Elevated serum ferritin level effectively discriminates severity illness and predicts prognosis of COVID-19 patients

During coronavirus disease 2019 (COVID-19) pandemic, medical resources in every country is in shortage. Ecacious indicators of discriminating severe illness and predicting outcome is in urgent need. We collected data and clinical records from 79 COVID-19 patients admitted between January 12, 2020 and February 21, 2020 at Wuhan Union hospital, China. Spearman’s correlation analysis, receiver operating characteristic (ROC) curve, logistic regression model, and Kaplan-Meier survival curves were employed in the analysis. Of 79 patients enrolled, 2 died in hospital, 8 were transferred to other hospitals, and 69 were discharged. Patients with elevated ferritin levels (> 200 ng/mL) had a higher incidence of severity illness when compared with those with normal ferritin levels ( ≤ 200 ng/mL) (50.0% vs 2.9%). In addition, severity illness manifested signicantly higher level of ferritin as compared with non-severe ones (median 921.3 vs 130.7 ng/mL, p < 0.001). Furthermore, ferritin could effectively discriminate severity and non-severity, with an area under the ROC curve (AUC) reaching 0.873 (sensitivity 96%, specicity 70%), larger than that of age (0.697), C-reactive protein (0.730) and lymphocytes% (0.717). Combined model incorporating multivariate revealed a similar manner with ferritin alone (p = 0.981). Furthermore, elevated ferritin group showed longer viral clearance time (median 16 vs 6 days, p < 0.001) and in-hospital length (median 18 vs 10 days, p < 0.001). Our results suggest that ferritin could act as a simple and ecacious complementary tool to identify severe COVID-19 patients at early stage and predict their outcome. This indicator would provide guidance for subsequent clinical practice, alleviate the medical stress and reduce the mortality. clinical characteristics, treatments and outcomes were acquired by the hospitalization management system. All the data were collected by well-trained researchers with a double-blind method. Data collection of laboratory results were dened using the rst-time examination at admission. The date of symptom onset, admission, negative detection of COVID-19 RNA, discharge and death were recorded accurately. The date of admission was used as the starting point of the virus clearance process, and the date of the negative detection of COVID-19 RNA was calculated as the ending point of viral clearance.


Introduction
The coronavirus disease 2019 (COVID- 19) pandemic has spread to 44 countries on all continents except Antarctica and the number of diagnosed cases worldwide is accelerating every day [1]. Medical resources in every country are in shortage or even overdrawn, so there is an urgent need of seeking indicators for disease severity and prognosis at early stage, which is believed to signi cantly reduce the medical stress and mortality rate. However, such e cient indicators have not been fully investigated. Currently, the de nition of severe cases mainly relies on the observation of clinical manifestation [2,3], and the prognosis of COVID-19 remains inconclusive.
Ferritin is the primary tissue iron-storage protein in the liver, it is also an acute-phase protein that can be induced in the setting of systemic in ammation [4]. Ferritin is able to act as an independent predictor of discriminating the severity of nonalcoholic fatty liver disease [5]. Recent studies related to COVID-19 have consistently discovered the abnormal level of ferritin in severe COVID-19 patients, but further exploration of its value is halted [6,7]. Herein, we aimed to assess the ability of ferritin in discriminating severe patients from non-severe ones on admission, and the potential of predicting viral clearance rate and hospitalization duration, to provide guidance for subsequent clinical practice.

Participants
From January 12, 2020 to February 21, 2020, a total of 147 consecutive patients were initially enrolled from the department of infectious diseases of Wuhan Union Hospital, all of which were con rmed cases of COVID-19 after examination of COVID-19 RNA by RT-PCR (upper respiratory throat swab samples) and chest computerized tomography (CT) scanning. One patient who died of traumatic brain injury with viral pneumonia was excluded. Patients without ferritin detection on admission were excluded. Therefore, 79 inpatients were enrolled in this study. By March 14, 2 patients died, 8 patients were transferred to other hospitals, and the remaining patients were discharged.

Data collection
The data of demographics, laboratory examinations, CT scan, clinical characteristics, treatments and outcomes were acquired by the hospitalization management system. All the data were collected by well-trained researchers with a double-blind method. Data collection of laboratory results were de ned using the rst-time examination at admission. The date of symptom onset, admission, negative detection of COVID-19 RNA, discharge and death were recorded accurately. The date of admission was used as the starting point of the virus clearance process, and the date of the negative detection of COVID-19 RNA was calculated as the ending point of viral clearance.

