Skip to main content
NCBI home page
F1000Research logoLink to F1000Research
. 2022 Nov 10;11:1285. [Version 1] doi: 10.12688/f1000research.125115.1

The role of IL-6, ferritin, and coagulopathy in COVID-19 clinical progression

Alvin Tagor Harahap 1, Cosphiadi Irawan 2,a, Adityo Susilo 3, Kuntjoro Harimurti 4, Dewi Gathmyr 4, Hamzah Shatri 4, Anna Mira Lubis 2, Leonard Nainggolan 4, Murdani Abdullah 4
PMCID: PMC10576189  PMID: 37841828

Abstract

Background

In COVID-19, the release of pro-inflammatory mediators in the cytokine storm, primarily interleukin-6 (IL-6), has been hypothesized to induce pulmonary intravascular coagulation. However, the relationship between IL-6 and coagulopathy remains unclear in COVID-19 progression. We aimed to investigate the correlation of IL-6 with D-dimer, fibrinogen, prothrombin time (PT), and ferritin. Furthermore, we also analyzed the effect of those parameters on the worsening of COVID-19 patients.

Methods

A prospective cohort study was conducted in moderate and severe COVID-19 patients from June 2020 to January 2021. A serial evaluation of IL-6, D-dimer, fibrinogen, ferritin, and PT was performed and correlated with the patient's condition at admission and on the 14th day. The outcomes (improvement, worsening, or discharged patients) were recorded during the study.

Results

Of 374 patients, 73 study subjects (61 severe and 12 moderate COVID-19) were included in this study. A total of 35 out of 61 severe and one out of 12 moderate illness subjects had experienced worsening. Spearman-rank correlation of IL-6 with with ferritin, D-dimer, fibrinogen, and PT was 0.08 ( p=0.5), −0.13 ( p=0.27), 0.01 ( p=0.91), and 0.03 ( p=0.77), respectively. In ROC analysis, D-dimer (74,77%) and IL-6 (71,32%) were the highest among other variables (>60%).

Conclusions

In COVID-19 patients, there was a correlation between elevated IL-6 and D-dimer levels with disease deterioration. There was no correlation between elevated IL-6 levels with ferritin, D-dimer, fibrinogen, and PT levels. Therefore, changes in IL-6 and D-dimer can predict worsening in moderate and severe COVID-19 patients.

Keywords: D-dimer, Ferritin, Fibrinogen, IL-6, Prothrombin Time

Introduction

The SARS-CoV-2 virus is a β-coronavirus with an enveloped, positive-sense, and single-stranded RNA. The COVID-19 pandemic due to SARS-CoV-2 virus poses a serious threat due to systemic inflammation and coagulopathy. Interleukin-6 (IL-6) is a cytokine that has a positive pleiotropic effect on inflammation. This cytokine can amplify the coagulation system to activate the epithelial cells, monocytes, and neutrophils, 1 that can inhibit anticoagulant protein S and antithrombin and enhance the activity of clotting factor VII, von Willebrand, and fibrinogen.

Previously considered, increased ferritin was associated with hemophagocytic lymphohistiocytosis/macrophage activation syndrome in COVID-19. However, the absence of five out of eight characteristics in the previous COVID-19 study explains the differences between the disease. 2 Several retrospective studies have been conducted to observe the relationship between IL-6, PT, fibrinogen, and ferritin with COVID-19 progression. 3 – 7 In this current prospective cohort study, we aimed to investigate the effectiveness of these parameters in COVID-19 progression.

Methods

Study design

In this prospective cohort study conducted from July 2020 to January 2021, COVID-19 patients aged ≥18 years at the Pertamina Central Hospital Modular Extension Simprug (Simprug Modular Extension Hospital, SMEH, Jakarta, Indonesia) were included. Patients with COVID-19 were detected based on nasopharyngeal and oropharyngeal swab examination by polymerase chain reaction (PCR). Inclusion criteria: aged 18 years or older; moderate and severe COVID-19 (according to COVID-19 WHO guideline confirmed by positive BALF PCR SARS-CoV-2); willingness to provide blood sample. Exclusion criteria: history of chronic bleeding; undergoing hemodialysis; undergoing plasma convalescent clinical trial, or taking immunomodulation therapy (particularly IL-6 and intravenous immunoglobulin therapy).

Sample collection and storage

Patients who fulfilled the criteria were observed on the first day of hospital admission, and blood samples ( i.e., first sample collection) were collected to analyze IL-6, D-dimer, ferritin, fibrinogen, and prothrombin time. The collected blood samples were stored in the fridge at -20° celcius at Clinical Pathology laboratory of Pertamina Central Hospital.

After collected, IL-6 was analyzed in the Integrated Laboratory of the Faculty of Medicine, Universitas Indonesia. During transferring IL-6 samples were maintained at constant temperature -20° celcius accoding to Guidance on regulations for the Transport of Infectious Substances Guidance on regulations for the Transport of Infectious Substances 2007. IL-6 was analyzed using an ELISA IL6 Vmax microplate reader. Complete blood count, D-dimer, fibrinogen, and prothrombin time (PT) were analyzed using Sysmex CS2100i. Ferritin was analyzed using Cobas 601 Roche in the Clinical Pathology laboratory of Pertamina Central Hospital.

Assessment patients’ severity

The severity of the illness was classified according to the WHO interim guidance (version 18th May 2020) 8 as moderate, severe, or critical illness. Patients were observed up to day 14th or considered completed when the patients improved, worsened, died, or were discharged earlier. Afterward, samples were collected at the end of the observation ( i.e., second sample collection). Worsening was defined as COVID-19 disease severity progression from moderate to severe, critical illness or death. All patients received antibiotics, antivirals, corticosteroids, and anticoagulants according to the standard hospital therapy (Fondaparinux, LMNH, or UFH).

Statistical analysis

All collected data were analyzed using Anaconda package program 9 and reported in the text, table, or figures. All p-values < 0.05 were considered statistically significant. Correlation test was used to analyze correlation between IL-6 with ferritin and coagulopathy. Method of cutoff points obtained using area under curve method. Cutoff points by receiver operator characteristic obtained by calculating the optimum sensitivity and specificity.

This study has been approved (approval date: June 29 th 2020) by the ethical committee, Universitas Indonesia (approval number: KET-650/UN2.F1/ETIK/PPM.00.02/2020), and Pertamina Central Hospital (approval number: 3315/B00000/2020-S8).

Results

During the study period from July 2020 to January 2021, 374 severe and moderate COVID-19 patients were treated at the SMEH. Of those, 117 patients were randomized, 42 were excluded due to incomplete data, one received convalescent plasma therapy, and 1 had lysis of blood samples. The remaining 73 patients were further analyzed.

The characteristics of the study subjects are summarized in Table 1. Of the 73 COVID-19 patients, 61 (84%) were classified as having severe COVID-19 and 12 (16%) were classified as having moderate COVID-19. The majority of the patients were male and the mean age of patients was 61 years (SD±12.74). Most of the patients were overweight, hospitalized at day 6 th of the illness with duration of 7 days. As the final observation outcomes, most of the subjects with moderate disease experienced improvement, and subjects with severe disease experienced worsening on day 14 of the illness. The majority of the patients had at least one comorbidity, with hypertension was the most common. All subjects were given the combination of remdesivir, favipiravir, and oseltamivir. They also received corticosteroids (dexamethasone or methylprednisolone) and anticoagulants (unfractionated heparin, enoxaparin, or fondaparinux).

