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Abstract

Objectives: The induction of an intense immune response and cytokine storm is proposed to be central in the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The study evaluated serum cytokine/chemokine profiles, and clinical and paraclinical data of COVID-19 deceased and recovered patients in Iran. Methods: The severity of disease, clinical data, and routine laboratory and inflammatory cytokine/chemokine responses were retrospectively explored in 60 in-hospital patients in northern Iran. Characteristics of those who deceased (n = 30) were compared to recovered (n = 30), and associations with serum levels of potential disease regulating pro- and anti-inflammatory mediators were studied. Results: The serum levels of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IP-10, MIP1-α, MCP1, RANTES, and TNF-α were upregulated in all COVID-19 patients when compared to healthy and gender-matched individuals (n = 30). Although with no significant difference between deceased and recovered cases, the serum levels of all cytokines/chemokines tended to be higher in the severely diseased non-surviving patients. Association analyses revealed that all cytokine/chemokine levels (except IL-10) significantly affect the disease outcome. Conclusion: This study provides more evidence for the association of cytokine/chemokine levels with the clinical course and outcome of COVID-19. More studies are needed to consider this measurement as an indicator of disease stage and strategy for treatment.

Keywords

coronavirus disease 2019 severe acute respiratory syndrome coronavirus 2 cytokine/chemokine profile clinical/paraclinical data

Introduction

The coronavirus disease 2019 (COVID-19), caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has swept across the globe, infected more than 535 million people, and killed over 6.31 million.¹ While most develop a mild to moderate disease, the infection appears lethal in a significant proportion of patients (with the range of 3.3–8.8%).^1,2 Growing evidence on the pathogenesis of SARS-CoV-2 indicates a dysregulated and intense immune response as the leading contributor to disease development.^3,4 Although lessons from the previous coronaviruses can be drawn, there is still much to explore on whether SARS-CoV-2 behaves similarly to its predecessors or is characterized by peculiar specificities.⁵

The immune response triggered by SARS-CoV-2 infection acts as a “double-edged sword”. A strong response is essential to eliminate viral pathogens, whereas a dysregulated and intense inflammatory response can damage the respiratory tract.^6,7 Studies show that underlying disease, old age, high viral titer, and sustained inflammation (known as cytokine storm) correlate with adverse outcomes of the virus infection.⁸ An increased number of innate immune cells such as macrophages/monocytes and neutrophils, and high concentrations of different cytokines and chemokines, were found in critically ill COVID-19 patients.^9–16 It is well hypothesized that the intensity of the cytokine storm in these patients is associated with disease severity and outcomes.

So far, no effective treatment for COVID-19 has been successfully developed.¹⁷ As declared, the intense and uncontrolled inflammation induced by SARS-CoV-2 leads to severe disease, increased morbidity, and mortality, so dampening and downregulating the inflammatory response and reducing its intensity could be a promising therapy.^18,19 Exploring broad with patients in different geographical areas and ethnical groups might pave the way toward controlling SARS-CoV-2 immunopathogenesis.²⁰ The challenges would be to increase knowledge and understand the physiopathology of COVID-19 and emerging mutants.

The hide-and-seek challenge of immune responses between the host and virus, understanding the viral-induced mechanisms that increase viral infectivity and lead to severe and fatal disease, and the associated intensity and character of the immune response need to be explored and understood. While many studies approved the association between serum cytokine profile and COVID-19 severity and outcome, there are no comprehensive studies from Iran. We aimed to find if any measure could reveal COVID-19 patients are at higher risk of dying.

Materials & Methods

This case-control study was performed on samples and data collected from patients hospitalized between February and December 2020 in the Golestan Province, north of Iran. Sixty confirmed COVID-19 cases, and 30 healthy subjects were enrolled in this study. The mean age for healthy, recovered, and deceased subjects were 40.00 ± 7.22, 56.97 ± 15.75, and 63.30 ± 13.71 years, respectively. The COVID-19 patients were confirmed positive by real-time RT-PCR assay targeting the SARS-CoV-2 nucleoprotein (N) and ORF1ab genes (Pishtazteb, Iran). Blood samples of patients were collected immediately after hospitalization. All COVID-19 patients were included in the severe group, with oxygen saturations <93% and arterial blood oxygen partial pressure (PaO2)/oxygen concentration (FiO2) ≤ 300 mm Hg and needed intubation and admission to the intensive care unit (ICU). Patients were followed and divided into recovered (n = 30) and deceased (n = 30) groups. Serum samples from 30 healthy individuals collected before the pandemic (during 2018) were used as a control group. Data of age, gender, clinical symptoms and signs, and routine laboratory tests were collected from patient records at admission. The study was approved by the Ethics Committee of Golestan University of Medical Sciences (IR.GOUMS.REC.1399.007) and performed under the declaration of Helsinki for medical research involving human subjects.²¹

Serum samples were stored at −80°C until cytokine and chemokine analysis. The cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, and TNF-α) and chemokines (IL-8, IP-10, MIP1-α, MCP1, and RANTES) levels were measured with commercial ELISA kits according to the manufacturer’s instruction (Invitrogen, USA). The sensitivity of detection for IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IP-10, MIP1-α, MCP1, RANTES and TNF-α were 4, 0.3, 9.1, 1.3, 1.5, 0.92, 5, 1, 2.1, 0.4, 2, 2, 2.3, 2, and 2.3 pg/mL, respectively. Demographic, clinical, and laboratory data and cytokine/chemokine levels were compared between groups and associations between groups done.

Statistical Analysis

Data were analyzed using SPSS22 software (SPSS Inc, Chicago, Illinois, USA). The normality status of the data was assessed with the Kolmogorov-Smirnov test. The Fisher exact test was used to distribute binary variables in the study groups. Comparing of the mean of continuous variables in the study groups was done using the Mann-Whitney U or Kruskal-Wallis tests, followed by paired comparison using the Tukey post-hoc test. Correlations were assessed by Spearman’s rank correlation coefficient. Graphs were produced using SPSS22 software. The results were considered statistically significant if the p-values were <0.05.

Results

Of all cases, 41 (45.6%) and 49 (54.4%) were males and females, respectively. The mean age for healthy, recovered, and deceased subjects were 40.00 ± 7.22, 56.97 ± 15.75, and 63.30 ± 13.71 years, respectively, with significant differences in mean age between healthy, recovered, and deceased subjects (p < .001). Clinical data such as fever (61.7%), cough (41.7%), dyspnea (41.7%), headache (38.4%), myalgia (36.7%), sputum (18.3%), diarrhea (16.7%), sore throat (15%), and vomiting (8.3%) were obtained among COVID-19 patients (recovered and deceased). There were differences in symptoms such as dyspnea (p = .001), myalgia (p < .001), and sputum (p = .02) between deceased and recovered COVID-19 cases. Demographic and clinical data are shown in Table 1.

Table 1.

Demographic and clinical data in healthy, recovered, and deceased cases.

