Predictors of mortality in patients with COVID-19 by flow cytometry

Abstract

Despite the great impact of severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2), we still lack techniques that allow us to anticipate the natural history of the disease in order to avoid or shorten the clinical period of the disease. The values of nine cytokines were measured in COVID-19+ patients admitted to the Hospital Universitario Reina Sofía (HURS) using flow cytometry. The cytokines measured are IL-1ß, IL-6, MCP-1, IP-10, IL-10, IL-8, IL-12, IFN-γ and TNF-α. Given the absence of previous studies on cytokine values in healthy patients using the flow cytometry technique, and the low availability of resources in the first waves of COVID-19, a control group was lacking, all resources were employed for monitoring sick patients. However, this study has revealed a greater increase in two specific cytokines, which are also found to be higher than the rest in healthy patients: MCP-1 and IP-10, which are mainly responsible for cytokine storm and post-disease thrombosis.

Introduction

COVID-19 disease is a bilateral pneumonia caused by the SARS-CoV-2 virus (acronym for Severe Acute Respiratory Syndrome due to Coronavirus 2) that emerged in Wuhan, Hubei Province, China, in December 2019. Severe respiratory pathology refers to a serious medical condition that affects the respiratory system and causes severe impairment of normal breathing function. Examples of severe respiratory pathologies include chronic obstructive pulmonary disease (COPD), severe asthma, pneumonia, lung cancer, and acute respiratory distress syndrome (ARDS). These conditions can cause difficulty breathing, coughing, chest pain, and other symptoms, and can be life-threatening if left untreated. According to the U.S. Center for Disease Control and Prevention (CDC), transmission of the virus is primarily through direct contact or Flügge droplets, usually requiring a distance to the source of transmission of less than 1.5 m, for more than 15 min. Airborne transmission by prolonged exposure in an enclosed space without proper ventilation has been shown to be evident.

This trial will highlight the important implication of cytokine levels in the evolution and prognosis of the disease as a marker to be considered in order to implement the most appropriate treatment, anticipating the fatal outcome of the disease. Cytokines are a broad group of proteins, peptides or glycoproteins that are secreted by specific cells of the immune system, mainly Th lymphocytes and/or macrophages. They have a relevant role as regulators of immunity, inflammation and hematopoiesis, i.e. they are mediator molecules in these processes that are synthesized in different types of cells of diverse embryonic origin. Cytokines are produced during the effective phases of natural and specific immunity and serve to regulate immune responses. At the onset of infection, proinflammatory cytokines predominate, but in the resolution phase the balance is reversed and anti-inflammatory cytokines predominate, returning the immune system to its normal pre-process stage. The cytokine storm is, in short, an uncontrolled state of the immune response in which the imbalance between proinflammatory and anti-inflammatory mediators feeds back.

Determination of inflammatory biomarkers with the flow cytometer allows simultaneous quantification of up to twenty-five cytokines in a single sample. It is also possible to use pre-designed panels of cytokines or create your own customized panel to detect and quantify the inflammatory response. Among the pro-inflammatory cytokines we find IL-1ß, IL-8, IL-12, TNF-α, IFN-γ, MCP-1 and IP-10; IL-10 is the main anti-inflammatory cytokine; IL-6 has mixed action. In this article, samples of COVID-19+ patients from HURS are analyzed, assessing the levels of cytokines they present at different stages of disease progression to see the possible correlation between levels and disease progression.