Table 1. Characteristics of the study subjects.

Total (n=73) Severe (n=61) Moderate (n=12)
Age (years), mean (±SD) 61.00 (±12.74) 61.52 (±12.77) 56.50 (±12.24)
Gender
 Male (%) 47 (64%) 38 (62%) 9 (75%)
 Female (%) 26 (36%) 23 (38%) 3 (25%)
Body Mass Index (kg/m 2), median (IQR) 26.28 (25.10–28.11) 26.17 (24.96–28.18) 27.01 (25.98–27.98)
Time from illness onset to admission (days), median (IQR) 6.00 (4.00–7.00) 6.00 (4.00–7.00) 6.00 (4.00–7.00)
Length of stay (days), median (IQR) 7 (5–8) 7 (5–8) 8 (7–11.75)
Final observation outcomes
 Improved (n) 37 26 11
 Worsening (n) 36 35 1
Number of comorbidities
 None 22 20 (33%) 2 (17%)
 One comorbidity 25 19 (31%) 6 (60%)
 Two comorbidities 20 17 (28%) 3 (25%)
 Three comorbidities 6 5 (8%) 1 (8%)
Therapy
 Oseltamivir (n) 1 1 0
 Favipiravir (n) 29 23 6
 Remdesivir (n) 42 36 6
 Meropenem (n) 52 46 6
 Levofloxacin (n) 35 31 4
 Azithromycin (n) 31 29 2
 Enoxaparin (n) 8 6 2
 UFH (n) 47 43 4
 Fondaparinux (n) 17 11 6
 Methylprednisolone (n) 63 55 8
 Dexamethasone (n) 8 5 3
Death 27 27 0
Survived 46 34 12
First sample collection
 D-dimer (mg/dL), median (IQR) 1.21 (0.60–2.84) 1.45 (0.62–2.93) 0.61 (0.48–1.04)
 Fibrinogen (mg/dL), median (IQR) 678.00 (497.00–778.00) 703.00 (553.00–806.00) 469.50 (345.75–617.25)
 Prothrombin time (seconds), median (IQR) 11.00 (10.00–11.00) 11.00 (10.00–11.00) 10.50 (10.00–11.00)
 Ferritin (ng/mL), median (IQR) 1505.00 (893.00–2334.00) 1505.00 (946.00–2334.00) 1433.00 (599.95–2058.00)
 IL-6 (pg/mL), median (IQR) 8.00 (2.07–56.21) 8.00 (2.07–63.47) 9.07 (2.40–29.53)
  SpO 2/ FiO 2 ratio, median (IQR) 170.18 (141.43–206.25) 148.48 (139.39–192.16) 395.24 (330.00–466.67)
Second sample collection
 D-dimer (mg/L), median (IQR) 1.39 (0.85–3.86) 1.51 (1.03–4.31) 0.41 (0.32–0.81)
 Fibrinogen (mg/dL), median (IQR) 426.00 (342.00–597.00) 431.00 (352.00–611.00) 381.00 (327.25–480.50)
 Prothrombin time (seconds), median (IQR) 11.00 (11.00–12.00) 11.00 (11.00–12.00) 10.50 (10.00–11.00)
 Ferritin (ng/mL), median (IQR) 1301.00 (813.00–2187.00) 1366.00 (933.00–2242.00) 1003.10 (361.75–1887.00)
 IL-6 (pg/mL), median (IQR) 7.39 (1.50–33.26) 11.63 (2.45–36.54) 2.20 (0.82–4.50)
  SpO 2/ FiO 2 ratio, median (IQR) 111.47 (94.00–394.64) 97.50 (93.97–247.50) 408.33 (408.33–411.46)
Open in a new tab

Notes: SD: Standard deviation, IQR: Interquartile range, n: number of samples, UFH: unfractionated heparin.

The variable distributions were analyzed according to disease severity and the worsening or improvement of patients' condition, as shown in Table 2.

Table 2. Variables' distribution according to disease severity and outcomes.

Severe Moderate
Improved Worsening Improved Worsening
Number of subjects 26 35 11 1
Age (years), median (IQR) 62.00 (47.00–71.75) 62.00 (55.00–69.00) 58.00 (50.50–64.00) 52.00 (52.00–52.00)
Body mass index (kg/m 2), median (IQR) 26.38 (25.17–28.19) 26.17 (24.85–27.85) 27.34 (26.18–28.03) 25.51 (25.51–25.51)
Day of worsening, median (IQR) 7.00 (6.00–7.75) 6.00 (5.00–7.50) 8.00 (7.00–10.00) 18.00 (18.00–18.00)
Time from illness onset to the worsening (days), median (IQR) 14 (13–17) 11 (10–14) 16 (12–18) 18
First sample collection
 D-dimer (mg/L), median (IQR) 1.34 (0.51–2.71) 1.50 (0.78–3.37) 0.63 (0.47–1.27) 0.60 (0.60–0.60)
 Fibrinogen (mg/dL), median (IQR) 709.50 (515.50–799.00) 678.00 (568.00–808.50) 442.00 (344.50–554.00) 766.00 (766.00–766.00)
 Prothrombin time (seconds), median (IQR) 11.00 (10.00–11.00) 11.00 (10.50–11.00) 11.00 (10.00–11.00) 10.00 (10.00–10.00)
 Ferritin (ng/mL), median (IQR) 1137.00 (779.75–1799.00) 1906.00 (1361.50–2643.00) 1326.00 (533.70–1747.00) 2436.00 (2436.00–2436.00)
 IL-6 (pg/mL), median (RIK) 6.27 (2.55–58.90) 10.78 (1.52–68.31) 12.19 (2.29–33.42) 3.93 (3.93–3.93)
  SpO 2/ FiO 2, median (IQR) 179.41 (148.48–196.08) 143.94 (134.85–171.05) 333.33 (330.00–466.67) 457.14 (457.14–457.14)
Second sample collection
 D-dimer (mg/L), Median (IQR) 1.19 (0.94–1.64) 3.08 (1.33–9.63) 0.38 (0.32–0.55) 1.63 (1.63–1.63)
 Fibrinogen (mg/dL), median (IQR) 406.00 (321.50–492.50) 498.00 (403.00–620.50) 361.00 (326.50–445.50) 491.00 (491.00–491.00)
 Prothrombin Time (seconds), median (IQR) 11.00 (10.25–11.00) 11.00 (11.00–12.00) 10.00 (10.00–11.00) 11.00 (11.00–11.00)
 Ferritin (ng/mL), median (IQR) 1030.00 (777.83–1452.25) 1938.00 (1154.00–2997.50) 719.20 (349.50–1743.00) 2163.00 (2163.00–2163.00)
 IL-6 (pg/mL), median (IQR) 6.40 (1.47–17.21) 16.93 (3.58–94.38) 1.94 (0.64–3.75) 5.41 (5.41–5.41)
  SpO 2/ FiO 2, Median (IQR) 247.50 (112.94–412.50) 95.00 (91.50–105.56) 408.33 (376.58–412.50) 267.57 (267.57–267.57)
Open in a new tab

Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT ( Table 3).

Table 3. Correlation between IL-6 and other variables.

IL-6 and Correlation coefficient p
Ferritin 0.08 0.50
D-dimer −0.13 0.27
Fibrinogen 0.01 0.91
Prothrombin time 0.03 0.77
Open in a new tab

Spearman correlation.