Variables	Healthy, N (%)	Recovered, N (%)	Deceased, N (%)	p-value
Gender
Female	16 (53.3)	18 (60)	15 (50)	0.73^*
Male	14 (46.7)	12 (40)	15 (50)	0.73^*
Age (Mean±SD)	40 ± 7.22	56.97 ± 15.75	63.30 ± 13.71	<0.001
Symptoms
Fever	—	17 (56.7)	20 (66.7)	0.63^**
Cough	—	13 (43.3)	12 (40)	0.50^**
Dyspnea	—	8 (26.7)	17 (56.7)	0.001^**
Headache	—	11 (36.7)	12 (40)	0.82^**
Myalgia	—	19 (63.3)	3 (10)	<0.001^**
Sputum	—	2 (6.7)	9 (30)	0.02^**
Diarrhea	—	3 (10)	7 (23.3)	0.16^**
Sore throat	—	5 (16.7)	4 (13.3)	0.500^**
Vomiting	—	2 (6.7)	3 (10)	0.641^**

*Chi square test, **Fisher exact test.

Laboratory data of WBC (White Blood Cells), RBC (Red Blood Cells), Hb (Hemoglobin), HCT (Hematocrit), MCV (Mean Corpuscular Volume), MCH (Mean Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration), PLT (Platelet Cells), RDW (Red Cell Distribution Width), MPV (Mean Platelet Volume), PDW (Platelet Distribution Width), P-LCR (Platelet-large cell ratio), ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase), ALP (Alkaline Phosphatase), CPK (Creatinine Phosphokinase), LDH (Lactate Dehydrogenase), Mg (Magnesium), PMN (Polymorph Nuclear Leukocytes), Lymph (Lymphocyte), and electrolytes were statistically analyzed in all groups. The results revealed that ALP (p < .001), Calcium (p < .001), Phosphorus, (p = .002), PMN (p = .003), and monocyte counts (p < .001) were significantly higher, and PLT (p = .036) was significantly lower in deceased versus recovered. Moreover, significant differences were observed between COVID-19 patients and healthy subjects. Details of laboratory data are shown in Table 2.

Table 2.

Laboratory data in healthy, recovered, and deceased cases.

Variables	Group	Mean ± SD	p-value	Group	p-value	Group	p-value	Group	p-value
WBC (μ.l)	Deceased	10.11 ± 4.93	.003	Deceased	.004	Deceased	.998	Healthy	.005
	Healthy	6.72 ± 1.93		Healthy		Recovered		Recovered
	Recovered	10.31 ± 5.46		Healthy		Recovered		Recovered
RBC (μ.l)	Deceased	4.13 ± 0.35	<.001	Deceased	.001	Deceased	.95	Healthy	<.001
	Healthy	4.67 ± 0.59		Healthy		Recovered		Recovered
	Recovered	4.10 ± 0.63		Healthy		Recovered		Recovered
Hb (mg.dl)	Deceased	11.49 ± 1.78	.493	Deceased	—	Deceased	—	Healthy	—
	Healthy	12.06 ± 1.72		Healthy		Recovered		Recovered
	Recovered	11.85 ± 2.7		Healthy		Recovered		Recovered
HCT (%)	Deceased	34.80 ± 4.95	.148	Deceased	—	Deceased	—	Healthy	—
	Healthy	37.27 ± 4.18		Healthy		Recovered		Recovered
	Recovered	35.57 ± 5.64		Healthy		Recovered		Recovered
MCV (FL)	Deceased	86.22 ± 5.43	<.001	Deceased	.002	Deceased	.803	Healthy	<.001
	Healthy	80.48 ± 7.43		Healthy		Recovered		Recovered
	Recovered	87.25 ± 5.87		Healthy		Recovered		Recovered
MCH (pg)	Deceased	27.96 ± 2.22	<.001	Deceased	.020	Deceased	.207	Healthy	<.001
	Healthy	26.18 ± 3.12		Healthy		Recovered		Recovered
	Recovered	29.06 ± 2.03		Healthy		Recovered		Recovered
MCHC (%)	Deceased	34.45 ± 1.89	<.001	Deceased	<.001	Deceased	.170	Healthy	.014
	Healthy	32.19 ± 1.40		Healthy		Recovered		Recovered
	Recovered	33.33 ± 1.27		Healthy		Recovered		Recovered
PLT (mm3.μl)	Deceased	155.57 ± 79.64	.001	Deceased	.001	Deceased	.036	Healthy	.44
	Healthy	239.57 ± 57.11		Healthy		Recovered		Recovered
	Recovered	212 ± 114.21		Healthy		Recovered		Recovered
RDW (μm)	Deceased	47.36 ± 4.18	<.001	Deceased	<.001	Deceased	.978	Healthy	<.001
	Healthy	12.88 ± 0.43		Healthy		Recovered		Recovered
	Recovered	47.15 ± 4.30		Healthy		Recovered		Recovered
MPV(fl)	Deceased	10.25 ± 1.08	.257	Deceased	—	Deceased	—	Healthy	—
	Healthy	NA		Healthy		Recovered		Recovered
	Recovered	9.94 ± 1.06		Healthy		Recovered		Recovered
PDW (%)	Deceased	13.64 ± 3.28	.612	Deceased	—	Deceased	—	Healthy	—
	Healthy	NA		Healthy		Recovered		Recovered
	Recovered	13.23 ± 2.88		Healthy		Recovered		Recovered
P-LCR (ng.ml)	Deceased	27.31 ± 7.79	.60	Deceased	—	Deceased	—	Healthy	—
	Healthy	NA		Healthy		Recovered		Recovered
	Recovered	26.30 ± 7.74		Healthy		Recovered		Recovered
Urea (mg.dl)	Deceased	59.37 ± 56.55	.008	Deceased	.035	Deceased	.909	Healthy	.011
	Healthy	30.77 ± 4.55		Healthy		Recovered		Recovered
	Recovered	64.07 ± 50.26		Healthy		Recovered		Recovered
Creatinine (mg.dl)	Deceased	2.01 ± 1.73	.012	Deceased	.009	Deceased	.153	Healthy	.478
	Healthy	1.07 ± 0.89		Healthy		Recovered		Recovered
	Recovered	1.43 ± 1.16		Healthy		Recovered		Recovered
AST (IU.L)	Deceased	60.43 ± 65.69	.119	Deceased	.248	Deceased	.931	Healthy	.127
	Healthy	22.66 ± 6.73		Healthy		Recovered		Recovered
	Recovered	68.83 ± 140.60		Healthy		Recovered		Recovered
ALT (IU.L)	Deceased	57.47 ± 39.59	<.001	Deceased	<.001	Deceased	.129	Healthy	.033
	Healthy	23 ± 8.04		Healthy		Recovered		Recovered
	Recovered	42.50 ± 31.66		Healthy		Recovered		Recovered
ALP (IU.L)	Deceased	264.97 ± 87.89	<.001	Deceased	<.001	Deceased	<.001	Healthy	.986
	Healthy	173.48 ± 40.25		Healthy		Recovered		Recovered
	Recovered	170.13 ± 77.89		Healthy		Recovered		Recovered
LDH (U.L)	Deceased	856.13 ± 735.36	.152	Deceased	—	Deceased	—	Healthy	—
	Healthy	NA		Healthy		Recovered		Recovered
	Recovered	645.80 ± 299.10		Healthy		Recovered		Recovered
CPK (U.L)	Deceased	413.97 ± 560.63	.013	Deceased	—	Deceased	—	Healthy	—
	Healthy	NA		Healthy		Recovered		Recovered
	Recovered	147.73 ± 82.80		Healthy		Recovered		Recovered
Mg (mEq.L)	Deceased	2.05 ± 0.55	.009	Deceased	—	Deceased	—	Healthy	—
	Healthy	NA		Healthy		Recovered		Recovered
	Recovered	1.74 ± 0.26		Healthy		Recovered		Recovered
Ca (mg.dl)	Deceased	8.05 ± 0.36	<.001	Deceased	<.001	Deceased	<.001	Healthy	.271
	Healthy	9.17 ± 0.36		Healthy		Recovered		Recovered
	Recovered	8.88 ± 0.72		Healthy		Recovered		Recovered
P (mg.dl)	Deceased	4.30 ± 0.60	.002	Deceased	<.001	Deceased	.002	Healthy	.889
	Healthy	3.87 ± 0.58		Healthy		Recovered		Recovered
	Recovered	3.78 ± 0.49		Healthy		Recovered		Recovered
Na (mEq.L)	Deceased	135.46 ± 23.12	.530	Deceased	—	Deceased	—	Healthy	—
	Healthy	NA		Healthy		Recovered		Recovered
	Recovered	138.16 ± 3.97		Healthy		Recovered		Recovered
K (mEq.L)	Deceased	3.96 ± 0.35	.418	Deceased	—	Deceased	—	Healthy	—
	Healthy	NA		Healthy		Recovered		Recovered
	Recovered	4.08 ± 0.72		Healthy		Recovered		Recovered
PMN (%)	Deceased	86.97 ± 5.98	<.001	Deceased	<.001	Deceased	.003	Healthy	<.001
	Healthy	56.97 ± 5.49		Healthy		Recovered		Recovered
	Recovered	81.07 ± 8.21		Healthy		Recovered		Recovered
Lymph (%)	Deceased	9.80 ± 6.08	<.001	Deceased	<.001	Deceased	.127	Healthy	<.001
	Healthy	38.63 ± 5.46		Healthy		Recovered		Recovered
	Recovered	13.17 ± 8.08		Healthy		Recovered		Recovered
Monocyte (%)	Deceased	1.60 ± 0.67	<.001	Deceased	<.001	Deceased	<.001	Healthy	.991
	Healthy	2.93 ± 1.01		Healthy		Recovered		Recovered
	Recovered	2.90 ± 1.26		Healthy		Recovered		Recovered
Eosinophil (%)	Deceased	1.53 ± 0.62	.135	Deceased	.947	Deceased	.262	Healthy	.147
	Healthy	1.47 ± 0.83		Healthy		Recovered		Recovered
	Recovered	1.87 ± 0.97		Healthy		Recovered		Recovered