Statistical Analysis
The data were presented as mean ± standard deviation (SD) or median (Inter Quartile Range, IQR). Intergroup comparisons between groups were made by the Student's t test (normally distributed continuous variables), Mann-Whitney U test (nonnormally distributed continuous variables). The categorical variables (shown by percentage) were compared by using Chi-square analysis. Spearman's correlation analysis was used to explore the coe cients of ferritin with other covariates. The performance of the discriminant model was characterized by estimating the area under the receiver operating characteristic (ROC) curve (AUC). The cutoff value of ROC was calculated based on the maximum Youden index, which was used to assess the global diagnostic effectiveness. Cumulative survival curves of hospitalization span and viral clearance time were estimated using the Kaplan-Meier estimation method for two groups with normal and elevated ferritin levels (ferritin ≤ 200 ng/mL and ferritin > 200 ng/mL) by log-rank test. All of the analyses were performed with the R software version 3.4.3 (http: //www.R-project.org, The R Foundation) and EmpowerStats version 2.20 (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA). A two-sided P value < 0.05 was determined as with statistically signi cant differences.

Baseline data
All participants were divided into two groups by normal and elevation ferritin levels (ferritin ≤ 200 ng/mL and ferritin > 200 ng/mL) according to clinical reference range. As shown in Table 1, severe cases were more frequently found in patients with higher ferritin levels, compared to those who with normal ferritin levels (50.0% vs 2.9%, p < 0.001). There were signi cant differences of sex between the two groups, with male patients accounting for the 68.2% in elevation ferritin group but only 17.1% in normal ferritin group (p < 0.001). The average age of elevation ferritin group was also signi cantly older than the normal ferritin group (49.9 vs 36.8 years old). The other characteristics including systolic blood pressure (SBP), diastolic blood pressure (DBP), respire, pulse, smoking, comorbidities and symptoms of both groups were recorded but no statistical difference was found.

Determination of ferritin and corresponding CT manifestation
As mentioned above, there existed signi cantly more severe cases in elevation ferritin group than the normal group. In addition, we compared the ferritin levels among severe and non-severe patients, nding that the ferritin level of severe group is signi cantly higher than that of nonsevere group (median 921.3 (IQR 440.0-1609.8) vs median 130.7 (IQR 58.8-320.4), p < 0.001) ( Fig. 2A). Furthermore, we recorded the ferritin levels which were calculated from the onset of symptom of each patient. Likewise, at every stage the ferritin level of sever group was markedly higher than their counterparts (Fig. 2B). On the other side, we observed the CT images that were corresponding to the ferritin values in three representative patients (Fig. 2C). The results demonstrated that the severity of pulmonary imaging was consistent with the ferritin level. Taken together, the ferritin level is able to reveal the severity of COVID-19.
In addition, we employed logistic regression analysis to assess the risk factors related with severity illness (Fig. 3C). The results showed that a 100 ng/mL increase in serum ferritin, the odds ratio (OR) of severity illness was 1.17 (95% CI: 1.03-1.33, p = 0.014). After adjusted for sex, age, CRP or LYM%, the OR of severity illness was 1.20 (95% CI: 1.02-1.41, p = 0.014). Moreover, Fig. 3D showed that the OR of severity illness for serum ferritin was 10.78 (95% CI: 1.13-102.73, p = 0.039) when serum ferritin was calculated as categorical variable (> 200 ng/mL vs ≤ 200 ng/mL) in crude model. After adjusted for age, sex, CRP and LYM%, the OR of severity illness for patients with elevated ferritin level (> 200 ng/mL) was 18.75 (95% CI: 1.28, 275.41, p = 0.032), as compared with patients with normal ferritin level (≤ 200 ng/mL). Therefore, we established a nomogram based on the above risk factors to improve the availability in clinical practice (Fig. 4).

