Commentary: Defining the Cytokine Storm Syndrome of COVID-19: Role of the Clinical Laboratory

Coronaviruses are typically single stranded RNA viruses. A novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), first reported in December, 2019 has now resulted in a pandemic with a high mortality referred to as COVID19 disease. This highly infectious disorder of COVID19 emanated from Wuhan, China and according to the Johns Hopkins University Resource Center at the time of submission (6/22/20) has affected 8.99 million persons globally with a mortality of 468,907. The US appears to endure the major brunt of this onslaught with over 2.3 million cases and a mortality of 119,997. Whilst the majority of patients contracting this virus have mild disease, around 20% appear to have severe disease affecting the lung which can progress from pneumonia to acute respiratory distress syndrome (ARDS) that could eventually require ventilator support and finally multi organ failure ensuing [1].

Studies suggest that SARS-CoV-2 enters human cells by attaching to a cell surface receptor called angiotensin converting enzyme II (ACE2) and utilizing proteases including transmembrane serine protease 2 (TMPRSS2). However, ARDS with cytokine storm starts to appear in the later phase of infection even when there might be a decreasing in viremia [2]. Thus, there must be additional pathobiological pathways responsible for the cytokine storm.

When encountering a pathogen the body usually mounts an acute phase response [3] and this also occurs with COVID19. However with COVID19 there appears to be a dysregulated inflammatory response comprising increasing number of neutrophils and other cells with a characteristic lymphopenia. Chen et al, in 21 hospitalized patients in Wuhan province, reported that the absolute numbers of T lymphocytes, CD4+ T cells, and CD8+ T cells as well as IFN-gamma production by CD4+ T cells were decreased in COVID-19 infected patients and were commensurate with the severity of disease [4]. These cellular impairments together with decreases in regulatory T cells may result in the aggravated and enhanced inflammatory response which results in the genesis of the Cytokine Storm Syndrome (CSS). It appears that COVID19 through certain pathogen-associated molecular pattern motifs is recognized by pattern recognition receptors such as retinoic acid-inducible gene I-(RIG-1)-Like receptors (RLRs) and certain Toll-like receptors (TLRs) [5]. The activation of RLR results in a signal transduction pathway culminating in the production of Type 1 interferons (IFN), alpha and beta. Type 1 interferons are key components of the immediate antiviral response, and low levels of systemic IFN levels are found in COVID 19 patients, and appear to correlate with severe COVID-19 disease [6]. All of these events results in exaggerated and exuberant pro-inflammatory responses due to the activation of numerous immune cells including macrophages, neutrophils, dendritic cells, lymphocytes etc [6]. Another possibility is that since decreased ACE2 receptors results in increase in angiotensin-II, which in turn can trigger a cascade of inflammation involving NFKb and STAT pathways promoting cytokines, including IL-6, resulting in excessive activation and CSS [2].

SARS-CoV-2 like other coronaviruses could also engage numerous TLRs including TLR2, TLR3, and TLR4 etc however, confirmatory data has not been published in humans. Engagement of certain TLRs result in an increase in nuclear factor-Kappa B (NFkB) with the secretion of multiple inflammatory biomediators seen in COVID19 such as Interleukin (IL-1), IL-6,IL-8(CXCL8), tumor necrosis factor alpha (TNFa), monocyte chemoattractant protein (MCP-1), IFN gamma induced protein (IP)-10 also referred to as CXCL10 [7]. It is important to emphasize that the patients with CSS do attempt to mount an anti-inflammatory response to COVID19 which manifests by increased levels if IL-10, IL-4, IL-RA etc.