WBC: White Blood Cells, RBC: Red Blood Cell, Hb: Hemoglobin, HCT: Hematocrit, MCV: Mean Corpuscular Volume, MCH: Mean Corpuscular Hemoglobin, MCHC: Mean Corpuscular Hemoglobin Concentration, PLT: Platelet Cells, RDW: Red Cell Distribution Width, MPV: Mean Platelet Volume, PDW: Platelet Distribution Width, P-LCR: Platelet-large cell ratio, AST: Aspartate Aminotransferase, ALT: Alanine Aminotransferase, ALP: Alkaline Phosphatase, CPK: Creatinine Phosphokinase, LDH: Lactate Dehydrogenase, Mg: Magnesium, Ca: Calcium, P: Phosphorus, Na: Sodium, K: Potassium, PMN: Polymorph nuclear leukocytes, Lymph: Lymphocyte, NA: Not Available.

ELISA analyses showed levels of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IP-10, MIP1-α, MCP1, RANTES, and TNF-α significantly higher in COVID-19 patients compared to healthy individuals (Figure 1 and Figure 2). Although with no significant difference between recovered and deceased cases, the serum levels of all cytokines/chemokines tended to be higher in the severely diseased non-surviving patients (supplementary File 1). Association analyses between cytokine/chemokine levels and clinical data revealed that all cytokine/chemokine levels (except IL-10) significantly affected the clinical course and outcome of COVID-19. Moreover, we found significant associations between MIP1-α with cough in the recovered group, IL-12 with dyspnea in the deceased group, MCP-1 with myalgia in recovered groups, IL-1β with dyspnea in the deceased and recovered groups, MIP1-α with headache in the deceased group, IL-6 and MIP1-α with diarrhea in the recovered group, and IP-10 with vomiting in deceased and recovered groups. Associations between cytokine/chemokine levels and clinical data are shown in Table 3.

Figure 1.

The levels of cytokines in healthy, recovered and deceased case. The serum concentration of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, and TNF-α from 60 COVID-19 patients and 30 controls were analyzed immediately after hospital admission. Median with range was presented.

Figure 2.

The levels of chemokines in healthy, recovered and deceased case. The serum concentration of IL-8, IP-10, MIP1-α, MCP1, and RANTES from 60 COVID-19 patients and 30 controls were analyzed immediately after hospital admission. Median with range was presented.

Table 3.

Association between cytokine/chemokine levels and clinical data.