Prognostic ability of ferritin
Median duration of viral clearance was 6 days (IQR 2-25) in normal ferritin group (≤ 200 ng/mL, but elevation ferritin group (> 200 ng/mL) was 16 days (IQR 2-47). The viral clearance events in in normal group and elevation ferritin group by using Kaplan-Meier survival analysis were displayed in Fig. 5A (p < 0.001).
Besides, we recorded the length of hospitalization of all patients except 2 deceased ones and 8 patients that transferred to other hospitals. Our result showed that patients with higher ferritin levels on admission stayed in hospital for a longer time (18 days, IQR 3-47), when compared with the ones with normal ferritin levels (10 days, IQR 5-29). The discharge events in in normal group and elevation ferritin group by using Kaplan-Meier survival analysis are displayed in Fig. 5B (p < 0.001).

Discussion
The number of diagnosed COVID-19 patients worldwide is increasing rapidly every day. Until now, there is no effective medicine available to treat COVID-19. Medical resources in every country are in shortage or even overdrawn, so there is an urgent need of identifying severe cases and poor prognosis cases at early stage of disease. Only in this way, can medical staffs make greater use of limited medical resources and reduce mortality. Recent clinical data have indicated that lymphocyte [9,10], CRP [11] and other factors may be related to the severity of COVID-19 [6]. However, the role of ferritin seems to be ignored even though clinical observation has discovered the abnormal ferritin levels in severe COVID-19 patients [6]. For the rst time, the present study shows the relationships between ferritin and severity and viral clearance in COVID-19 patients.
Herein, the data of 79 patients with COVID-19 pneumonia were analyzed, the baseline characteristics of patients in the severe and non-severe groups were described and compared, and the dynamic changes of ferritin and imaging were demonstrated. Our results showed that ferritin levels differed signi cantly between severity and non-severity illness. Ferritin variations were also consistent with CT manifestation, presenting the ability to act as a sensitive and accurate indicator. Then, the diagnostic value of ferritin for the incidence of severe illness was compared to that of age, sex, CRP and LYM%, which had been recognized as the risk factors of COVID-19 [9,[12][13][14]. Our results showed that the AUC of ferritin ROC were signi cantly larger than other risk factors, and the multivariable combined model manifested similar ability compared with ferritin alone, demonstrating that ferritin was an independent easy-to-use predictor. In addition, we compared the viral clearance rate and in-hospital length between elevation ferritin group and normal group, nding that ferritin was also a discriminated indictor for prognosis of COVID-19 patients. Viral clearance is the golden standard for de ning the recovery of COVID-19 infections and predicting inhospital length is extremely vital in COVID-19 pandemic due to the lack of medical resource [15]. Thus, we suppose ferritin could act as both an effective discriminator for severity illness and a predictor for prognosis.
Ferritin is an acute phase protein which can be released from damaged hepatocytes [16]. Hyperferritinemia has been previously recognized in abnormal liver function conditions or metabolic syndrome [5,17,18]. In our study, the severe patients on admission manifested slight risk of liver injury and dyslipidemia. Other observations also nd that liver injury is prevalent in severe cases of COVID-19 [19]. Therefore, we recommend that patients should pay close attention to the risk of developing secondary liver diseases or metabolic syndrome after they are discharged. Intensive surveillance and regular comprehensive medical examination are necessary, especially for the severe patients.
However, there were some limitations in the study. First, this was a single-center retrospective study with limited size. Second, some cases had incomplete biochemistry determinations. Third, the present study failed to predict the mortality of COVID-19 patients due to the insu cient number of deaths.
In summary, the ndings indicate that early examination of ferritin in COVID-19 patients could effectively discriminate severity illness and predict the prognosis. Therefore, ferritin could act as a simple complementary tool to help guiding clinical decision and facilitating appropriate treatment. Patients with elevated admission ferritin level should be provided with strengthened attention and treatment. More studies are needed to con rm these ndings and to explore exact pathological mechanisms. Further understanding of pathological signi cance of serum ferritin elevation in severe COVID-19 patients is warranted to help clinicians make reasonable decisions to decrease the risks of adverse outcome.

Conclusion
In conclusion, serum ferritin might be an independent risk factor for severity illness and predictor for prognosis of COVID-19 patients. The authors declare no con icts of interest.

Consent for publication
Not applicable.

Availability of data and materials
The raw data required to reproduce these ndings cannot be shared at this time as the data also forms part of an ongoing study.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupplementalTable.docx