Figure 1 and Table 4 displayed the analysis of the receiver operating characteristic (ROC) curve (AUC) for correlation between elevated IL-6, ferritin, fibrinogen, D-dimer, and PT levels with COVID-19 patients' deterioration.

Figure 1. Receiver Operator Characteristic Curve of variable differences and COVID-19 patients’ deterioration.

Figure 1.

Open in a new tab

Notes: (1) dDdimer: D-dimer difference, dFibrinogen: Fibrinogen difference, dFerritin: Ferritin difference, dProthrombin Time: Prothrombin Time difference, dIL-6: IL-6 difference. (2) Difference is defined as the value at the end of observation – value at initial observation.

Table 4. Area under ROC curve for the variable differences and COVID-19 patients’ deterioration.

Variables AUC 95% CI
D-dimer 74.77% 63.48–86.07%
Fibrinogen 50.15% 36.81–63.49%
Prothrombin Time 47.67% 34.36–60.99%
Ferritin 48.42% 35.10–61.75%
IL-6 71.32% 59.48–83.17%
Open in a new tab

Notes: AUC: Area under the ROC curve, ROC: Receiver operator characteristic, CI: Confidence interval.

We found no correlation between IL-6 and other variables. Thus, as one of the disease severity index components, we aimed to investigate the correlation between the oxygen saturation ( SpO 2)/fraction of inspired oxygen ( FiO 2) ratio and IL-6, ferritin, fibrinogen, D-dimer, and PT ( Figures 2 and 3). As a result, our study demonstrated the correlation between IL-6, ferritin, fibrinogen, D-dimer, and PT and SpO 2/ FiO 2 ratio as the severity determinants.

Figure 2. Correlation between IL-6, ferritin, D-dimer, and fibrinogen variables with SpO 2/ FiO 2 ratio at the initial observation.

Figure 2.

Open in a new tab

Spearman correlation test. CRP: C-reactive protein.

Notes: A–D: correlation between SpO 2/ FiO 2 ratio and inflammatory markers CRP, IL-6, ferritin, and fibrinogen with a statistical significance was found between SpO 2/ FiO 2 ratio and inflammatory markers CRP, ferritin, and fibrinogen, but not with IL-6. Figure E and F: correlation between SpO 2/ FiO 2 ratio and coagulation markers D-dimer and prothrombin Time with a statistical significance was found between SpO 2/ FiO 2 ratio and D-dimer, but not with prothrombin time.

Figure 3. Correlation between IL-6, ferritin, D-dimer, and fibrinogen variables with SpO 2/ FiO 2 ratio at the end of observation.

Figure 3.

Open in a new tab

Spearman correlation test. CRP: C-reactive protein.

Notes: A–D: correlation between SpO 2/ FiO 2 ratio and inflammatory markers CRP, IL-6, ferritin, and fibrinogen with a statistical significance was found between SpO 2/ FiO 2 ratio and inflammatory markers CRP and ferritin, but not with IL-6 and fibrinogen. Figure E and F: correlation between SpO 2/ FiO 2 ratio and coagulation markers with a statistical significance was found between SpO 2/ FiO 2 ratio and D-dimer and prothrombin time.

Discussion

Characteristics of the study subjects

Males were predominant in this study (64%) with the age range 52–68 years old. 10 , 11 Hypertension (44.4%) and diabetes (31.8%) were the most common comorbidities, and similar results were also reported in other studies from China. 12 , 13 This study found that the comorbidity was not associated with the patient's deterioration. Subjects with hypertension tend to experience worsening, as stated by Li Yongheng et al. 13

In this study, most subjects with moderate disease experienced improvement. Despite the standard therapy, subjects with severe disease experienced worsening on day 14 of the illness. Meanwhile, all the subjects with moderate illness and 55.7% with severe illness survived ( Table 2).

The courses of the COVID-19 disease have been reported comprehensively, with mild symptoms occurring mainly on the 5th day until the 10th day of illness (phase II disease). 14 , 15 In this phase, most patients begin to feel shortness of breath accompanied by hypoxia. Our study subjects visited the emergency department during the mild symptom phase on the sixth day of hospitalization. Furthermore, they showed increased IL-6 and APR of ferritin and fibrinogen. Subjects who visited the emergency department for more than 6 days (mean 7.5 days) had almost three times greater risk of worsening than those who came for less than 6 days (mean 4 days).

Subjects with severe disease had higher levels of D-Dimer ( Table 1), which is consistent with previous studies conducted by Chen et al, Zhou et al, and Guan et al. 16 – 18 Additionally, levels of D-dimer were higher in the worsening compared with the improved group. This study reports that levels of D-Dimer tend to increase at the second sample collection ( Table 2).

According to Chen et al. 16 and Zhou et al., 17 non-survivor COVID-19 subjects had higher ferritin levels than the survivors. At admission, this study shows that subjects with moderate and severe conditions had higher ferritin levels ( Table 1).

Levels of IL-6 at the first sample collection ( Table 1) were similar to the previous study. 17 In contrast to the study by Jin Zhang et al. 10 and Awasthi et al., 19 study subjects did not show a significant difference in IL-6 levels at the first measurement between moderate to severe illness.

Correlation between increased levels of IL-6 and ferritin

In acute phare response (APR), IL-6 is a pro-inflammatory cytokine that increases and determines infection-associated ferritin levels. 20 , 21 However, we found no correlation between ferritin levels and IL-6 in the first and second sample collection ( Figure 3).

Although subjects did not experience an inflammatory process at admission, we found an increase in ferritin and fibrinogen levels, indicating that inflammation occurred in most study subjects. Furthermore, other factors contributing to increased ferritin levels, such as epithelial damage, were also considered. Since a decrease in the SpO 2/ FiO 2 ratio was due to an acute respiratory distress syndrome (ARDS) manifestation, 13 , 22 the correlation between SpO 2/ FiO 2 ratio with ferritin and fibrinogen was investigated to determine whether the epithelial damage affected IL-6 and other the inflammatory variables. Our result showed a statistically significant correlation between SpO 2/ FiO 2 ratio with ferritin and fibrinogen ( Figure 2). This correlation showed that inflammation affected the SpO 2/ FiO 2 ratio from the onset of the illness. However, since the increase of IL-6 was not correlated with SpO 2/ FiO 2 ratio, we assumed that inflammation and ferritin release was not affected by IL-6. 7 , 23 According to a study by Zhi et al., several possibilities cause the increase of ferritin: 1) pro-inflammatory cytokines stimuli ( i.e., IL-1β, tumour necrosis factor α (TNF-α), and IL-6) caused an inflammatory reaction that damaged the cells, 2) intracellular ferritin leakage due to cell damage and inflammation, and 3) the ferritin leakage from the injured cells further triggers the damage of other cells via Fenton and Haber–Weiss's reaction. 24

Correlation between levels of IL-6 with D-dimer, fibrinogen, and PT

Inflammation will trigger the coagulation system characterized by changes in the value of D-dimer, fibrinogen, and PT at the time of viral entry. 25 Thus, we investigated the correlation between levels of IL-6 and markers of coagulopathy and inflammation.

Since there was no correlation found between IL-6, ferritin, and the incidence of coagulopathy, we concluded that coagulopathy in COVID-19 patients could occur without the role of IL-6 and ferritin. Such explanations for this finding are: 1) ARDS pathophysiology in the infected subjects with moderate and severe illness is related to a massive loss of the angiotensin-converting enzyme (ACE)-2 enzyme, causing damage to the alveolar epithelium and vascular endothelium, 26 2) compartmentalization of the inflammatory cascade, as described by Chow and Tisoncik et al., 27 , 28 and 3) an acute phase response (APR) increase may be due to other pro-inflammatory cytokines, such as TNF-α or IL-1, 23 , 25 , 29 – 31 and the aforementioned vicious cycle of cellular destruction.