Variables		IFN-γ	IL-1β	IL-2	IL-4	IL-5	IL-6	IL-8	IL-10	IL-12	IL-17	IP-10	MCP-1	MIP-1α	RANTES	TNF-α
Outcome
Deceased		30.66 ± 14.98	2.47 ± 1.84	18.45 ± 12.79	7.26 ± 4.13	7.12 ± 4.65	2.32 ± 1.49	9.65 ± 6.59	2.99 ± 2.25	6.59 ± 4.58	2.78 ± 2.17	7.13 ± 5.30	8.39 ± 4.41	7.23 ± 4.33	30.85 ± 15.59	12.92 ± 6.91
Healthy		3.99 ± 2.22	0.64 ± 0.90	9.15 ± 5.91	1.53 ± 1.73	1.67 ± 0.89	0.47 ± 0.41	5.17 ± 2.05	1.73 ± 1.47	2.37 ± 2.15	1.07 ± 0.89	2.22 ± 1.42	4.39 ± 2.28	4.10 ± 2.14	7.25 ± 5.88	6.42 ± 2.55
Recovered		26.19 ± 13.8	1.90 ± 1.55	14.94 ± 11.69	5.98 ± 3.20	6.09 ± 3.88	1.76 ± 1.20	8.05 ± 4.31	2.63 ± 2.10	5.02 ± 3.77	2.17 ± 1.91	5.85 ± 5.06	7.29 ± 480	6.24 ± 3.76	26.09 ± 15.50	10.35 ± 5.12
p-value		<.001	<.001	.004	<.001	<.001	<.001	.004	.058	<.001	.001	.001	<.001	.004	<.001	<.001
Deceased	Male	29.43 ± 12.76	2.51 ± 1.98	21.61 ± 13.14	8.84 ± 3.80	7.73 ± 4.63	2.27 ± 1.71	9.80 ± 7.59	2.92 ± 2.09	5.61 ± 3.83	2.74 ± 2.29	6.35 ± 4.89	7.32 ± 4.20	6.95 ± 4.49	29.35 ± 13.68	10.72 ± 5.61
Deceased	Female	31.90 ± 17.29	2.42 ± 1.75	15.30 ± 12.04	5.68 ± 3.95	6.52 ± 4.75	2.37 ± 1.30	9.50 ± 5.69	3.06 ± 2.48	7.58 ± 5.16	2.83 ± 2.12	7.92 ± 5.74	9.46 ± 4.50	7.50 ± 4.31	32.34 ± 17.64	15.11 ± 7.56
p -value		.693	.88	.394	.29	.29	.547	.64	.91	.299	.83	.289	.218	.575	.589	.056
Healthy	Male	3.52 ± 1.89	0.79 ± 1.19	9.76 ± 6.30	1.46 ± 1.14	1.92 ± 1.02	0.50 ± 0.47	5.46 ± 2.65	1.96 ± 1.73	2.42 ± 2.11	1.20 ± 0.92	2.00 ± 1.20	3.75 ± 1.24	4.42 ± 2.35	8.69 ± 6.33	6.77 ± 2.42
Healthy	Female	4.40 ± 2.45	0.50 ± .57	8.61 ± 5.71	1.59 ± 2.16	1.44 ± 0.72	0.44 ± 0.36	4.91 ± 1.38	1.53 ± 1.23	2.33 ± 2.24	0.96 ± 0.88	2.41 ± 1.60	4.96 ± 2.83	3.81 ± 1.96	6 ± 5.34	6.11 ± 2.69
p -value		.252	.80	.983	.69	.21	.917	.63	.80	.917	.21	.771	.389	.545	.453	.33
Recovered	Male	21.85 ± 12.01	1.81 ± 1.58	13.45 ± 10.78	6.01 ± 3.32	6.35 ± 4.48	1.73 ± 1.03	6.62 ± 3.06	3.05 ± 2.43	6.05 ± 4.18	2.49 ± 2.37	6.15 ± 5.25	7.1 ± 5.01	5.74 ± 3.41	27.51 ± 17.35	10.90 ± 6.45
Recovered	Female	29.08 ± 14.47	1.96 ± 1.57	15.93 ± 12.47	5.96 ± 3.22	5.91 ± 3.55	1.78 ± 1.32	9.01 ± 4.81	2.36 ± 1.87	4.34 ± 3.42	1.95 ± 1.58	5.64 ± 5.08	7.42 ± 4.79	6.57 ± 4.03	25.14 ± 14.59	9.98 ± 4.19
p -value		.362	.91	.672	.83	.81	.735	.35	.45	.290	.73	.932	.671	.687	.983	.58
Fever
Deceased	Yes	32.38 ± 16.65	2.76 ± 1.94	17.12 ± 12.80	6.73 ± 3.92	7.14 ± 4.80	2.30 ± 1.47	9.41 ± 6.45	3.13 ± 2.13	7.13 ± 4.93	3.15 ± 2.45	6.99 ± 5.19	8.24 ± 4.23	7.72 ± 4.60	31.05 ± 15.94	13.20 ± 7.03
Deceased	No	27.2 ± 10.88	1.88 ± 1.54	21.13 ± 13	8.34 ± 4.54	7.10 ± 4.58	2.36 ± 1.62	10.14 ± 7.20	2.71 ± 2.57	5.52 ± 3.77	2.05 ± 1.26	7.41 ± 5.79	8.70 ± 4.98	6.25 ± 3.78	30.45 ± 15.70	12.35 ± 7.01
p-value		.538	.194	.523	.32	.93	.930	.89	.64	.367	.28	.965	.723	.209	.965	.71
Recovered	Yes	28.30 ± 13.63	1.95 ± 1.3	15.12 ± 11.80	5.18 ± 3.11	6.35 ± 3.31	2.35 ± 1.40	8.96 ± 4.55	2.63 ± 2.10	5.13 ± 2.93	2.17 ± 1.91	5.99 ± 5.19	6.40 ± 4.23	6.72 ± 4.65	26.05 ± 11.94	10.35 ± 5.12
Recovered	No	26.8 ± 12.88	1.90 ± 1.55	14.13 ± 9.21	5.98 ± 3.20	6.09 ± 3.88	1.36 ± 1.62	8.05 ± 4.31	2.45 ± 2.11	4.62 ± 3.75	1.98 ± 1.75	5.41 ± 4.79	5.70 ± 3.98	6.25 ± 3.88	25.45 ± 11.70	9.86 ± 4.96
p-value		.621	.43	.498	.61	.56	.846	.67	.95	.695	.45	.951	.965	.469	.841	.44
Cough
Deceased	Yes	26.53 ± 12.23	2.20 ± 2.12	13.92 ± 12.54	7.31±4.64	5.16 ± 3.45	1.99 ± 1.13	8.15 ± 5.53	2.45 ± 2.03	6.74 ± 4.51	2.96 ± 2.39	5.50 ± 5.20	8.26 ± 4.28	8.83 ± 4.18	30.37 ± 16.03	14.79 ± 7.88
Deceased	No	33.42 ± 16.31	2.64 ± 1.66	21.47 ± 12.37	7.23 ± 3.90	8.43 ± 4.96	2.54 ± 1.69	10.65 ± 7.19	3.34 ± 2.38	6.50 ± 4.75	2.67 ± 2.07	8.22 ± 5.22	8.47 ± 4.62	6.16 ± 4.21	31.16 ± 15.75	11.67 ± 6.10
p-value		.299	.26	.054	.76	.09	.656	.58	.24	.916	.62	.175	.815	.051	.899	.25
Recovered	Yes	22.87 ± 13.33	1.64 ± 0.64	11.77 ± 10.58	6.04 ± 3.73	5.11 ± 3.30	1.39 ± 0.673	8.22 ± 4.62	2.56 ± 2.01	5.01 ± 4.64	2.02 ± 1.85	4.47 ± 3.98	7.37 ± 4.65	7.86 ± 3.90	25.26 ± 14.98	12.35 ± 6.86
Recovered	No	28.73 ± 14	2.10 ± 1.49	17.37 ± 12.23	5.93 ± 2.85	6.83 ± 4.21	2.05 ± 1.43	7.83 ± 4.03	2.68 ± 2.23	5.04 ± 3.10	2.28 ± 2.01	6.90 ± 5.65	7.22 ± 5.05	5 ± 3.22	26.72 ± 16.32	8.81 ± 2.56
p-value		.315	.39	.187	.86	.35	.544	.95	.96	.476	.98	.335	.801	.016	.917	.18
Sputum
Deceased	Yes	28.16 ± 16.71	2.13 ± 1.66	15 ± 13.95	5.85 ± 4.69	5.12 ± 4.15	2.38 ± 1.51	9.77 ± 7.29	4.11 ± 2.58	7.75 ± 4.83	3.32 ± 2.09	6.69 ± 5.33	8.20 ± 4.99	7.70 ± 4.40	26.50 ± 16.50	12.76 ± 5.11
Deceased	No	31.73 ± 14.49	2.61 ± 1.93	19.93 ± 12.31	7.87 ± 3.83	7.98 ± 4.67	2.29 ± 1.52	9.60 ± 6.46	2.51 ± 1.97	6.11 ± 4.49	2.56 ± 2.21	7.32 ± 5.41	8.47 ± 4.27	7.02 ± 4.41	32.71 ± 15.21	12.98 ± 7.67
p-value		.455	.61	.341	.23	.10	0964	.92	.18	.287	.27	.856	.802	.454	.330	.54
Recovered	Yes	15 ± 0.01	1.75 ± 1.20	9.55 ± 10.53	3.90 ± 4.24	6.60 ± 8.06	2.75 ± 2.47	4.60 ± 2.40	6.60 ± 2.96	6.31 ± 4.66	2.25 ± 1.90	9.21 ± 7.49	10 ± 9.89	5.61 ± 4.94	32.5 ± 28.99	9.10 ± 2.68
Recovered	No	26.99 ± 13.95	1.91 ± 1.59	15.33 ± 11.85	6.13 ± 3.16	6.05 ± 3.71	1.69 ± 1.11	8.30 ± 4.33	2.35 ± 1.78	4.93 ± 3.79	2.16 ± 1.95	5.61 ± 4.96	7.1 ± 4.53	6.28 ± 3.77	25.63 ± 14.96	10.43 ± 5.27
p-value		.318	.73	.647	.36	.86	.739	.27	.37	.454	.83	.261	.616	.647	.835	.73
Myalgia
Deceased	Yes	33.54 ± 15.67	2.45 ± 1.90	19.43 ± 12.64	7.46 ± 3.78	7.47 ± 4.60	2.25 ± 1.48	10.49 ± 7.39	2.44 ± 1.73	8.01 ± 4.86	3.15 ± 2.20	7.59 ± 4.78	7.92 ± 4.11	7.82 ± 4.53	29.33 ± 14.12	13.384 ± 7.51
Deceased	No	25.71 ± 12.89	2.49 ± 1.82	16.76 ± 13.48	6.92 ± 4.86	6.53 ± 4.89	2.44 ± 1.58	8.20 ± 4.90	3.93 ± 2.79	4.16 ± 2.83	2.15 ± 2.05	6.34 ± 6.26	9.21 ± 5.01	6.21 ± 3.96	33.47±18.27	12.11 ± 6.05
