Diagnosis of Coronavirus disease by measuring serum concentrations of IL-6 and blood Ferritin

Objectives: Coronavirus disease 2019 (COVID-19) associated by infection and high death rate. The unresolved questions about the fatality rate of COVID-19 is most probably related to cytokine storm syndrome. There is currently no speci�c medication. Understanding the pathogenic pathway of this disease will lead to production of treatment and decreases of death rate. The aim of this study is to investigate changes of peripheral blood parameters (Interleukin-6 and Ferritin) in COVID-19 patients, which may be bene�cial in the management of patients. Methods: In this case-control study, we collected data of 270 subjects in two groups including 133 patients with severe type COVID-19 (case) and 137 patients with nonsevere (control) between March 20 and May 21, 2020, and the clinical symptoms and in�ammatory indications of patients diagnosed by laboratory test in Shahid Jalil hospital of Yasuj University of medical Sciences were collected to explore potential markers for disease monitoring. The data were analyzed by SPSS software version 20. Descriptive statistics, T-test and bivariate correlation tests were used to analyze. Results: The enrolled COVID-19 patients consisted of 53.4% males and 46.6% females with the medium age of 45.56±18.55 years and there were 50.04% males and 49.6% females with the medium age of 45.59±17.0 years for non COVID-19 patients. There was no signi�cant difference in the age and sex ratio between two population under study. The proportion interstitial abnormalities evidenced

Coronavirus binds to the angiotensin-converting enzyme2 (ACE2) which is present on no immune cells, such as respiratory and endothelial cells and immune cells such as alveolar monocytes/macrophages (5), and can induce endogenous stress signals or mutations in the host genome and recruitment of in ammatory cells(6).In the defensive line, innate immunity senses microorganisms and release of cytokines, Chemokines and in ammatory mediators by immune cells, epithelial cells and etc. in pathological conditions (7).The increase in speci c cytokine production can be linked to an activation cascade and uncontrolled cytokine release; the cytokine storm(8).Although cytokines expression is strictly controlled by transcriptional and posttranscriptional mechanisms, but high-level concentrations of cytokines for a long time leads to chronic in ammatory diseases and widespread tissue damage including ARDS (9,10).
During a virus infection, the plasma concentration Interleukin (IL)-6, IL-1 and IL-10 were increased (11), especially, IL-6, which promptly stimulate the production of acute phase proteins in response to infections and tissue injuries (7).IL-6 alterations, re ect the presence and severity of in ammation, and they have long been used as a clinical Guideline for diagnosis and management of diseases.(11,12).Also, high levels of lactate dehydrogenase (LDH), and ferritin are suggestive due to the cytokine storm in Laboratory ndings.Also, elevated Ferritin and Lactate dehydrogenase (LDH) levels due to cytokine storm syndrome have been reported in severe COVID-19 patients (13).In hospitalized patients, serum ferritin testwhich is widely available and affordable-is a good quali ed screening tool for informing physicians of cytokine storm syndrome for COVID-19 patients (14).
In general, COVID-19 in infected people of all ages, can be implications on different organs as lungs, brain and nervous system and eventually, may lead to death (15).Therefore, diagnose and treatment of cytokine storm and infection by measuring serum concentrations of IL-6 and blood ferritin has become an important part of rescuing severe patients.

Samples Data collection
In this case-control study, we collected data of 270 subjects in two groups including 133 patients with severe type COVID-19 (case) and 137 patients with non-severe (control), between March 20 and May 21, 2020, and the clinical characteristics and in ammatory indications of patioents which diagnosed by laboratory test in Shahid Jalil hospital of Yasuj University of medical Sciences, were collected to explore potential markers for disease monitoring.
In this study, General and speci c biosafety guidelines for diagnosis of COVID-19 was accordance with the WHO guidance.A con rmed positive case for COVID-19, was assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) test from the nasopharyngeal or oropharyngeal swabs specimens.lonely laboratory con rmed cases were contained in this study, while disease diagnosed based on clinical presentation and CT imaging ndings were excluded.
In this study, demographic data, clinical symptoms, imaging examinations, previous nucleic acid test results, and other laboratory ndings, CT Scanning, whole blood cell count and blood chemistry of patients were collected from electronic medical records using data collection forms.In addition, ferritin, lymphocyte subset and cytokine tests was analyzed in severe patients by Flow cytometry and ELISA detection.