An analysis was carried out on the second blood collection after patients had a worsening during treatment to determine whether the incidence of coagulopathy correlates with IL-6 levels, in which the effect of IL-6 and cytokine storm was the utmost. The data from the second blood collection showed a significant correlation between the SpO 2/ FiO 2 ratio and the D-dimer, ferritin, PT, and IL-6 ( Figure 3). Furthermore, the D-dimer, fibrinogen, and PT were correlated with ferritin, while there was no correlation with IL-6. Our result demonstrated that inflammation correlates with coagulopathy, but conceivably not via a direct IL-6 pathway. To explain this finding, according to a study described by Sinha et al. that the interaction of mediators and pathways involved is not always constantly linear or uniform. 32

Worsening subjects showed significant changes in D-dimer, fibrinogen, and PT. Since there was no correlation between IL-6, ferritin and PT, fibrinogen, and D-Dimer, we determined the changes in the variables' mean values between the first and second blood collections. Furthermore, Wilcoxon's non-parametric test was performed since the data was not normally distributed ( Figure 4).

Figure 4. Variable alterations between the first and second sample collection in the worsening group.

Figure 4.

Open in a new tab

Notes: Ddimer1, Fibrinogen1, Prothrombin Time1, Ferritin1, IL-61: D-dimer, fibrinogen, prothrombin time, ferritin, and IL-6 at the first sample collection; Ddimer2, Fibrinogen2, Prothrombin Time2, Ferritin2, IL-62: D-Dimer, fibrinogen, prothrombin time, ferritin, and IL-6 at the second sample collection.

In worsening subjects, the second IL-6 blood collection had a higher value than the first one, albeit not statistically significant. Possible reasons include: 1) cytokines other than IL-6 affect the increase in D-dimer, fibrinogen, and ferritin levels, or 2) external factors, such as administration of anti-inflammatory corticosteroids and heparin, also have anti-inflammatory effects, 33 resulting in an insignificant increase of IL-6 and ferritin. During the study period, corticosteroids and heparin were administrated following the standard therapy for moderate and severe COVID-19 patients, or 3) large amounts of soluble IL-6 receptors production binds free IL-6 to reduce the concentration, as described by Garbers et al., 34 or 4) the presence of other cytokines (IL-6 cytokine family), including IL-11, 35 that also has a pro-inflammatory effect. 36

The disease may be influenced by the period between the onset and the patient's admission to the hospital. However, we found no differences between severe and moderate illness subjects. This result demonstrated that the disease onset was not correlated with the disease severity.

The disease severity was determined using WHO criteria for oxygen saturation, respiratory rate, and radiological findings. Most subjects were admitted to the emergency department with severe illness (83%), with a median SpO 2/ FiO 2 ratio was 97. In moderate illness, there were no differences in IL-6 at the baseline.

Our study found a negative correlation between D-dimer, fibrinogen, and PT levels with the SpO2/FiO2 ratio, reflecting the disease severity. This finding demonstrated that coagulopathy had a role in the patient's deterioration even though SpO2/FiO2 ratio showed no correlation with IL-6. The worsening lung function was due to pre-existing inflammation before the increase in IL-6 may explain the correlation with the coagulation system.

Sinha, 32 and Leisman 31 proposed that the pathophysiology of moderate and severe COVID-19 ARDS differs from typical ARDS, where the inflammatory system activation marked by increased IL-6 occurred only in a few COVID-19 ARDS patients. Our study showed that only 15.1% of subjects had IL-6 elevation at the initial examination, with an increase of 10-times the lower standard limit. Furthermore, if COVID-19 ARDS is considered a hyperinflammatory type, the IL-6 will be much lower than the value found in studies of the hyperinflammatory type that showed an increase of more than 100-times the lower standard limit.

The second blood collection sample showed that IL-6, ferritin, D-dimer, and PT had a statistically significant negative correlation with the SpO 2/ FiO 2 ratio, suggesting that deteriorating lung function is also correlated with inflammation and coagulopathy. However, we found no statistically significant correlation between IL-6 with D-dimer, fibrinogen, PT, and ferritin. As mentioned previously, this phenomenon may be caused by administering anti-inflammatory drugs that can reduce APR levels.

Fibrinogen is a soluble glycoprotein synthesized by the liver, and the activation can produce insoluble fibrin in the plasma. This process occurs via intrinsic and extrinsic pathways, and the activity is assessed by measuring PT.

Fibrinogen level is elevated in inflammatory conditions. 23 In patients who experienced worsening, we found that the fibrinogen levels at admission were higher than normal, with no changes in PT values. Thachil argued that the initial increase of fibrinogen would regulate inflammation. However, when the D-dimer levels continue to elevate with decreased fibrinogen levels, the protective role of fibrinogen ceases, and thrombus formation begins. 37 In our finding, the initial fibrinogen levels in moderate and severe COVID-19 were increased but decreased at the second blood collection sample analysis, which showed a tendency towards normal compared to the worsening group. This result demonstrated that hypercoagulable conditions might have a role in the pathology of the worsening group, a condition that differs from the hypothesis as reported by Thachil. 37 The hypercoagulable state can result from neutrophils’ subsequent pulmonary capillary endothelial activation, namely the formation of neutrophil extracellular traps (NETs). 38 – 42

The correlation between ferritin levels with D-dimer, fibrinogen, and PT

As part of APRs, ferritin was negatively correlated with the SpO 2/ FiO 2 ratio ( Figure 2). We assumed that the inflammation process had occurred prior to the patients' hospitalization, which explains the presence of a correlation between ferritin and the disease severity (moderate and severe). In the early phase, ferritin levels did not correlate with D-dimer, PT, and fibrinogen levels, which unlikely indicate that inflammation triggers coagulopathy.

On the initial observation, there was an elevation in ferritin levels in the subjects with moderate and severe illness ( Table 1). In the second blood collection sample, ferritin levels increased in the worsening but decreased in the improved group. Finally, we found the most significant changes in the improved group of moderate illness subjects ( Table 2), as also reported by Zhi et al. 24 To explain this finding, we hypothesized that 1) pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 can increase ferritin synthesis, 2) damage to the cellular level due to inflammation will release intracellular ferritin, 30 and 3) in acidosis, increased production of reactive oxygen species induce the release of iron from the ferritin. In addition, Fenton and Haber–Weiss reaction increases the concentration of hydroxyl radicals and eventually damages the cells. Overall, this process will initiate a vicious cycle of cell damage by increasing ferritin concentration.