p-value		.212	.98	.635	.62	.54	.714	.63	.10	.020	.14	.518	.729	.211	.651	.73
Recovered	Yes	25.61 ± 12.78	3.03 ± 2.84	13.1 ± 21.59	4.43 ± 2.89	4.46 ± 2.91	1.81 ± 0.41	10.30 ± 6.12	1.13 ± 1.0	4.31 ± 2.81	1.80 ± 0.87	1.51 ± 0.55	13.33 ± 0.57	5.73 ± 3.72	27.11 ± 21.22	13.26 ± 8.45
Recovered	No	26.21 ± 14.13	1.77 ± 1.37	15.15 ± 10.78	6.15 ± 3.24	6.27 ± 3.97	1.76 ± 1.26	7.80 ± 4.14	2.80 ± 2.13	5.10 ± 3.90	2.21 ± 2.02	6.33 ± 5.12	6.62 ± 4.58	6.30 ± 3.83	25.98 ± 15.28	10.02 ± 4.76
p-value		.756	.46	.468	.53	.72	.316	.51	.20	.945	.86	.067	.026	.782	.890	.53
Dyspnea
Deceased	Yes	34.03 ± 14.56	3.08 ± 2.11	16.84 ± 12.66	6.96 ± 3.84	6.11 ± 3.78	1.89 ± 1.04	9.96 ± 7.53	2.65 ± 1.97	7.07 ± 4.64	2.54 ± 2.39	7.51 ± 5.21	8.91 ± 4.24	7.94 ± 4.25	31.57 ± 14.78	13.33 ± 5.79
Deceased	No	26.26 ± 14.93	1.66 ± 1.01	20.56 ± 13.16	7.66 ± 4.61	8.44±5.46	2.88 ± 1.83	9.25 ± 5.40	3.43 ± 2.59	5.97 ± 4.61	3.11±1.89	6.63 ± 5.59	7.73 ± 4.71	6.31 ± 4.43	29.91 ± 17.18	12.60 ± 7.82
p-value		.149	.044	.489	.75	.32	.285	.95	.43	.391	.23	.276	.255	.216	.769	.52
Recovered	Yes	28 ± 13.40	2.98 ± 1.02	17.56 ± 10.23	4.56 ± 2.36	6.51 ± 35	2.06 ± 1.30	8.15 ± 3.98	2.63 ± 2.10	6.86 ± 3.21	1.63 ± 0.42	5.21 ± 4.25	6.98 ± 3.64	6.95 ± 3.20	27.85 ± 1.41	10.35 ± 5.12
Recovered	No	26.19 ± 13.80	1.01 ± 1.15	14.94 ± 11.69	5.98 ± 3.20	6.09 ± 3.88	1.76 ± 1.21	8.0567 ± 4.31	1.96 ± 1.45	5.02 ± 3.77	2.17 ± 1.91	5.85 ± 5.16	7.29 ± 4.81	6.24 ± 3.76	26.09 ± 15.52	9.88 ± 4.13
p-value		.458	.050	.647	.31	.41	.397	.42	.28	.632	.41	.436	.541	.841	.541	.85
Sore throat
Deceased	Yes	31.52 ± 18.26	3.05 ± 1.74	14.87 ± 16.31	3.77 ± 1.34	7.22 ± 6.71	2.27 ± 1.12	6.45 ± 5.53	3.12 ± 3.00	3.77 ± 1.34	1.92 ± 1.75	4.42 ± 5.03	7.72 ± 4.59	3.52 ± 0.95	39.75 ± 18.83	10.40 ± 3.19
Deceased	No	30.53 ± 14.84	2.38 ± 1.87	19.01 ± 12.47	7.80 ± 4.16	7.11 ± 4.43	2.33 ± 1.56	10.15 ± 6.70	2.96 ± 2.19	7.03 ± 4.75	2.92 ± 2.22	7.55 ± 5.31	8.49 ± 4.47	7.81 ± 4.38	29.4 ± 14.99	13.30 ± 7.28
p-value		.976	.39	.669	.54	.83	.783	.20	.85	.189	.44	.328	.539	.076	.285	.56
Recovered	Yes	34.61 ± 17.27	2.70 ± 1.14	14.30 ± 12.89	4.10 ± 1.37	5.84 ± 3.15	1.99 ± 1.14	8.16 ± 6.13	2.66 ± 2.18	5.36 ± 3.38	2.66 ± 2.23	4.58 ± 4.37	4.68 ± 3.11	3.94 ± 1.24	30.31 ± 16.43	10.92 ± 3.002
Recovered	No	24.51 ± 12.76	1.74 ± 1.59	15.07 ± 11.72	6.36 ± 3.34	6.14 ± 4.06	1.72 ± 1.22	8.03 ± 4.02	2.63 ± 2.13	4.96 ± 3.91	2.07 ± 1.88	6.11 ± 5.24	7.81 ± 4.95	6.71 ± 3.93	25.25 ± 15.53	10.23 ± 5.49
p-value		.172	.97	.978	.14	.98	.597	.80	.91	.636	.50	.802	.154	.253	.559	.32
Headache
Deceased	Yes	32.21 ± 14.71	2.73 ± 2.35	15.83 ± 12.91	7.44 ± 4.11	5.64 ± 3.29	2.17 ± 1.36	11.86 ± 7.89	3.25 ± 1.96	7.32 ± 4.47	2.97 ± 2.55	6.99 ± 5.01	8.29 ± 4.64	9.46 ± 4.43	25.61 ± 12.23	14.05 ± 8.57
Deceased	No	29.49 ± 15.53	2.26 ± 1.37	20.45 ± 12.71	7.12 ± 4.27	8.26 ± 5.28	2.43 ± 1.62	7.97 ± 5.01	2.78 ± 2.49	6.04 ± 4.71	2.64 ± 1.90	7.24 ± 5.66	8.47 ± 4.37	5.52 ± 3.49	34.86 ± 17.12	12.05 ± 5.45
p-value		.691	.73	.335	.72	.18	.850	.17	.50	.267	.96	.675	.768	.007	.154	.63
Recovered	Yes	34.25 ± 5.31	4.00 ± 2.68	10 ± 9.89	7.90 ± 2.82	6.95 ± 1.48	1.15 ± 0.63	8.55 ± 4.87	2.9 ± 0.01	3.9 ± 2.82	0.55 ± 0.21	9.55 ± 6.29	10 ± 8.48	8.45 ± 6.43	22 ± 1.41	7.90 ± 0.14
Recovered	No	25.62 ± 14.08	1.75 ± 1.40	15.31 ± 11.89	5.84 ± 3.23	6.02 ± 4.0	1.81 ± 1.22	8.02 ± 4.36	2.61 ± 2.17	5.10 ± 3.86	2.28 ± 1.93	5.58 ± 5.01	7.11 ± 4.63	6.08 ± 3.64	26.38 ± 16.03	10.52 ± 5.26
p-value		.506	.12	.708	.33	.50	.405	.80	.61	.901	.061	.228	.428	.559	.967	.53
Diarrhea
Deceased	Yes	33.12 ± 13.40	2.64 ± 2.02	18.80 ± 13.28	7.67 ± 3.81	7.87 ± 5.44	2.89 ± 1.79	8.74 ± 7.99	2.32 ± 1.67	8.50 ± 3.94	2.62 ± 2.28	10.01 ± 3.20	7.28 ± 3.83	7.85 ± 5.33	37.85 ± 16.62	14.37 ± 8.89
Deceased	No	29.91 ± 15.63	2.41 ± 1.82	18.35 ± 12.94	7.14 ± 4.30	6.90 ± 4.49	2.15 ± 1.39	9.93 ± 6.29	3.19 ± 2.40	6.01 ± 4.68	2.83 ± 2.18	6.25 ± 5.56	8.73 ± 4.61	7.03 ± 4.11	28.71 ± 15	12.47 ± 6.37
p-value		.462	.75	.864	.62	.75	.269	.31	.44	.105	.82	.202	.388	.863	.134	.86
Recovered	Yes	29.33 ± 13.86	1.30 ± 1.66	18.91 ± 11.54	9.56 ± 2.15	10.16 ± 7.52	3.61 ± 1.96	8.33 ± 7.55	0.96 ± 0.11	4.21 ± 4.16	1.73 ± 1.69	4.78 ± 6.25	4.81 ± 2.98	2.66 ± 0.58	31 ± 22.11	8.23 ± 1.49
Recovered	No	25.84 ± 14.015	1.96 ± 1.55	14.50 ± 11.84	5.58 ± 3.07	5.63 ± 3.21	1.56 ± 0.93	8.02 ± 4.03	2.82 ± 2.13	5.11 ± 3.80	2.22 ± 1.96	5.97 ± 5.05	7.57 ± 4.92	6.64 ± 3.75	25.54 ± 15.08	10.58 ± 5.34
p-value		.729	.32	.604	.61	.25	.049	.55	.14	.678	.67	.350	.331	.011	.489	.40
Vomiting
Deceased	Yes	36.6 ± 21.40	3.20 ± 2.30	21.36 ± 20.07	6.34 ± 6.79	5.71 ± 5.65	1.29 ± 0.53	12.06 ± 6.54	4.20 ± 1.57	12.16 ± 7.20	4.56 ± 3.30	13.66 ± 1.52	9.66 ± 4.04	7.76 ± 3.02	31.83 ± 16.60	12.63 ± 2.56
Deceased	No	30.01 ± 14.51	2.38 ± 1.81	18.13 ± 12.26	7.37 ± 3.92	7.28 ± 4.62	2.43 ± 1.53	9.38 ± 6.67	2.85 ± 2.30	5.97 ± 3.92	2.59 ± 2.01	6.41 ± 5.07	8.25 ± 4.50	7.17 ± 4.51	30.74 ± 15.80	12.95± 7.26
p-value		.489	.44	.678	.55	.51	.146	.28	.29	.084	.31	.019	.602	.510	.972	.44
Recovered	Yes	16 ± 7.07	0.56 ± 0.21	27 ± 18.38	4.05 ± 0.07	6.95 ± 4.03	0.98 ± 0.12	10.50 ± 0.98	1.80 ± 1.41	7.3 ± 3.11	0.85 ± 0.07	14.5 ± 0.70	12 ± 0.0	9.15 ± 2.61	22.25 ± 0.35	7.45 ± 3.46
Recovered	No	26.92 ± 13.94	1.99 ± 1.56	14.08 ± 11.08	6.12 ± 3.27	6.02 ± 3.94	1.82 ± 1.22	7.88 ± 4.41	2.69 ± 2.14	4.86 ± 3.81	2.26 ± 1.95	5.23 ± 4.65	6.95 ± 4.79	6.03 ± 3.77	26.36 ± 16.03	10.5 ± 5.20
p-value		.298	.10	.280	.33	.80	.170	.27	.45	.261	.26	.027	.210	.182	.934	.47