Methods

The study was carried out on a cohort of patients admitted for severe respiratory pathology at the Hospital Universitario Reina Sofía (HURS) in Córdoba (Spain), between March 15, 2021 and June 10, 2021, with an average of six patients per day, giving a total of 384 patients. The subjects of the base cohort are COVID-19+ patients diagnosed by quantitative Polymerase Chain Reaction (qPCR) technique. More than 90% of the patients were older than 60 years, admitted to the Intensive Care Unit (ICU). The proportion of males was 55%, with a mean age of 65–70 years. EDTA-anticoagulated blood samples were centrifuged at 10,000 rpm for 10 min to separate the cell fraction from the plasma to be used for determination. The samples were obtained by peripheral blood. Each patient underwent periodic analytical follow-up with the determination of the cytokines IL-1ß, IL-6, MCP-1, IP-10, IL-10, IL-8, IL-12, IFN-γ and TNF-α by means of a specific flow cytometry panel. A panel of nine cytokines common to all COVID-19+ patients was performed and the positivity of the cytokines examined was assessed. The cytokines were analyzed daily, together with three controls (two positive and one negative) per batch. The technique used is based on a DAS sandwich ELISA. The BD CBA Human Soluble Protein Flex Set Capture Bead kit, by BD Biosciences, contains beads coated with monoclonal antibodies specifically directed against a particular cytokine. The beads also contain a unique mixture of two fluorochromes (APC and APC—Cy7) [1]. The kit too contains monoclonal antibodies directed against a particular cytokine conjugated to the PE fluorochrome (phycoerythrin). The fluorescence emitted by the fluorochrome mixture of the capture beads allows identification of which cytokine is involved, while the fluorescence emitted by the PE conjugated to the monoclonal antibody provides a quantitative measure of the amount of cytokine present in the sample. For this purpose, the data obtained were extrapolated using analysis software to a previously performed standard curve, named FCAP Array multiplex assay software. The values obtained on the cytometer, using the FCAP Array multiplex assay analysis software, are extrapolated to conventional units of measurement, most commonly fg/mL. The FCAP Array software can determine the existence of up to 72 analytes per sample, plus positive and/or negative controls. FCAP Array is a powerful, scientific analysis application designed for Cytometric Bead Array (CBA) data. The app contains tools for all of the jobs you need to easily and efficiently perform CBA data analysis. It enables laboratories to perform bioassays with an already extensive list of microbead applications far more easily and quickly than with other systems. The software is highly flexible as the technology can be customized to the user's specific bioassay needs. When FCAP Array is first installed, the bead library is empty. For BD CBA reagents, we recommend adding beads to the bead library by importing the XML file. After analysis in the software, a calibration curve is obtained (Fig. 4), which allows us to know the concentration of each cytokine in each patient, within the limit values, of each cytokine in each patient. The commercial house BD biosciences provides the sensitivity range which is from 274 to 200,000 fg/mL, marking the detection limit of the technique, concentrations lower or higher than these would not be reliable.

Fig. 4.

Calibration curve for IL-10. On the X-axis we observe the different tubes (Std001 for blank, and Std010 for TOP). On the Y-axis, the concentration in fg/mL of IL-10 for each tube is shown.

Calibration curves are obtained by serial 1:2 dilutions starting from the TOP" tube up to dilution 1:256 (Fig. 5).

Fig. 5.

Graphical representation of the performance of a calibration curve according to the protocol established by BD Bioscience. A TOP tube is shown in which the standards of the beads present in the kit and 4 mL of Assay Diluent will be added, with subsequent homogenization. Add 0.5 mL of Assay Diluent to each Eppendorf except the TOP. Transfer 1 mL from the TOP tube to the Eppendorf TOP, homogenize and transfer 0.5 mL from the Eppendorf TOP to the Eppendorf 1:2. Then transfer 0.5 mL from the Eppendorf 1:2 to the Eppendorf 1:4. Continue successively until the Eppendorf 1:256 is reached. Subsequently, transfer 25 μL to each Falcon tube, previously numbered, with its respective dilution. The WHITE tube should contain only Assay Diluent. Then, make a Mix Beads and Mix PE and carry out the protocol as if it were a normal sample.

The process of extrapolating data from a FCAP array multiplex assay software typically involves the following steps:

• 1Data import: The first step is to import the data from the FCAP array into the software. This can be done by selecting the appropriate file format, such as .fcs or .txt, and then importing the data into the software.
• 2Data analysis: Once the data is imported, it can be analyzed using various tools provided by the software. This may include data normalization, compensation, and gating to identify the specific populations of interest within the data.
• 3Data visualization: The software may provide various visualization tools, such as histograms, scatter plots, and heat maps, to help visualize and interpret the data. These tools can help identify trends, patterns, and outliers within the data.
• 4Data extraction: Once the data has been analyzed and visualized, it can be extracted and saved for further analysis or for use in other applications. This may involve exporting the data to a specific file format, such as .csv or .xlsx, or it may involve copying and pasting the data into another application.

It is important to note that the exact process of extrapolating data from a FCAP array multiplex assay software may vary depending on the specific software used, so it is important to consult the software's manual or documentation for specific instructions and guidelines.

Although the original protocol of the commercial company recommends obtaining a minimum of 300 events (beads) for each sample, in this study we have used a minimum of 1000 events per cytokine, in order to reduce the possibility of error in the extrapolation of the data and the mean fluorescence intensity values.

After obtaining the histograms and extrapolating them using calibration curves (Fig. 4), the negativity or positivity of each cytokine can be assessed independently. If positive, a quantitative value (fg/mL in the case of the kit used) was also obtained.