Rosario, 43 explained that ferritin has immunosuppressant and pro-inflammatory effects. The immunosuppressant properties were found in the ferritin H fraction, supressing B lymphocyte antibodies' production. Additionally, ferritin also reduces granulocytes' phagocytosis, regulates the production of granulocytes-monocytes, and induces the production of IL-10 in lymphocytes. Moreover, it also inhibits the function of CXCR4 (chemokine receptor 4), an activator of the mitogen-activated protein kinase system that plays a role in the proliferation, differentiation, and migration of the cells. The role of ferritin in the inflammatory process has been described in hepatic stellate cells. For example, increased nuclear factor κB can promote the expression of inflammatory mediators, including IL-1β, and induce the production of nitric oxide synthase. This immunomodulatory process belongs to ferritin H and L properties. However, the role of ferritin as an inducer of inflammation or anti-inflammatory in COVID-19 infection has not been proven since the elevated ferritin type is still unknown. 20 , 30 , 43

The correlation between ferritin with fibrinogen, D-dimer, and PT in COVID-19 patients is recognized as a damage-associated molecular pattern that can modulate IL-6 concentrations. 30

There was no correlation between decreased SpO 2/ FiO 2 ratio and IL-6 in the first blood collection sample, but SpO 2/ FiO 2 ratio was correlated with acute-phase protein ferritin and fibrinogen. Albeit D-dimer, fibrinogen, and PT were correlated with the SpO 2/ FiO 2 ratio, we found no correlation between ferritin and those. Based on this finding, we demonstrated that the increase in APR does not correlate with the incidence of coagulopathy in COVID-19 since the condition has different mechanisms, including epithelial damage due to the entry of the SARS-CoV-2 virus and the increased production of NETs by neutrophils, as previously described.

Alteration of IL-6, ferritin, fibrinogen, PT, and prediction of COVID-19 severity

Theoretically, inflammatory (IL-6 and ferritin) and coagulopathy (D-dimer, fibrinogen, and PT) markers are correlated with patients' deterioration. Interleukin-6 regulates the inflammatory process, and the elevation is associated with the worsening of COVID-19 patients. This study found that increased levels of IL-6 can predict the deterioration of subjects, but it is insufficient to justify it as a routine examination. Additionally, uncorrelated IL-6 with coagulopathy markers, perhaps due to the ability of IL-6 to activate the inflammatory cascade in multiple body systems, 32 inducing amplification of the cascade.

Inflammation due to COVID-19 can activate epithelium, endothelium, macrophages, and neutrophils. Massive recruitment of neutrophils to the lungs and release of NETs regulates coagulation and IL-6 activation. 40 , 44 We found that alteration in D-dimer levels can predict the patients' deterioration. Therefore, the availability of D-dimer in hospitals can justify its function as a routine examination.

Although the alteration of ferritin and fibrinogen can not predict the patient's deterioration, these APR markers can be elevated in inflammatory conditions. 20 , 23 Thus, administration of corticosteroid therapy or anti-inflammatory agents, such as heparin, can reduce the inflammation process, 33 , 45 as marked in our study by the unaltered ferritin and decreased fibrinogen levels ( Table 2).

Prothrombin time is the period required for plasma to clot after adding tissue factor. The reaction velocity is the result of coagulation factors activation that consist of coagulation factors XII to X. Furthermore, Yu Zhang et al. found that PT had a sensitivity of 83.54% and specificity of 65.22% for sepsis patients at the cut-off 20. 46 Our study showed a 75% quartile of 12 in severe COVID-19 subjects, describing that levels of PT were insensitive to measure patient's deterioration. A similar finding was also reported in a retrospective study by Long et al., 47 where the initial measurement of PT was not correlated with the disease severity. This phenomenon may demonstrate the effect of hypercoagulation as the dominant pathophysiology in the early phase of the disease, in contrast to DIC in sepsis. 48 , 49

Our study limitation is that the IL-6's diurnal variation cannot be eliminated since the use of a consecutive sampling method and the disease phase factor.

Conclusion

In moderate and severe COVID-19 patients, there was a correlation between elevated IL-6 and D-dimer levels with disease deterioration. There was no correlation between elevated IL-6 levels with ferritin, D-dimer, fibrinogen, and PT levels.

Informed consent statement

Written informed consent from the patient/patient’s family for the use and publication of the patient’s data was obtained from all subjects involved in the study. Informed consent was conceptualized according to local ethics committee, and hospital review committee.

Funding Statement

The author(s) declared that no grants were involved in supporting this work.

[version 1; peer review: 1 approved with reservations

Data availability statement

Underlying data

Underlying data cannot be shared due to privacy concerns. Data will be made available to readers and reviewers on request from Alvin Tagor Harahap ( alvinharahap@gmail.com).