Significant correlations between cytokine/chemokine levels and laboratory data were identified. In the deceased group, there were several correlations; IFN-γ with PDW, urea and ALP; IL-1β with LDH; IL-2 with Mg and eosinophils; IL-4 with MCHC, urea and Potassium; IL-5 with urea, creatinine, and Mg, IL-6 with Sodium; IL-8 with RDW, MPV, and LDH; IL-12 with MPV, PDW, P-LCR, and Calcium; IL-17 with PLT and Potassium; IP-10 with ALP; MCP-1 with Potassium; MIP1-α with PLT, MPV, PDW, P-LCR and Potassium; RANTES with MCHC and RDW, and TNF-α with RDW. In the recovered group, there was a significant correlation between levels of IFN-γ with PMN and eosinophils; IL-1β with PLT; IL-2 with Potassium and ALP; IL-4 with PLT; IL-5 with PLT and Lymph; IL-6 with RDW, AST, ALP, and LDH; IL-8 with PLT and CPK; IL-10 with age and Sodium; IL-12 with MCV and MCH; IL-17 with HCT, PLT, and Sodium; IP-10 with ALT; MCP-1 with Phosphorus; MIP1-α with Sodium; RANTES with MCHC; TNF-α with WBC and Mg. The correlation between cytokine/chemokine levels and laboratory data is shown in Table 4.