Statistical parameters

We start from an open and dynamic base cohort, in which the effect has already occurred (COVID-19 disease). The analytical values of these patients were measured and are expressed in fg/mL concentration. The epidemiological parameters used are the ODDs ratio and prevalence ratio, which assess the likelihood of a health condition or disease occurring in one population group versus the risk of it occurring in another.

Results

We propose IL-6 and IL-10 as possible indicators of the degree of dysregulation of the immune system, which translates into an uncontrolled and generalized inflammatory response that feeds back positively and can seriously damage various organs of our body, up to a fatal outcome. In Fig. 2, we can appreciate the histograms of positivity of the nine cytokines, highlighting the negativity (they are not elevated) of the anti-inflammatory interleukins (IL-6 and IL-10), which suggests a favorable result. In the cytometer we will obtain histograms, which allow us to know the negativity (cytokine values are low or normal) and/or positivity (cytokine values are elevated). The main limitation of this method is that it does not differentiate between a normal or low value, both are considered negative values.

Fig. 2.

Histograms of positivity and negativity of the panel of nine cytokines by flow cytometry. Interleukins IP-10, IL-8 and MCP-1 are positive, i.e. they are elevated. In contrast, interleukins IL-1ß, IL-6, IL-10, IL-8, IL-12, IFN-γ and TNF-α are negative (their values are normal or low).

Patients who required ICU admission presented higher concentrations of IP-10, MCP-1 and TNF-α, responsible for the cytokine storm and thrombosis (Fig. 1, Fig. 2 ); therefore, it can be hypothesized that those patients whose cytokine levels remain stable will be less likely to be admitted to the ICU and have a better prognosis. Most of the patients analyzed presented an upward maintenance of IP-10, MCP-1 and IL-8 values. IP-10 (or CXCL10) inhibits endothelial tissue healing, independently of other inflammatory factors. This is the reason why an anti-CXCL10 monoclonal antibody (Eldelumab) is being investigated as a possible treatment to prevent the cytokine storm.

Fig. 1.

Panel of 9 cytokines in the flow cytometer of a COVID-19+ patient. A) From left to right: selection of singletons (antibody-labeled beads) by forward scatter (FSC, x-axis) and side scatter (SSC, y-axis). APC and APC—Cy7 are then confronted to obtain the localization of each cytokine separately, and labeled. B) Event count of each cytokine.

This demonstrates the importance of controlling serum cytokine values and the existence of a clear positive relationship between COVID-19 severity and serum IP-10 levels, being one of the main biomarkers of an unfavorable evolution. In these patients with unfavorable prognosis, dexamethasone (a corticosteriod with anti-inflammatory effect)  was also used to prevent the severe cytokine storm characteristic of COVID-19 patients with pneumonia and, along with it, ICU saturation. IL-10 negativity or, its downward maintenance, is significant of a favorable prognosis, given that its concentration is correlated with IL-6, as a rapid response to the accumulation of pro-inflammatory cytokines in serum. IL-8 was positive in most patients coming from the ICU.

Discussion

It can be concluded that SARS-CoV-2, despite proliferating in the airways, is capable of causing disseminated infection that can lead to sepsis and death from multi-organ failure.  This sepsis triggers an exaggerated inflammatory response driven by cytokine storm. The release of cytokines, which in principle is a physiological and beneficial event for infection control, can become uncontrolled by the release of interferons and tumor necrosis factor that activate macrophages, with the consequent release of pro-inflammatory cytokines that, instead of slowing down, would feed back the inflammatory process [2].

The quantitative determination of cytokines by flow cytometry is essential to evaluate the possible development of the disease and to make clinical decisions, in view of the possible prognosis of the disease according to the degree of the inflammatory response evaluated by cytometry. A clear example would be IL-6, an increase in this cytokine is closely related to respiratory failure and, therefore, implies worse prognosis. The quantification of cytokines is performed by means of a DAS sandwich ELISA and subsequent analysis of the data through a calibration curve, previously performed (Fig. 3, Fig. 4 ). Likewise, the technique uses beads coated with monoclonal antibodies with a unique mixture of APC-A and APC—Cy7, which allow the identification and quantification of the interleukin. The beads are differentiated by their sizes and fluorescence intensity captured by flow cytometry.

Fig. 3.

Histograms of positivity and negativity of the TOP tube used for the calibration curve. The TOP tube corresponds to the one with the highest concentration, which in turn corresponds to the upper limit value of the technique. The homogeneity in the positivity of the nine cytokines can be seen.