References

  • 1. Yoshikawa T, Hill T, Li K, et al. : Severe acute respiratory syndrome (SARS) coronavirus-induced lung epithelial cytokines exacerbate SARS pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells. J. Virol. 2009;83(7):3039–3048. 10.1128/JVI.01792-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. McGonagle D, O'Donnell JS, Sharif K, et al. : Immune mechanisms of pulmonary intravascular coagulopathy in COVID-19 pneumonia. Lancet Rheumatol. 2020;2(7):e437–e445. 10.1016/S2665-9913(20)30121-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Zhu Z, Cai T, Fan L, et al. : Clinical value of immune-inflammatory parameters to assess the severity of coronavirus disease 2019. Int. J. Infect. Dis. 2020;95:332–339. 10.1016/j.ijid.2020.04.041 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. McGonagle D, Sharif K, O'Regan A, et al. : The Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage Activation Syndrome-Like Disease. Autoimmun. Rev. 2020;19(6):102537. 10.1016/j.autrev.2020.102537 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Marietta M, Ageno W, Artoni A, et al. : COVID-19 and haemostasis: a position paper from Italian Society on Thrombosis and Haemostasis (SISET). Blood Transfus. 2020;18(3):167–169. 10.2450/2020.0083-20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Jin Y, Yang H, Ji W, et al. : Virology, Epidemiology, Pathogenesis, and Control of COVID-19. Viruses. 2020;12(4). 10.3390/v12040372 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Luo W, Zhang JW, Zhang W, et al. : Circulating levels of IL-2, IL-4, TNF-α, IFN-γ, and C-reactive protein are not associated with severity of COVID-19 symptoms. J. Med. Virol. 2021;93(1):89–91. 10.1002/jmv.26156 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. World Health Organization: Clinical management of COVID-19: interim guidance, 18 May 2020. Geneva: World Health Organization;2020. [Google Scholar]
  • 9. Anaconda Software Distribution: Anaconda Inc. 2020. Reference Source
  • 10. Zhang J, Hao Y, Ou W, et al. : Serum interleukin-6 is an indicator for severity in 901 patients with SARS-CoV-2 infection: a cohort study. J. Transl. Med. 2020;18(1):406. 10.1186/s12967-020-02571-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Zhang L, Yan X, Fan Q, et al. : D-dimer levels on admission to predict in-hospital mortality in patients with Covid-19. J. Thromb. Haemost. 2020;18(6):1324–1329. 10.1111/jth.14859 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Huang C, Wang Y, Li X, et al. : Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395(10223):497–506. 10.1016/S0140-6736(20)30183-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Wang Y, Lu X, Li Y, et al. : Clinical Course and Outcomes of 344 Intensive Care Patients with COVID-19. Am. J. Respir. Crit. Care Med. 2020;201(11):1430–1434. 10.1164/rccm.202003-0736LE [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Simonnet A, Chetboun M, Poissy J, et al. : High Prevalence of Obesity in Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Requiring Invasive Mechanical Ventilation. Obesity (Silver Spring). 2020;28(7):1195–1199. 10.1002/oby.22831 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Lariccia V, Magi S, Serfilippi T, et al. : Challenges and Opportunities from Targeting Inflammatory Responses to SARS-CoV-2 Infection: A Narrative Review. J. Clin. Med. 2020;9(12). 10.3390/jcm9124021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Chen T, Wu D, Chen H, et al. : Clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study. BMJ. 2020;368:m1091. 10.1136/bmj.m1091 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Zhou F, Yu T, Du R, et al. : Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020;395(10229):1054–1062. 10.1016/S0140-6736(20)30566-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Guan W-j, Ni Z-y, Hu Y, et al. : Clinical Characteristics of Coronavirus Disease 2019 in China. N. Engl. J. Med. 2020;382(18):1708–1720. 10.1056/NEJMoa2002032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Awasthi S, Wagner T, Venkatakrishnan AJ, et al. : Plasma IL-6 levels following corticosteroid therapy as an indicator of ICU length of stay in critically ill COVID-19 patients. Cell Death Dis. 2021;7(1):55. 10.1038/s41420-021-00429-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Kernan KF, Carcillo JA: Hyperferritinemia and inflammation. Int. Immunol. 2017;29(9):401–409. 10.1093/intimm/dxx031 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Cappanera S, Palumbo M, Kwan SH, et al. : When Does the Cytokine Storm Begin in COVID-19 Patients? A Quick Score to Recognize It. J. Clin. Med. 2021;10(2). 10.3390/jcm10020297 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Riviello ED, Kiviri W, Twagirumugabe T, et al. : Hospital Incidence and Outcomes of the Acute Respiratory Distress Syndrome Using the Kigali Modification of the Berlin Definition. Am. J. Respir. Crit. Care Med. 2016;193(1):52–59. 10.1164/rccm.201503-0584OC [DOI] [PubMed] [Google Scholar]
  • 23. Davidson SJ, editor. Inflammation and Acute Phase Proteins in Haemostasis. 2013.
  • 24. Lin Z, Long F, Yang Y, et al. : Serum ferritin as an independent risk factor for severity in COVID-19 patients. J. Infect. 2020;81(4):647–679. 10.1016/j.jinf.2020.06.053 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Ince C, Mayeux P, Nguyen T, et al. : The Endothelium in Sepsis. Shock (Augusta, Ga). 2016;45:259–270. 10.1097/SHK.0000000000000473 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Imai Y, Kuba K, Rao S, et al. : Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature. 2005;436(7047):112–116. 10.1038/nature03712 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Tisoncik JR, Korth MJ, Simmons CP, et al. : Into the eye of the cytokine storm. Microbiol. Mol. Biol. Rev. 2012;76(1):16–32. 10.1128/MMBR.05015-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Chow AW, Liang JF, Wong JS, et al. : Polarized secretion of interleukin (IL)-6 and IL-8 by human airway epithelia 16HBE14o- cells in response to cationic polypeptide challenge. PLoS One. 2010;5(8):e12091. 10.1371/journal.pone.0012091 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Gulhar R, Ashraf MA, Jialal I: Physiology, Acute Phase Reactants. StatPearls. Treasure Island (FL): StatPearls Publishing;2022. Copyright © 2022, StatPearls Publishing LLC. [PubMed] [Google Scholar]
  • 30. Moreira AC, Mesquita G, Gomes MS: Ferritin: An Inflammatory Player Keeping Iron at the Core of Pathogen-Host Interactions. Microorganisms. 2020;8(4). 10.3390/microorganisms8040589 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Leisman DE, Deutschman CS, Legrand M: Facing COVID-19 in the ICU: vascular dysfunction, thrombosis, and dysregulated inflammation. Intensive Care Med. 2020;46(6):1105–1108. 10.1007/s00134-020-06059-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Sinha P, Matthay MA, Calfee CS: Is a “Cytokine Storm” Relevant to COVID-19? JAMA Intern. Med. 2020;180(9):1152–1154. 10.1001/jamainternmed.2020.3313 [DOI] [PubMed] [Google Scholar]
  • 33. Mousavi S, Moradi M, Khorshidahmad T, et al. : Anti-Inflammatory Effects of Heparin and Its Derivatives: A Systematic Review. Adv. Pharmacol. Sci. 2015;2015:507151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Garbers C, Rose-John S: Genetic IL-6R variants and therapeutic inhibition of IL-6 receptor signalling in COVID-19. Lancet Rheumatol. 2021;3(2):e96–e97. 10.1016/S2665-9913(20)30416-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Kerr R, Stirling D, Ludlam CA: Interleukin 6 and haemostasis. Br. J. Haematol. 2001;115(1):3–12. 10.1046/j.1365-2141.2001.03061.x [DOI] [PubMed] [Google Scholar]
  • 36. Cook SA, Schafer S: Hiding in Plain Sight: Interleukin-11 Emerges as a Master Regulator of Fibrosis, Tissue Integrity, and Stromal Inflammation. Annu. Rev. Med. 2020;71:263–276. 10.1146/annurev-med-041818-011649 [DOI] [PubMed] [Google Scholar]
  • 37. Thachil J: The protective rather than prothrombotic fibrinogen in COVID-19 and other inflammatory states. J. Thromb. Haemost. 2020;18(8):1849–1852. 10.1111/jth.14942 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Arcanjo A, Logullo J, Menezes CCB, et al. : The emerging role of neutrophil extracellular traps in severe acute respiratory syndrome coronavirus 2 (COVID-19). Sci. Rep. 2020;10(1):19630. 10.1038/s41598-020-76781-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Nicolai L, Leunig A, Brambs S, et al. : Immunothrombotic Dysregulation in COVID-19 Pneumonia Is Associated With Respiratory Failure and Coagulopathy. Circulation. 2020;142(12):1176–1189. 10.1161/CIRCULATIONAHA.120.048488 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Middleton EA, He XY, Denorme F, et al. : Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome. Blood. 2020;136(10):1169–1179. 10.1182/blood.2020007008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Li T, Zhang Z, Li X, et al. : Neutrophil Extracellular Traps: Signaling Properties and Disease Relevance. Mediat. Inflamm. 2020;2020:9254087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Borges L, Pithon-Curi TC, Curi R, et al. : COVID-19 and Neutrophils: The Relationship between Hyperinflammation and Neutrophil Extracellular Traps. Mediat. Inflamm. 2020;2020:8829674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Rosário C, Zandman-Goddard G, Meyron-Holtz EG, et al. : The hyperferritinemic syndrome: macrophage activation syndrome, Still's disease, septic shock and catastrophic antiphospholipid syndrome. BMC Med. 2013;11:185. 10.1186/1741-7015-11-185 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Mozzini C, Girelli D: The role of Neutrophil Extracellular Traps in Covid-19: Only an hypothesis or a potential new field of research? Thromb. Res. 2020;191:26–27. 10.1016/j.thromres.2020.04.031 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Susilo A, Rumende CM, Pitoyo CW, et al. : Coronavirus disease 2019: Tinjauan literatur terkini. Jurnal penyakit dalam Indonesia. 2020;7(1):45–67. 10.7454/jpdi.v7i1.415 [DOI] [Google Scholar]
  • 46. Zhang Y, Khalid S, Jiang L: Diagnostic and predictive performance of biomarkers in patients with sepsis in an intensive care unit. J. Int. Med. Res. 2019;47(1):44–58. 10.1177/0300060518793791 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Long H, Nie L, Xiang X, et al. : D-Dimer and Prothrombin Time Are the Significant Indicators of Severe COVID-19 and Poor Prognosis. Biomed. Res. Int. 2020;2020:6159720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Iba T, Nisio MD, Levy JH, et al. : New criteria for sepsis-induced coagulopathy (SIC) following the revised sepsis definition: a retrospective analysis of a nationwide survey. BMJ Open. 2017;7(9):e017046. 10.1136/bmjopen-2017-017046 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Simmons J, Pittet JF: The coagulopathy of acute sepsis. Curr. Opin. Anaesthesiol. 2015;28(2):227–236. 10.1097/ACO.0000000000000163 [DOI] [PMC free article] [PubMed] [Google Scholar]
F1000Res. 2023 May 22. doi: 10.5256/f1000research.137385.r168232

Reviewer response for version 1

Gopal Krishna 1

The authors have conducted a good study. However, the parts of article are not properly organized. There is extensive overlap of results and discussion. The write up needs extensive revision. Some important aspects to be addressed:

Introduction:

  1. The introduction is very brief. Basic concepts regarding pathophysiological aspects of inflammation in COVID-19 may be described here. The role of parameters included in metadata and their behaviour in COVID-19 may be elucidated. 