ELISA detection and Blood Count Test
In this study, Complete Blood Count (CBC) Test, were measured using a Sysmex KX21 Hematology Analyzer (Block Scienti c, Bohemia, NY, USA).
Serum concentrations of IL-6 were also measured in in all COVID-19 and non COVID-19 samples.IL-6 levels were measured by the enzyme-linked immunosorbent assay (ELISA) kit (Gen-Probe, Diaclone France) as per the manufacturer's instructions.
Human serum ferritin concentrations were determined by using a diagnos-tic kit from Abbott Laboratories (Abbott Park, IL).All tests were performed according to the product manual.

Statistical analysis
All data were analyzed by descriptive statistics include: Chi-squared for categorical variables and averages and standard deviations for continuous data.Continuous variables were compared using the Student Ttest between groups.The Pearson's correlation coe cient (r) was used to understand the correlations between various laboratory ndings.Data were carried out by SPSS software version 20.For all statistical analysis, p-value less than 0.05 (typically ≤ 0.05) is statistically signi cant.

Ethical considerations
Since, there was no threat for participants and its results were only used to improve diagnisis planning of covid19 ; so we obtained informed consent.It should also be noted that in this study, we avoided mentioning any names, titles or characteristics that cause the loss of privacy of the participants, and the participants were clearly informed of the possible uses of the results.
There was no statistical evidence for different association in the ages and sex between two case and control groups (Table 1).
In addition, data analysis revealed that there was non-signi cant correlation in Lymphocyte with WBC (r = 0.118, p = 0.052), Neutrophil (r=-0.071,p = 0. 243) and Ferritin (r=-.076,p = 0.211) (Table 4).(22).According to the latest guidelines released by the Chinese government, Chest CT scan is a common imaging tool for pneumonia diagnosis, and the key indicator for the con rmation of the diagnosis of COVID-19 is based on RT-PCR or sequencing of the gene for respiratory or blood specimens (20,22).However, a rate of 10-40% RT-PCR false-negative results made this technique insu cient for proper detection (20,23).We suggest a major obstacle for this low e cacy might be related to sampling errors, markedly inappropriate timing of sampling, which is re ected by variations in viral load in upper versus lower respiratory tract.
In a recent study on patients with COVID-19 in K&B province, our data reported a higher rate of IL-6 in patients with infection than in the general population.Probably, this ndings indicate that IL-6 cytokine was positively associated with the severity of COVID-19.In accordance with present study, Coomes et al. suggested that in patients with COVID-19, IL-6 levels are signi cantly elevated and associated with noxious clinical subsequences including: ICU admission, asthma, and death (24).In the analysis by Zhang et al.IL-6 level in patients with COVID-19 have dramatically enhanced (25).Similarly, in research by Ruan et al.
reported higher levels of IL-6 in COVID-19 patients compared to non-patients(26).Overall, elevations in IL-6 levels between patients with COVID-19 were identi ed in all previous studies (13,(27)(28)(29).This suggests that dynamic changes in level of IL-6 can be used as a diagnosis factor in patients with severe COVID-19 and also, the progression of COVID-19 to complexed disease may be the result of an over-response of the immune system to in ammatory mediators.
In our study cohort, data analysis revealed that there was a high signi cant levels of ferritin in COVID- We have some limitations in this study.There were not complete clinical information for all patients with COVID-19 diseases admitted to Jalil hospital during the study term.The study was conducted with limited sample size, therefore, we were unable to do a multivariate analysis and generalize the results.There may also be a selection bias when identify factors that in uence the clinical outcomes.
We have an opinion that the ndings of this epidemiological study, in a region in southeast of Iran with a high number of COVID-19 cases, was one of the strengths of the present study.