Table 4.

Correlation between cytokine/chemokine levels and laboratory data.

Outcome	Variables	IFN-γ	IL-1β	IL-2	IL-4	IL-5	IL-6	IL-8	IL-10	IL-12	IL-17	IP-10	MCP-1	MIP-1α	RANTES	TNF-α
Deceased	WBC (μ/l)	r = 0.219	0.325	−0.232	−0.058	0.023	−0.069	0.031	0.046	0.193	0.112	0.009	−0.035	0.355	−0.044	0.064
	WBC (μ/l)	p = 0.245	0.080	0.218	0.761	0.905	0.716	0.869	0.810	0.307	0.555	0.960	0.853	0.054	0.818	0.737
	RBC (μ/l)	r = 0.136	0.020	0.126	−0.103	−0.096	−0.246	0.055	0.286	0.178	−0.107	−0.169	0.108	0.184	−0.218	0.272
	RBC (μ/l)	p = 0.474	0.918	0.505	0.586	0.612	0.189	0.773	0.125	0.347	0.574	0.371	0.570	0.330	0.247	0.145
	Hb (mg/dl)	r = 0.280	0.300	−0.012	−0.199	0.017	−0.073	0.013	−0.033	0.085	0.069	0.271	0.027	0.119	0.180	−0.199
	Hb (mg/dl)	p = 0.134	0.107	0.952	0.291	0.930	0.700	0.945	0.863	0.654	0.719	0.147	0.886	0.531	0.340	0.292
	HCT (%)	r = 0.268	0.225	0.047	−0.112	−0.051	−0.048	0.103	0.001	0.066	−0.059	0.242	0.115	0.187	0.231	−0.219
	HCT (%)	p = 0.152	0.232	0.804	0.554	0.790	0.802	0.588	0.996	0.728	0.757	0.199	0.544	0.322	0.219	0.245
	MCV (fl)	r = 0.130	−0.053	0.255	−0.015	−0.087	0.007	0.324	−0.345	−0.051	0.006	−0.153	0.329	0.184	0.028	0.185
	MCV (fl)	p = 0.495	0.782	0.174	0.937	0.646	0.971	0.081	0.062	0.791	0.975	0.419	0.076	0.331	0.881	0.327
	MCH (pg)	r = 0.275	−0.074	0.281	0.002	0.085	−0.033	0.348	−0.060	0.087	0.253	0.253	−0.184	0.010	−0.234	0.060
	MCH (pg)	p = 0.142	0.698	0.133	0.991	0.657	0.861	0.059	0.754	0.649	0.177	0.178	0.331	0.956	0.214	0.751
	MCHC (%)	r = 0.002	0.096	0.048	−0.578**	0.045	0.116	0.000	0.147	0.149	0.162	0.145	0.128	−0.223	0.371*	−0.027
	MCHC (%)	p = 0.993	0.615	0.801	0.001	0.813	0.540	0.998	0.437	0.431	0.393	0.444	0.501	0.235	0.044	0.889
	PLT (mm3/μl)	r = 0.254	0.182	−0.087	−0.037	−0.182	0.092	−0.016	0.018	0.319	0.394*	0.125	0.048	0.387*	−0.183	0.302
	PLT (mm3/μl)	p = 0.176	0.337	0.646	0.845	0.336	0.630	0.931	0.924	0.086	0.031	0.509	0.802	0.035	0.334	0.104
	RDW (μm)	r = −0.058	−0.197	0.054	0.245	0.001	−0.242	0.324	0.149	0.164	0.088	0.106	−0.135	0.242	−0.398*	0.373*
	RDW (μm)	p = 0.761	0.298	0.776	0.192	0.997	0.198	0.080	0.431	0.387	0.643	0.576	0.478	0.197	0.029	0.042
	MPV (fl)	r = 0.328	−0.037	−0.223	−0.177	−0.289	−0.090	0.364*	−0.042	0.415*	0.298	0.098	0.160	0.568**	−0.102	0.312
	MPV (fl)	p = 0.077	0.844	0.237	0.348	0.121	0.635	0.048	0.825	0.022	0.109	0.606	0.398	0.001	0.593	0.094
	PDW (%)	r = 0.407*	−0.051	−0.101	−0.099	−0.131	−0.051	0.266	−0.054	0.461*	0.196	0.115	0.186	0.510**	0.006	0.296
	PDW (%)	p = 0.026	0.790	0.595	0.603	0.490	0.790	0.155	0.777	0.010	0.299	0.545	0.325	0.004	0.975	0.112
	P-LCR (ng/ml)	r = 0.355	−0.095	−0.105	−0.071	−0.148	−0.155	0.325	−0.032	0.484**	0.230	0.119	0.158	0.590**	−0.055	0.293
	P-LCR (ng/ml)	p = 0.054	0.618	0.582	0.709	0.435	0.414	0.080	0.866	0.007	0.220	0.533	0.404	0.001	0.774	0.116
	Urea (mg/dl)	r = 0.416*	0.130	0.063	0.425*	0.389*	−0.080	0.167	−0.242	−0.065	0.037	−0.097	−0.291	0.030	−0.161	−0.136
	Urea (mg/dl)	p = 0.022	0.493	0.742	0.019	0.034	0.674	0.377	0.198	0.732	0.848	0.610	0.118	0.876	0.396	0.474
	Cr (mg/dl)	r = 0.267	0.186	0.272	0.177	0.405*	−0.054	0.348	0.002	−0.061	−0.088	−0.032	0.072	0.000	0.044	−0.280
	Cr (mg/dl)	p = 0.153	0.325	0.146	0.350	0.026	0.778	0.059	0.990	0.749	0.644	0.865	0.705	0.999	0.816	0.134
	AST(IU/L)	r = −0.206	−0.220	−0.030	0.215	−0.100	−0.217	−0.092	0.083	−0.034	0.132	0.027	−0.049	−0.324	−0.240	−0.025
	AST(IU/L)	p = 0.274	0.243	0.877	0.255	0.600	0.250	0.627	0.661	0.860	0.485	0.889	0.798	0.081	0.202	0.896
	ALT (IU/L)	r = 0.051	0.175	0.065	0.072	−0.070	−0.012	−0.115	−0.049	0.116	−0.325	0.069	0.139	0.062	−0.108	0.320
	ALT (IU/L)	p = 0.789	0.355	0.735	0.704	0.715	0.952	0.546	0.796	0.543	0.080	0.716	0.465	0.746	0.569	0.084
	ALP (IU/L)	r = −0.376*	−0.075	−0.114	−0.004	−0.040	−0.010	−0.086	−0.110	−0.281	−0.172	−0.415*	0.227	−0.195	0.143	0.191
	ALP (IU/L)	p = 0.040	0.692	0.547	0.985	0.834	0.960	0.651	0.564	0.132	0.364	0.023	0.229	0.303	0.452	0.311
	LDH (U/L)	r = −0.013	0.380*	−0.168	0.174	−0.115	0.116	−0.434*	−0.094	−0.098	0.145	−0.038	0.046	−0.243	−0.127	−0.013
	LDH (U/L)	p = 0.944	0.039	0.374	0.359	0.545	0.543	0.016	0.622	0.606	0.445	0.842	0.811	0.196	0.505	0.944
	CPK (U/L)	r = −0.236	−0.238	0.081	0.233	0.338	−0.225	0.043	−0.272	−0.114	−0.095	−0.340	0.049	0.072	0.184	−0.222
	CPK (U/L)	p = 0.210	0.206	0.670	0.216	0.068	0.231	0.822	0.146	0.549	0.618	0.066	0.799	0.706	0.331	0.237
	Ca (mg/dl)	r = 0.062	−0.006	−0.187	0.096	0.248	−0.212	0.084	0.247	0.514**	0.140	0.210	0.121	−0.088	0.092	−0.140
	Ca (mg/dl)	p = 0.750	0.976	0.332	0.622	0.195	0.269	0.664	0.196	0.004	0.469	0.274	0.533	0.650	0.634	0.469