  2. The treatment protocol pertaining to corticosteroids, low molecular weight heparin or UFH, and antiviral medications e.g. remdesivir etc. should be mentioned. And the modifications of these drugs according to the severity of disease.

Methods 

  1. The exclusion criteria should explain whether patients on oral anticoagulants, liver dysfunction were included or not. As a large size of study population had diabetes and hypertension, underlying coronary artery disease is likely to be present. Such population may have been on oral anticoagulant therapy prior to COVID-19.

  2. C-reactive protein have been also studied and detailed in result section. It is a new parameter which is described by authors in results bearing no mention in methods.

  3. The criteria of worsening of patients need to be described well according to citation used or modification, if any.

Results:

  1. "The patients were randomized" is not correct as selection of patients and their classification was the decision of authors.

  2. The part of discussion, "Fig 4.Variable alterations between the first and second sample collection in the worsening group" should be explained in the result section.

  3. With using terms like "most" and "majority", the percentages must be mentioned next to them.

  4. Repetition of many result parameters in discussion e.g., "males were most commonly affected in this study" is not appropriate.

  5. Attempt may be made to include data on patients who worsened.

  6. The normal reference laboratory values should be given for all parameters used in the study.

Discussion:

  1. This section needs to be rewritten as there are many grammatical errors. The authors must seek professional help.

  2. Many lines and paragraphs are not correct. e.g., ”This study found that increased levels of IL-6 can predict the deterioration of subjects, but it is insufficient to justify it as a routine examination. Additionally, uncorrelated IL-6 with coagulopathy markers, perhaps due to the ability of IL-6 to activate the inflammatory cascade in multiple body systems, 32  inducing amplification of the cascade".

  3. The part, “Although subjects did not experience an inflammatory process at admission" is not acceptable. Subjects do not experience inflammatory process they suffer with symptoms.

  4. In subheading "Correlation between levels of IL-6 with D-dimer, fibrinogen, and PT", "Since there was no correlation found between IL-6, ferritin, and the incidence of coagulopathy, we concluded. The word "concluded" may be changed to "assumed". Authors should mention e.g., "in the first sample analysis..." and then start elaboration. The entire part  does not fit well with subsequent portions of  discussion.  Authors are advised to write in flow oriented manner.

  5. The authors are also advised to discuss the results and its implications with a statistician. 

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

No

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Neuroendocrine disorders, Coagulation disorders in Neurosurgery, Neurospinal disorders, vascular Neurology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

F1000Res. 2023 Sep 15.
Cosphiadi Irawan 1

Thank you for your constructive review. We have made some corrections in respond to your feedback.

Introduction 

1. The introduction is very brief. Basic concepts regarding pathophysiological aspects of inflammation in COVID-19 may be described here. The role of parameters included in metadata and their behaviour in COVID-19 may be elucidated. 

  • We have reconstructed the introduction part to be more comprehensive and narrative. We included the concept of pathophysiological aspect of inflammation in Covid 19 and the reason we did the study

2. The treatment protocol pertaining to corticosteroids, low molecular weight heparin or UFH, and antiviral medications e.g. remdesivir etc. should be mentioned. And the modifications of these drugs according to the severity of disease. 

  • We have added the protocol of covid 19 management used in this study

Methods 

1. The exclusion criteria should explain whether patients on oral anticoagulants, liver dysfunction were included or not. As a large size of study population had diabetes and hypertension, underlying coronary artery disease is likely to be present. Such population may have been on oral anticoagulant therapy prior to COVID-19

  • We have added more detailed criteria of exclusion in the methods section and corrected ambiguous statement in the methods. The population with prior anticoagulant therapy was excluded

2. C-reactive protein have been also studied and detailed in result section. It is a new parameter which is described by authors in results bearing no mention in methods  

  • We have added explanation of CRP analysis in this study in method section. CRP was not the main focus of this study thus we did not describe CRP comprehensively in the method section.

3. "The patients were randomized" is not correct as selection of patients and their classification was the decision of authors.

  • we agreed and have revised the grammar

4. The part of discussion, "Fig 4.Variable alterations between the first and second sample collection in the worsening group" should be explained in the result section.

  • We added more explanations about fig 4

5. With using terms like "most" and "majority", the percentages must be mentioned next to them.

  • We agreed and added percentages next to them

6. Repetition of many result parameters in discussion e.g., "males were most commonly affected in this study" is not appropriate.

  • we reconstructed the statements and reduced repetitions

7. Attempt may be made to include data on patients who worsened.

  • we added some narratives about worsened patients in discussion

8. The normal reference laboratory values should be given for all parameters used in the study.

  • We added normal reference laboratory values as footnotes below the table 

Discussion:

1. This section needs to be rewritten as there are many grammatical errors. The authors must seek professional help.

  • We already consulted with professional translator and we made corrections in discussion. We hope the corrected version will convey the study result more clearly

2. Many lines and paragraphs are not correct. e.g., ”This study found that increased levels of IL-6 can predict the deterioration of subjects, but it is insufficient to justify  it as a routine examination. Additionally,  uncorrelated IL-6 with coagulopathy markerss due to the ability of IL-6 to activate the inflammatory cascade in multiple body systems, 32  inducing amplification of the cascade".,

  • we reconstructed the statements

3. The part, “Although subjects did not experience an inflammatory process at admission" is not acceptable. Subjects do not experience inflammatory process they suffer with symptoms.

  • we reconstructed the statement

4. In subheading "Correlation between levels of IL-6 with D-dimer, fibrinogen, and PT", "Since there was no correlation found between IL-6, ferritin, and the incidence of coagulopathy, we concluded. The word "concluded" may be changed to "assumed". Authors should mention e.g., "in the first sample analysis..." and then start elaboration. The entire part  does not fit well with subsequent portions of  discussion.  Authors are advised to write in flow oriented manner.

  • we tried to reconstruct the structure and add more conjunctive narrations. 

5. The authors are also advised to discuss the results and its implications with a statistician.

F1000Res. 2023 Mar 13. doi: 10.5256/f1000research.137385.r164119

Reviewer response for version 1

Azlan Bin Husin 1

Overall writing of this manuscript requires extensive language and grammatical improvement. Subject matter of interest were properly introduced but some of methodology parts were not defined. Hence, there is a missing connection between the subject of interest, methodology, results and discussion.

Introduction

The introduction needs to be revised to better contextualize on the work performed.

  1. Provide rationale of evaluating only prothrombin time and D-dimer instead of adding partial thromboplastin time too whereas it was mentioned in introduction that von Willebrand activity also enhanced.