In the study by Hina Chaudhry and Juhua Zhour (2013), the importance of cytokine storm in the severity of various diseases was highlighted [3]; currently, the relevance of interleukins in COVID-19 can be appreciated. Cytokines are a double-edged sword in sepsis, since their action is critical to eliminate the infection, although their overproduction can cause tissue damage, the most typical example being the generation of scar tissue in the lungs and the senescence of alveolar cells [4], and can also lead to acute renal failure [5]. In the study by Ali A. Rabaan et al. (2021), it is detailed how the signs and symptoms of the disease are aggravated when the cytokine storm occurs, and can become fatal [6]. Similarly, Huang et al. (2020), concluded that prognosis worsens with high levels of inflammatory biomarkers and ARDS symptoms. IL-1, IL-6, MCP-1 and IP-1 are related to thrombosis, pointing out that MCP-1 collaborates in the recruitment of monocytes to arterial walls during the formation of atheroma plaques, so assessing the levels of these cytokines may be essential in avoiding a fatal outcome. In 2020, Anastasios Kollias et al. already mentioned that the incidence of thromboembolism in COVID-19+ patients without anticoagulants was 25%, with a mortality of 40% [7]. However, the Mexican study by Samuel Treviño reveals a higher concentration of pro-inflammatory cytokines in obese patients and in older patients, so that old age leads to a greater lack of control of the immune system and a greater tendency towards the lack of control of inflammatory processes (a process known as inflammaging) [8,9]. This could explain why the cytokine storm is more dangerous in obese and/or elderly patients, given that this subpopulation starts from a basal inflammation, requiring more frequent admission to the ICU and making it difficult to apply artificial ventilation [10]. In our case, IP-10 and MCP-1 values were found to be elevated in all ICU patients, as was the case in the different trials mentioned above, and a higher incidence was also observed in older patients. Likewise, in a study carried out by the resident Paula Álvarez Romero in the cellular immunology laboratory of the HURS, in Córdoba (Spain), in which the values of various cytokines were measured by the same technique in healthy patients at different ages, a higher concentration of these two cytokines was observed in both groups despite their good health.

Inflammatory phenomena occur in response to a wide variety of pathologies, not only infectious ones such as COVID-19+. Flow cytometry can be used, and has been used in HURS, in other types of diseases such as Kawasaki syndrome or other autoinflammatory diseases, in which cytokine storm contributes to systemic inflammation [11]. Cytometry constitutes an objective and highly sensitive technique that allows multiparametric analysis of different macromolecules and/or cells. Despite its usefulness, it is a technique that is not fully implemented in laboratories as routine analysis, due to the high cost of reagent cytometers, as well as the need for highly qualified personnel. Fluorescent anti-cytokine and anti-chemokine monoclonal antibodies have become very useful for multiparametric flow cytometry analysis of individual cytokines using coated beads providing a high resolution method to identify the nature and assess their concentration. In addition, it allows highly specific and sensitive measurements of several individual parameters, simultaneously, so this method has the ability to perform rapid analysis of a large number of samples required for statistically significant measurements.

For all these reasons, cytometry will probably be the reference technique in the future for routine analysis of multiple inflammatory and infectious pathologies and, in this article, we have attempted to highlight some of its advantages in the diagnosis and prognosis of COVID-19.

The classical method of cytokine analysis, so far, is ELISA (enzyme-linked immunosorbent assay), which also allows a quantitative measurement with lower sensitivity than flow cytometry, with a detection limit of 0.5 pM (500 fg/mL) [12]. Given the greater objectivity of cytometry (it is independent of the color that the human eye appreciates in the reaction), its clear superiority in sensitivity, specificity and versatility, makes it more advantageous along with the possibility of performing a multiparametric analysis. All this makes cytometry a more advanced and useful technique in the field of research and hospital routine.

It is important to remember that the values of the concentrations used with this technique may not be considered high or normal by other techniques, given that there are no studies in healthy population and that the values of each cytokine vary according to the analytical method used. Also, it should be considered that the sensitivity range of the flow cytometry measurement (fg/ml) is much higher than that of the usual classical ELISA measurement techniques (pg/ml). Cytokines can also be quantified by PCR mRNA transcripts, although both techniques are considered restrictive, due to the time required and their sensitivity and specificity problems. Similarly, mRNA transcripts and ELISAs cannot identify actual cytokine production by not taking into account cytokines consumed by cells and low sensitivity for detecting cytokines produced in autocrine microenvironments [13].

Although there are no normality ranges, for IL-6 the Spanish Agency of Medicines and Health Products (AEMPS) recommends implementing drugs that control the eventual appearance of a cytokine storm when its value exceeds 40 pg/mL (measured by ELISA technique). Following the protocol supplied by the commercial company, this technique has only been validated for use with EDTA-anticoagulated plasma.

Data Availability

• I have shared our data research as a word called "Research data"