Conclusion
In

Consent for publication
Informed consent was obtained from all Subjects.This Informed Consent Form had two parts: Information Sheet (to share information about the study).Certicate of Consent (for signatures if participants choose to participate).
conclusion, in ammatory markers especially IL-6 and Ferritin and hematological parameters (WBC, Lymphocyte, Neutrophil, Platelet and Hb) were positively correlated with the severity of COVID-19.Measurement of IL-6, Ferritin and hematological indices might be workable tests to diagnosis, monitoring and prognosis of patients with COVID-19.Declarations Ethics approval and consent to participate The Research project (No. 990000) approved by the Ethic Committee of Yasuj University of medical sciences; IR.YUMS.REC.1399.003.Informed consent form was obtained from all subjects.

Table 2
Comparison of the laboratory ndings of patients with COVID-19 and non-COVID-19.

Table 4 .
The Correlation between IL-6 and Ferritin and hematological parameters in COVID-19 patients.PCR should be used for symptomatic and hospitalized covid19 patients.In the study by Tao et al, in the epidemic area, chest CT scan in comparison with RT-PCR, maybe a more reliable, practical, and rapid method to diagnose COVID-19 (21)ents with nonsevere and severe COVID-19 in Kohgiluyeh and Boyer Ahmad (K&B) province.In our data set of COVID-19, we revealed signi cant differences in positive chest CT scans and positive RT-PCR results, when comparing subjects with severe COVID-19 and hyper in ammation with those who did not show expression of hyper in ammation.The results from this review in similarly to other studies from Duan et al(19), Poortahmasebi et al(20), and Udugama et al.(21), indicate that the chest CT scan and RT- (35)tients compared to that of non-COVID-19 patients.Similar observations have been reported by scientists from China, Italian and the United States.In Yehuda et al. study, high levels of Ferritin (hyperferritinemia) has been associated with increased illness severity and adverse outcomes, including COVID-19(30).In the study with Guan et al, Ferritin was signi cantly elevated in more severe cases of COVID-19 Compared with control group(19).Accordance with retrospective cohort study by Zhou, et al, serum ferritin levels have been increased in non-survivors patients with COVID-19 from Wuhan as compared with survivors.(31).About ferritin, Wu et al. reported that higher serum ferritin was related with ARDS development and was able to predict an increased risk of COVID-19 illness(28).Recently, some scienti c secrets by Ruscitti et al reported.theydiscoveredtherole of the H-chain of ferritin in activating macrophages (macrophage activating syndrome; MAS) to increase the secretion of in ammatory cytokines and cytokine storms in COVID-19 patients.(32).Overall, in accordance with previous studies, it turns out that elevated ferritin concentrations play a critical role in innate immunity and associated with an increase in production of special signaling molecules of the body(13).These studies complete our understanding of the pathogenesis of the high levels of ferritin including the infection with Covid-19, and may contribut clinicians to apply more aggressive treatment for those patients.Several studies have shown the common presenting hematological manifestations of COVID-19.Wu et al. retrospectively demonstrated risk factors for the clinical outcomes of COVID-19 pneumonia and death in China patients.The study showed that several factors related to the development of disease which included, neutrophils, lymphocyte and etc (28).Guan et al. found that lymphocyte had signi cantly increased in most patients of COVID-19, while platelets had decreased(19).In Huang et al. research, COVID-19 infection is associated with alterations in the WBC and lymphocyte count(33).In Dawei et al study, Compared COVID-19 patients received ICU care with non-ICU patients who had signi cantly elevated neutrophil and WBC count, as well as lymphocyte were signi cantly decreased(34).Interestingly, Hu Yun et al. showed that among Covid-19 Positive Patients, the platelet count during the disease course were decreased(35).Similarity, Lippi et al. was showed that a low platelet count correlated with higher disease severity(36).So, the Results of this study reported the changes in hematological markers in covid-19 patients might help to developed the pathophysiology Knowledge of this disease and provide early guids to diagnosis of coronavirus based on routine laboratory tests.
In the our study, COVID-19 severe cases had elevated levels of hematological indices (except for Platelet), compared with non-severe cases.The results showed, the COVID-19 can cause some hematological indices changes between cases.