There is clearly a cytokine storm syndrome in patients with COVID19 which appears to correlate with disease severity as reviewed recently by England et al based on the original reports from China [8]. From the published literature there appears to a plethora of biomediators of inflammation including Interleukin(IL)-1, IL-2, soluble IL-2 receptor, IL-6, IL-7, IL-8, IL-10, IP-10, IL-17, TNF, G-CSF, MCP-1, MIP-1 alpha that have been reported [4,8–11]. Some of these appear to correlate with disease severity including ARDS, admission to the Intensive Care Unit and death [4,8–11]. Two well characterized downstream proteins of the pro-inflammatory response, C-reactive protein and ferritin, are also elevated in COVID19 and predict disease severity and progression of ARDS [8–11]. Interestingly, a biomarker of coagulation, D-dimer is also elevated and predicts progression to ARDS and disease severity [8–11].

New York City was a major epicenter of COVID19 in the United States. It is interesting that the major comorbidities reported in a study by Richardson et al [12] in hospitalized patients with COVID19 in New York included hypertension (57%), obesity (42%) and diabetes (34%). As we have reported previously, all three conditions are associated with a pro-inflammatory state and could have possibly primed the CSS but this data was not reported by Richardson et al.

Whilst there might be overlap with both secondary hemophagocytic lymphohistiocytosis (sHGH) and macrophage activation syndrome there are clear differences in the manifestation of CSS in COVID19 patients including a proclivity for the lung, neutrophilia, thrombosis, and absence of classical organomegaly of these syndromes [8]. However genetic studies in patients with COVID19 with CSS might prove very instructive about the pathobiology of CSS of COVID-19 and potential novel therapeutic strategies.

CSS appears to have an important role in ARDS, multi-organ failure and death. The adjunctive role of available focused immunotherapies targeting these biomediators of inflammation is very enticing. However given at the CSS stage the patients with COVID19 are very sick, we are in agreement with the National Institute of Health (NIH) guidelines [13] that controlled clinical trials need to be conducted showing benefit and safety before they are considered mainstream therapies. In this regard, strategies against IL-6 appear to be most favored based on emerging data with the monoclonal antibody against the IL-6 receptor, Tocilizumab. Also the race for a viable vaccine appears to be gaining momentum and Remdesivir has emerged as an antiviral that has some benefit in a controlled clinical trial and is now approved as a therapy [14]. In pediatrics, there is the emergency of a multisystem inflammatory syndrome in older kids with a Kawasaki disease like symptomatology progressing to shock and multiorgan failure and associated late onset inflammation like that seen in adult CSS, but clearly needs further study [15].

How as clinical pathologists can we provide a working definition of CSS of COVID19 for laboratorians and clinicians caring for these patients in hospital? In this regard, England et al [8] have provided a working model for COVID19-CSS; ferritin >1000ug/L and a CRP>100 mg/L coupled with the clinical presentation. We believe that the levels of both CRP and ferritin are too high and based on the published literature will possibly miss early CSS. Given the issues with the standardization of the surfeit of cytokines and chemokines reported in COVID19, we would add only an elevated IL-6 by the Roche assay (which is FDA approved) and D-dimer (>0.5 mg/L), a routine laboratory test in clinical laboratories. The importance of D-dimer should be no surprise given the nexus between inflammation and thrombosis.

In conclusion, we submit based on the limited data and adhering to stringent criteria required in our clinical laboratories for test reporting that in addition to the clinical presentation including lymphopenia our laboratory tests for a working diagnosis for COVID19-CSS includes elevated levels of CRP, ferritin, IL-6 (FDA-approved assays only) and D-dimer levels. In Table 1 we present concentrations we believe that need to be exceeded to qualify for a diagnosis of CSS of COVID-19[16]. Obviously as more data emerges especially from Europe and the USA our working diagnosis will need to be refined.

Table 1.

Laboratory Abnormalities Supporting a Working Diagnosis of Cytokine Storm Syndrome of COVID-19.

Parameter	Concentration
C-Reactive Protein	>50 mg/L
Ferritin	>700 ug/L
D-Dimer	>0.5 mg/L
Interleukin-6 (FDA approved assay)	Above the reference range (>7pg/ml)