Thus, cytokine biomarkers might be useful for predicting the prognosis of recovery from some traumatic brain injuries, and guide patient treatment.

Bao et al. (2023) tested the association between a positive diagnosis of autism spectrum disorder and an inflammatory-pertinent protein panel. In a study of 31 people with autism and 33 controls, they found that abnormal levels of four proteins, such as STAMPB, corresponded to good diagnostic accuracy for autism. STAMPB is a deubiquitinase that modulates cytokine secretion through the NLRP3 inflammasome, an essential component of the innate immune system. Applying these results to early-stage autism diagnosis that does not involve behavioral analysis requires further study, as does confirming that autism corresponds to inflammatory dysfunction.

Two main factors have hindered commercial applications of cytokine research. One, the complex biology of cytokines often hinders development of safe therapeutics (Saxton et al., 2023). Two, research tends to focus on a rather limited set of cytokines and cell systems. Thus, predicting cytokine signaling cascades and identifying new roles for cytokines from omics data remains challenging (Jiang et al., 2021). We recommend that you consult with established practitioners for up-to-date developments and modern industry procedures.