Evaluation of ferritin level in COVID-19 patients and its inflammatory response.

The importance of ferritin as an inflammatory marker is well recognized. However, it is unknown whether this differs between Covid-19 and non-Covid-19 patients. The blood levels of ferritin, white blood cells (WBC), C-reactive protein (CRP), and lactate dehydrogenase may all be measured to check whether there is a difference. The researchers want to see if the inflammatory process changes between these two kinds (LDH). Methodology: Blood samples were collected from 119 COVID-19 patients in the hospital and 50 healthy persons. Corona virus was discovered when a nasopharyngeal swab was collected and tested using the RT-PCR technique. Ferritin, LDH, WBC, and CRP were also tested using Min Vidus, AccEnT 200, Ruby system, and Latx in that sequence. The study revealed that COVID-19 patients had higher levels of ferritin, WBC, CRP, and LDH in their blood than healthy people, with values of 539,08 ng/mL, 44.7109/L, 22.95 mg/L, and 403.95 U/L for COVID-19 patients versus 77.103 ng/mL, 4.9.4109/L, 6.53 mg/L, and 171.56 U/L for healthy people. According to the existing data, males are more likely to be infected with COVID-19 (81%) than females (32%), and females had greater ferritin, CRP, WBC, and LDH levels than males. Because they are related to an optimum test for predicting COVID-19 infection, the recommended cut-off values for ferritin, WBC, CRP, and LDH are 109.8 ng/mL, 14.9109/L, 10.15 mg/L, and 229.33 U/L, respectively. Finally, an increase in ferritin levels in the inflammatory response to COVID-19 is linked to an increase in inflammatory markers including CRP, WBC, and LDH, which may assist in the diagnosis of COVID-19 infection. © King Abdulaziz City for Science and Technology 2021.

Introduction

Coronavirus disease 19 (COVID-19) is a multisystemic disorder. The majority of COVID-19 patients, according to clinical data and statistics from the literature, have an abnormal inflammatory response to the viral infection, which leads to multi-organ failure and death (He et al. 2020; Wang et al. 2020a). Clinical symptoms in COVID-19 patients have been linked to a condition described as a “cytokine storm” (Tisoncik et al. 2012). As a result, when the host’s immune response to the virus is inadequate, the main response against the infection develops. To start the process, COVID-19 penetrates cells and binds with surface receptors (angiotensin-converting enzyme 2 receptor—ACE2-) (Guan et al. 2020; Wang et al. 2020b). The bulk of these receptors, located in upper respiratory epithelial type 2 cells, deliver damage-associated chemicals and activate the innate immune system (Xu et al. 2020; Cao 2020). Epithelial and endothelial cells, as well as macrophages, are then activated with the aid of monocytes and T cells, releasing IL-6, IP-10 (interferon gamma-induced protein-10), MIG chemokine, and monocyte chemoattractant protein-1 mediators and causing further inflammation. According to a recent study, iron metabolism may fluctuate drastically, and that these variations can be used to predict death in critical care patients. In COVID-19 patients, serum ferritin has recently been shown to be a predictor of death (Chen et al. 2020a; Spiezia et al. 2020; García 2020). As a result, we decided to compare ferritin levels in hospitalized COVID-19 patients with acute respiratory syndrome admitted in the Emergency Department versus non-COVID-19 patients to see if serum ferritin might be used as an inflammatory marker. Early detection of COVID-19 patients with poor prognostic features might be quite useful in deciding the treatment strategy (Zhang et al. 2020; Martinez-Outschoorn et al. 2011), preventing catastrophic effects and death. Inflammatory markers such as white blood cell count, lactate dehydrogenase, C-reactive protein, fibrinogen, and d-dimer are commonly employed in clinical practice to monitor sepsis progression (Yuan et al. 2020; Nienhold et al. 2020; Tan et al. 2020; Poggiali et al. 2020). In adult COVID-19 patients, white blood cell, lymphocyte, and cytokine levels can be used as important immune-inflammatory indicators (Faroogh et al. 2021b; Gao et al. 2020). Lymphocytes and their subtypes are essential for the immune system's proper functioning. Viral infections, like other infectious and immunological diseases, can cause lymphocyte and subgroup dysregulation (Abosaooda et al. 2021; Chan et al. 2004).