  2. The term "effectiveness of parameters" in Introduction, para 2 is inappropriate.

  3. Provide rationale of evaluating SpO2/FiO2 ratio as the chosen severity index to compared with selected inflammatory markers.

Methods

This section requires substantial revision and improvement 

  1. Define the term "BALF" in Study design. 

  2. Clarify what happen if enrolled patients received IL-6 inhibitor or Ig later part of hospital stay? Example before day 14. Were they withdrawn or included in the analysis?

  3. CRP was not mentioned in the sample collection and tables but is showed in the graphs.

  4. Provide rationale and appropriate reference of using cut off point 14 days for assessment in the Assessment of patient's severity. What if patient stay longer than that and changed in clinical status?

  5. Revise the term "or considered complete" used in Assessment of patient's severity. Perhaps use 'censored'?

  6. Provide reference and appropriate citations used for definition of COVID-19 disease severity progression or worsening.

  7. Provide more information on the standardized approach of administering antibiotics, antivirals, corticosteroids and anticoagulant in the study center. This is to understand the different types of these agents used and possible interaction with the variables assessed.

  8. The term "coagulopathy" used in Statistical analysis, para 1 should be defined earlier.

  9. The term "randomized" in Results, para 1 is incorrect because the study population was not randomized. Perhaps 'randomly selected' is more suitable?

Results

  1. For Table 1; should add normal reference range as foot note.

  2. Statement "subjects with severe disease experienced worsening on day 14..." should be "subjects with severe disease experienced worsening within 14 days of..."

  3. Percentage of hypertension should be reflected in the table 1; can be as footnote.

  4. The statement "All subjects were given the combination of remdesivir, favipiravir, and oseltamivir" is not consistent with the actual number of patients received it.

  5. Author should describe the approach or basis of how different types of antibiotics given to these patients. This important to understand factors may influence the inflammatory markers evaluated in different category of patients.

  6. Statement "Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT" should be, "Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT levels".

  7. Disease severity index was not mentioned in introduction and methodology.

Discussion;  Characteristics of the study subjects

  1. Para 3, line 2: statement "In this phase, most patients begin to feel shortness of breath accompanied by hypoxia" is this refers to all mild cases? If so, then it is incorrect statement.

Discussion:  Correlation between increased levels of IL-6 and ferritin

  1. Para 2, line 1: statement should be "...patient did not show raised inflammatory markers...".

  2. The argument using Zhi et al., findings was rather contradicting to author's finding of no correlation with IL-6.

Discussion: Correlation between levels of IL-6 with D-dimer, fibrinogen, and PT

  1. It is difficult to analyze and to explain what really happened among worsening patients as no clear definition of this event and what type of treatment they received?

Conclusion

  1. Perhaps should include differences in inflammatory and hemostatic response between initial presentation and as patient became worse.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

No

Reviewer Expertise:

Clinical hematology, malignant hematology, benign hematology, autologous hematopoietic stem cell transplant

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

F1000Res. 2023 Sep 17.
Cosphiadi Irawan 1

Dear Azlan Bin Husin

We thank you for your constructive feedback. We made revisions in our article and hope you would not mind to review once again our article if it is eligible for indexing.

Introduction

The introduction needs to be revised to better contextualize on the work performed.

1. Provide rationale of evaluating only prothrombin time and D-dimer instead of adding partial thromboplastin time too whereas it was mentioned in introduction that von Willebrand activity also enhanced.

  • we added explanations in the introduction

2. The term "effectiveness of parameters" in Introduction, para 2 is inappropriate.

  • we fixed the grammar

3. Provide rationale of evaluating SpO2/FiO2 ratio as the chosen severity index to compared with selected inflammatory markers.

  • we added explanations in the introduction

Methods

This section requires substantial revision and improvement 

1. Define the term "BALF" in Study design. 

  • we added some explanations

2. Clarify what happen if enrolled patients received IL-6 inhibitor or Ig later part of hospital stay? Example before day 14. Were they withdrawn or included in the analysis?

  • the subjects were exempted. We revised the statement regarding the exclusion criteria

3. CRP was not mentioned in the sample collection and tables but is showed in the graphs.

  • We added some explanations in sample collection but we 

4. Provide rationale and appropriate reference of using cut off point 14 days for assessment in the Assessment of patient's severity. What if patient stay longer than that and changed in clinical status?

  • We used 14 days as cutoff based on studies reported the mean duration of disease progression typically occur at 7-14 days after onset of symptoms

Revise the term "or considered complete" used in Assessment of patient's severity. Perhaps use 'censored'?

  • We agreed with your feedback. we fixed the sentence

Provide reference and appropriate citations used for definition of COVID-19 disease severity progression or worsening.

  • We added references and explanations regarding Covid-19 disease severity

Provide more information on the standardized approach of administering antibiotics, antivirals, corticosteroids and anticoagulant in the study center. This is to understand the different types of these agents used and possible interaction with the variables assessed.

  • We added explanations of management protocol used in our center, but mostly were based on clinical judgement

The term "coagulopathy" used in Statistical analysis, para 1 should be defined earlier.

  • We added definition of coagulopathy in introduction

The term "randomized" in Results, para 1 is incorrect because the study population was not randomized. Perhaps 'randomly selected' is more suitable?

  • We revised the sentence like you suggested

Results

For Table 1; should add normal reference range as foot note.

  • We added normal reference in table footnote

Statement "subjects with severe disease experienced worsening on day 14..." should be "subjects with severe disease experienced worsening within 14 days of..."

  • We revised the statement

Percentage of hypertension should be reflected in the table 1; can be as footnote.

  • We decided to not mention any comorbidities percentage

The statement "All subjects were given the combination of remdesivir, favipiravir, and oseltamivir" is not consistent with the actual number of patients received it.

  • We revised the statements

Author should describe the approach or basis of how different types of antibiotics given to these patients. This important to understand factors may influence the inflammatory markers evaluated in different category of patients.

  • We added explanations in results section regarding the selection of type of antibiotics given to the patients

Statement "Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT" should be, "Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT levels".

  • We revised the sentence like you suggested

Disease severity index was not mentioned in introduction and methodology.

  • We added explanations regarding disease severity index in introduction

Discussion;  Characteristics of the study subjects

Para 3, line 2: statement "In this phase, most patients begin to feel shortness of breath accompanied by hypoxia" is this refers to all mild cases? If so, then it is incorrect statement.

  • We revised the statement. What we meant was most patients begin to feel chest discomfort and in moderate or severe cases, begin to feel shortness of breath.

Discussion:  Correlation between increased levels of IL-6 and ferritin

Para 2, line 1: statement should be "...patient did not show raised inflammatory markers...".

The argument using Zhi  et al., findings was rather contradicting to author's finding of no correlation with IL-6.

  • We reconstructed the statements.

Discussion:  Correlation between levels of IL-6 with D-dimer, fibrinogen, and PT

It is difficult to analyze and to explain what really happened among worsening patients as no clear definition of this event and what type of treatment they received?

  • We tried to explain the management protocol used in the result section and explain what happened in worsening group based on the markers analysed

Conclusion

Perhaps should include differences in inflammatory and hemostatic response between initial presentation and as patient became worse

  • We added more explanations regarding the difference in inflammatory and coagulation marker between initial and later sample.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Availability Statement

    Underlying data

    Underlying data cannot be shared due to privacy concerns. Data will be made available to readers and reviewers on request from Alvin Tagor Harahap ( alvinharahap@gmail.com).

    Articles from F1000Research are provided here courtesy of F1000 Research Ltd

    RESOURCES