Lactate dehydrogenase is an enzyme that converts lactate to pyruvate and so contributes to energy production. It can be found in virtually every cell in the body, with the heart, liver, lungs, muscles, kidneys, and blood cells having the greatest concentrations. LDH is an inflammatory marker that may be used to determine if tissue damage is acute or persistent (Holmes and Goldberg 2009; Moghadasi et al. 2021). LDH levels have been reported to rise after acute and severe lung injury, and higher LDH levels have been identified in several interstitial lung infections (Shi and Pinto 2014). CRP is a good indicator of bodily inflammation. CRP is a liver protein whose transcription is influenced by the cytokines IL-6 and IL-1 (Black et al. 2004). Iron metabolism has been linked to several pathogenic disease processes, including infections and a number of hematological and immunological disorders. (Black et al. 2004). Despite the fact that ferritin is classified as an acute-phase protein, nothing is known about its involvement in monitoring inflammation, therefore, it is rarely employed for clinical evaluation. Despite the absence of a clear interpretation, iron metabolism is becoming more important in COVID-19 infection, and our data corroborate this.

Patients and methods

Study design and samples collection

Blood samples were obtained from 119 hospitalized COVID-19 patients at one hospital in China, from February 22, 2020, to June 23, 2020, as well as 50 healthy persons as a control group. After gaining consent from all participants, blood samples were taken and data were gathered. The questionnaire asked for participant’s (both patient and the healthy control) name, age, and gender. Furthermore, the study followed the hospital's ethics (Alhayani and llhan 2021; Alhayani et al. 2021; Zhang et al. 2014; Widjaja et al. 2021).

Laboratory tests

A nasopharyngeal swab was used to identify the presence of Corona virus in the Virology and Immunology Laboratory at the hospital, and the virus was examined using the RT-PCR method. Medical examination and X-rays were also performed to track the progress of the patient's health and determine the severity of the illness (Jalil et al. 2021). In addition, LDH, WBC, and CRP were measured in the hematology lab utilizing Min Vidus, AccEnT 200 (Kaplan and Meier-Kriesche 2002; Patschan et al. 2006; Failed 2020), Ruby system, and Latx, respectively.

Statistical analysis

Results with a probability value less than 0.05 were deemed statistically significant. The analysis was performed using Statistical Package for Social Sciences version 20 (SSPS20) computer program and Microsoft Excel 2010.

Results

The current study included blood samples of 119 patients infected with the Coronavirus. All the patients were aged between19 and 85 years, with a mean age of 51.36 ± 7.83 years. All these patients were clinically diagnosed by an RT-PCR examination. The control group included 50 uninfected healthy individuals their ages ranged between 18 and 86 years, with a mean age of 49.88 ± 9.11 years. No significant differences were reported when comparing the ages and genders of patients with healthy people, as in Table 1. On the other hand, the results of the current study showed that the number of COVID-19-infected males 81(68%) is more than COVID-19-infected females 38(32%).

Table 1

Comparison of patients and healthy control group according to age mean and gender

Age/year	Patients	Control	p value
Range	19–85	18–86
Mean ± SD	51.36 ± 7.83	49.88 ± 9.11	0.729 [NS]
SE	0.718	1.29

NS No Significant (p > 0.05), SD Standard Deviation, SE Standard Error, N Number

The results of the current study showed a high level of ferritin, WBC, CRP, and LDH in the blood of individuals infected with the Corona virus (539,08 ng/ml, 44.7 × 109/L, 22.95 mg/L, and 403.95 U/L, respectively) compared to the healthy group (77.103 ng/mL, 4.9.4 × 109/L, 6.53 mg/L, and 171.56 U/L, respectively). Moreover, this difference was found to be statistically significant (p = 0.002, 0.0018, 0.20, 0.025, respectively) as in Table 2.

Table 2

Comparison between the mean of serum/blood level of ferritin, WBC, CRP, and LDH of cases and controls

Inflammatory parameters	Patients	Control	p value
Ferritin	539.08 ng/mL	77.103 ng/mL	0.002*
WBC	44.7 × 109/L	4.9.4 × 109/L	0.0018*
CRP	22.95 mg/L	6.53 mg/L	0.020*
LDH	403.95 U/L	171.56 U/L	0.025*

*Significant association (p < 0.05)

On the other hand, the serum ferritin, blood WBC, and LDH showed a significant increase (p = 0.0322, 0.048 and 0.0379) among females (598.74 ng/mL, 47.9 × 109/L and 468.51 U/L, respectively) compared to males (511.21 ng/mL, 35.3 × 109/L and 373.66 U/L, respectively) except for the CRP appeared with a close concentration in both sexes (23.06 mg/L and 21.69 mg/L, respectively) with a small increase in females, which was not accompanied by statistical differences (p = 0.371) as in Table 3.

Table 3

Comparison between the mean of serum/blood level of ferritin, WBC, CRP, and LDH of COVID-19 infected females and males

Inflammatory parameters	Male	Female	p value
Ferritin	511.21 ng/mL	598.74 ng/mL	0.0322*
WBC	35.3 × 109/L	47.9 × 109/L	0.048*
CRP	21.69 mg/L	23.06 mg/L	0.371
LDH	373.66 U/L	468.51 U/L	0.0379*

*Significant association (p < 0.05)

As shown in Table 4, the optimum cut-off values for serum ferritin, WBC, CRP, and LDH are ≥ 109.8 ng/mL, ≥ 14.9 × 109/L, ≥ 10.15 mg/L, and ≥ 229.33 U/L, respectively. Moreover, increased values were associated with a perfect test for predicting COVID-19 infection so positive serum ferritin, WBC, CRP, and LDH at these cut-off values are 100% sensitive and specific and can establish a possible diagnosis of COVID-19 infection with 100% confidence. At the same time, testing negative (blood ferritin, WBC, CRP and LDH are < 109.8 ng/mL, < 14.9 × 109/L, < 10.15 mg/L and < 229.33 U/L, respectively) can exclude a possible diagnosis of Covid-19 infection with 100% confidence.

Table 4

Validity parameters for the optimal cut-off value for selected quantitative indices when used as a test to diagnosis in COVID-19 infection differentiating it from healthy control

Positive if ≥ cut-off value	Sensitivity%	Specificity %	Accuracy%	PPV%	DP%
Ferritin ≥ 109,8 ng/mL	100.0	100.0	100.0	100.0	50.00
WBC ≥ 14.9 × 109/L	100.0	100.0	100.0	100.0	50.00
CRP ≥ 10.15 mg/L	100.0	100.0	100.0	100.0	50.00
LDH ≥ 229.33 U/L	100.0	100.0	100.0	100.0	50.00

PPV positive predictive value, DP disease prevalence

To determine the pathological course of ferritin and its relationship to the studied inflammatory indicators, a linear Pearson correlation was conducted, which showed a strong positive linear correlation between ferritin and CRP (r = 0.601, p < 0.0001) while the relationship between ferritin and WBC was a linear moderate positive relationship with significant differences (r = 0.541, p < 0.0001), while the relationship between ferritin and LDH was linear positive (r = 0. 407, p < 0.0001) but weaker than CRP and WBC as shown in Table 5.

Table 5

Linear correlation between ferritin and studied inflammatory parameters

Inflammatory parameters	Inflammatory parameters range	Ferritin range (ng/mL)	Pearson correlation coefficient (r)	p value
CRP	5–35 mg/L	20–2000	0.601	< 0.0001*
LDH	92–1240 U/L	20–2000	0.407	< 0.0001*
WBC	10 × 109/L–77 × 109/L	20–2000	0.541	< 0.0001*

*Significant association (p < 0.05)

Discussion

Increasing evidence suggests that older age and male gender are important variables linked with a considerably higher risk of COVID-19-related incidents and mortality (Richardson et al. 2020; Onder et al. 2020; Guan et al. 2020). The present study found that males are more likely to be infected with the Coronavirus and that the number of deaths increases with age, which is consistent with numerous previous studies from other nations. In the Lombardy area of Italy, 82% of the 1591 patients admitted to ICU were male (Grasselli et al. 2020). Several other variables have been proposed to explain the gender discrepancy in COVID-19, including smoking and drinking habits, social and psychological characteristics, and the comorbidity profile of the sexes (Wenham et al. 2020). An inflammatory process' involving acute-phase response is characterized by a set of physiological and metabolic changes that occur shortly after tissue damage (Lucena et al. 2020). The change in the quantities of different plasma proteins, known as “acute-phase proteins,” is one of the many systemic symptoms of this acute-phase response (Lagadinou et al. 2020). Ferritin, C-reactive protein, amyloid material serum A, haptoglobin, fibrinogen, and LDH are the most commonly used markers in clinical practice (Lagadinou et al. 2020). Although serum ferritin has long been studied as a biomarker of iron metabolism (Faroogh et al. 2021a; Sajjad et al. 2021) it has recently gained prominence as a biomarker for inflammation in the context of COVID-19 development, as demonstrated by the current work and previous research in this field (Kell and Pretorius 2014; Cheung et al. 2020). As a result, a connection between ferritin and CRP, WBC, and LDH was identified in the current study. Ferritin is an acute-phase protein that is commonly elevated in inflammatory responses of various kinds (Henry et al. 2019).

The initial examination includes a complete blood count, ferritin, CRP, LDH, liver function tests, and other tests to detect the laboratory abnormalities most commonly linked with cytokine storm syndromes. The majority of healthcare institutions perform these tests and hence they are readily available in every health care setting. Furthermore, as Kell pointed out in 2014, ferritin can be a direct indication of cellular damage, especially when levels exceed 600 ng/mL (Kell and Pretorius 2014; Cheung et al. 2020; Banchini et al. 2021), indicating a link between organ damage and ferritin production. When this process becomes too vigorous, it results in cell death, known as ferroptosis. Acute respiratory distress syndrome is caused by this pneumonia, which is similar to COVID pneumonia (Sarjito et al. 2021) (ARDS). These findings suggest that the typical inflammatory process begins with SRIS-SEPSIS and then impacts iron metabolism. Iron deficiency, on the other hand, is the first sign in COVID patients, followed by SIRS and perhaps severe sepsis.

Ferritin may also be used as an indirect marker to determine if surgical patients with a negative SARS-CoV-2 swab were infected with COVID during their hospitalization, as well as an indication of severe sepsis (Alunno et al. 2020; Colafrancesco et al. 2020; Perricone et al. 2020). The number of white blood cells, as well as CRP and LDH levels, were all high in the blood samples of COVID-19 patients, especially in women. According to the current study, it was reported that in COVID-19 patients a high CRP concentration can be found. However, the concentration of WBC and LDH differed. The difference in inflammatory markers between males and females infected with COVID-19 might be due to hormonal changes linked to pregnancy and the menstrual cycle (Ruscitti and Giacomelli 2020; Bataille et al. 2020; Zhou et al. 2020a). The liver produces large amounts of acute-phase proteins (APPs), such as CRP, in response to infections (Khalil and Al-Humadi 2020; Kamal et al. 2019).

Acute inflammatory protein (Sproston and Ashworth 2018) is a very sensitive biomarker for inflammation, tissue damage, and infection. The intensity of inflammation has been associated with CRP levels (Rainer et al. 2009). CRP activates the complement system, which stimulates phagocytosis (Gershov et al. 2000). In other words, CRP attaches to bacteria and helps phagocytosis to remove them (Povoa et al. 2009). The non-severe and severe COVID groups had no statistically significant difference in CRP levels in the Chen et al. trial, however, the severe group's mean CRP level was greater than the non-severe group (Chen et al. 2020b). It was reported previously that certain patients with COVID-19 had increased white blood cell and neutrophil counts, as well as a decreased lymphocyte count. Moreover, it was also reported that the neutrophil-to-lymphocyte ratio can be utilized as a biomarker to predict how the infection will progress. Other studies, on the other hand, claim that the specific roles and subtypes of white blood cells in severe COVID-19 remain unclear (Guan et al. 2020; Zhou et al. 2020b). The researchers added that the frequency of different cell types might be influenced by several factors, including age, gender, sickness, and medications.

According to research (Mao et al. 2020; Saleh et al. 2020), White WBC count and peripheral blood lymphocytes are normal or slightly decreased in the early stages of COVID-19 infection, when patients have no symptoms, but these markers may change as the disease progresses. In a study of 140 hospitalized patients diagnosed with COVID-19 based on CT scan results, Zhang et al. found that the leukocyte count was within normal limits in 68.1% of patients diagnosed with COVID-19, which is increased in 12.3% of patients, and decreased in 19.6% of patients.

Increased LDH level indicated that multiple organ damage may have a bigger role in deciding clinical outcomes in patients with COVID-19. Cytokines and LDH production can induce tissue damage, which can lead to serious infections (Jalil et al. 2020). Patients with severe COVID-19 infections can expect to produce more LDH into the blood since LDH is present in lung tissue (isozyme 3), as the disease is characterized by a severe kind of interstitial pneumonia that generally leads to acute respiratory distress syndrome. However, the contribution of different LDH isoenzymes to the observed LDH rise in COVID-19 has yet to be established. Thrombotic microangiopathy, which is associated with renal failure and heart injury and may be caused by COVID-19 infection, also has elevated LDH levels (Kwekha-Rashid et al. 2021; Zhang et al. 2020; Martinez-Outschoorn et al. 2011).

The small number of patients enrolled, and the lower incidence of which was primarily abnormal serum ferritin tests in the early period is one of the major shortcomings of this study. Moreover, the retrospective nature of the study, which needs a bigger analysis to reveal current results is another limitation of the present study.

Conclusions and recommendations

The current study showed that most of the COVID-19-infected patients in the intensive care unit were males, and laboratory tests showed an elevated serum level of ferritin, which was accompanied by a high percentage of WBC, CRP, and LDH, especially in females. The optimum cut-off values for serum ferritin, WBC, CRP, and LDH are ≥ 109.8 ng/ml, ≥ 14.9 × 109/L, ≥ 10.15 mg/L, and ≥ 229.33 U/L, respectively, and can be included as a possible diagnosis of COVID-19 infection with 100% confidence, and these levels in COVID-19 patients should be closely monitored for any signs of disease progression or decompensation.