	P (mg/dl)	r = −0.254	0.049	−0.208	0.065	0.055	−0.055	−0.025	0.071	0.020	−0.277	−0.071	−0.374*	−0.336	−0.030	−0.259
	P (mg/dl)	p = 0.176	0.798	0.270	0.731	0.775	0.772	0.896	0.710	0.918	0.139	0.709	0.042	0.070	0.876	0.167
	Mg (mEq/L)	r = −0.174	0.290	0.540**	−0.335	−0.613**	−0.076	−0.053	−0.025	0.064	0.062	0.292	0.170	0.101	−0.179	0.182
	Mg (mEq/L)	p = 0.357	0.120	0.002	0.070	0.000	0.690	0.782	0.896	0.738	0.747	0.118	0.369	0.597	0.343	0.337
	Na (mEq/L)	r = −0.234	0.167	0.165	0.087	−0.002	−0.410*	−0.025	0.209	0.230	0.169	0.200	0.112	0.065	0.177	−0.265
	Na (mEq/L)	p = 0.212	0.376	0.385	0.648	0.992	0.024	0.898	0.268	0.220	0.372	0.290	0.556	0.732	0.351	0.157
	K (mEq/L)	r = 0.124	0.222	−0.353	0.391*	0.121	0.062	0.002	0.233	0.090	0.369*	0.292	0.178	−0.422*	0.005	−0.345
	K (mEq/L)	p = 0.515	0.238	0.056	0.032	0.523	0.744	0.993	0.215	0.637	0.045	0.117	0.348	0.020	0.979	0.062
	PMN (%)	r = 0.048	0.166	0.010	0.110	−0.159	0.058	−0.051	−0.019	0.131	0.341	0.044	0.127	−0.047	0.073	−0.180
	PMN (%)	p = 0.802	0.380	0.957	0.564	0.402	0.760	0.790	0.920	0.491	0.065	0.818	0.504	0.805	0.701	0.342
	Lymph (%)	r = −0.152	−0.158	0.082	−0.079	0.142	−0.049	−0.003	0.056	−0.185	−0.335	−0.109	−0.143	0.185	−0.024	0.213
	Lymph (%)	p = 0.422	0.405	0.668	0.679	0.454	0.799	0.986	0.768	0.328	0.071	0.567	0.451	0.328	0.900	0.257
	Monocyte (%)	r = −0.029	0.225	0.099	−0.076	−0.028	−0.135	0.001	0.047	0.203	−0.126	−0.038	−0.123	−0.294	−0.242	0.192
	Monocyte (%)	p = 0.878	0.231	0.602	0.691	0.882	0.478	0.997	0.803	0.281	0.507	0.843	0.518	0.115	0.198	0.309
	Eos (%)	r = 10.000	−0.049	−0.379*	0.002	0.106	0.012	0.155	−0.179	−0.236	0.183	0.032	−0.093	−0.069	−0.137	0.018
	Eos (%)	p = <00.001	0.796	0.039	0.993	0.579	0.948	0.414	0.343	0.209	0.333	0.868	0.624	0.717	0.469	0.926
Healthy	WBC (μ/l)	r = 0.082	0.149	0.056	−0.462*	−0.222	0.190	−0.058	0.123	−0.061	−0.394*	0.100	0.055	0.210	0.193	−0.300
	WBC (μ/l)	p = 0.667	0.432	0.770	0.010	0.239	0.316	0.760	0.517	0.747	0.031	0.597	0.771	0.265	0.306	0.107
	RBC (μ/l)	r = −0.207	0.211	−0.246	−0.049	−0.061	−0.364*	0.065	0.076	0.029	−0.125	0.151	0.074	0.158	−0.014	−0.299
	RBC (μ/l)	p = 0.272	0.263	0.191	0.797	0.749	0.048	0.733	0.689	0.881	0.511	0.427	0.697	0.404	0.942	0.108
	Hb (mg/dl)	r = 0.041	0.048	−0.245	−0.005	−0.087	0.076	−0.118	0.191	0.028	−0.028	0.133	−0.050	0.167	0.089	−0.373*
	Hb (mg/dl)	p = 0.828	0.800	0.193	0.978	0.648	0.689	0.535	0.312	0.882	0.882	0.484	0.793	0.379	0.641	0.042
	HCT (%)	r = 0.000	0.044	−0.285	0.011	−0.125	−0.012	−0.113	0.102	0.050	−0.013	0.188	0.033	0.072	0.085	−0.359
	HCT (%)	p = 0.998	0.817	0.127	0.952	0.509	0.950	0.554	0.593	0.791	0.946	0.320	0.862	0.705	0.656	0.051
	MCV (fl)	r = 0.183	−0.370*	0.317	0.046	0.076	0.475**	−0.081	0.086	0.066	0.290	0.062	0.052	−0.142	0.097	0.197
	MCV (fl)	p = 0.334	0.044	0.087	0.810	0.692	0.008	0.672	0.650	0.730	0.120	0.746	0.784	0.453	0.609	0.297
	MCH (pg)	r = 0.241	−0.230	−0.125	−0.131	−0.182	0.582**	−0.082	0.052	0.177	−0.077	−0.017	0.008	−0.082	0.336	−0.212
	MCH (pg)	p = 0.199	0.221	0.510	0.491	0.335	0.001	0.666	0.784	0.350	0.687	0.931	0.965	0.666	0.070	0.260
	MCHC (%)	r = −0.084	0.050	−0.110	−0.019	−0.082	0.207	−0.207	0.151	0.165	−0.024	0.037	−0.109	−0.029	0.209	−0.259
	MCHC (%)	p = 0.661	0.794	0.561	0.921	0.667	0.273	0.273	0.427	0.384	0.898	0.844	0.567	0.879	0.268	0.167
	PLT (mm3/μl)	r = 0.005	0.109	0.198	−0.188	−0.165	0.246	0.041	0.128	−0.078	−0.200	0.044	−0.045	0.125	0.079	−0.104
	PLT (mm3/μl)	p = 0.979	0.565	0.294	0.319	0.384	0.190	0.832	0.501	0.682	0.290	0.819	0.815	0.509	0.678	0.586
	RDW (μm)	r = −0.704	0.595	0.036	0.000	0.667	0.821*	−0.334	0.306	−0.519	−0.750	0.000	0.164	0.357	−0.071	−0.036
	RDW (μm)	p = 0.077	0.159	0.939	10.000	0.102	0.023	0.465	0.504	0.233	0.052	10.000	0.726	0.432	0.879	0.939
	MPV (fl)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	MPV (fl)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	PDW (%)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	PDW (%)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	P-LCR (ng/ml)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	P-LCR (ng/ml)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	Urea (mg/dl)	r = 0.372*	−0.256	0.067	−0.256	−0.315	0.123	0.008	−0.074	−0.397*	−0.031	−0.127	−0.091	−0.074	−0.116	0.149
	Urea (mg/dl)	p = 0.043	0.171	0.725	0.172	0.090	0.516	0.966	0.698	0.030	0.870	0.503	0.631	0.697	0.543	0.431
	Cr (mg/dl)	r = 0.053	−0.008	−0.237	−0.125	−0.299	−0.206	−0.032	0.005	−0.187	−0.037	−0.039	−0.039	0.119	−0.067	−0.188
	Cr (mg/dl)	p = 0.781	0.967	0.206	0.511	0.108	0.274	0.866	0.980	0.323	0.846	0.840	0.838	0.530	0.726	0.320
	AST (IU/L)	r = −0.274	0.381*	−0.414*	0.145	0.290	−0.060	−0.290	0.066	−0.189	−0.164	0.084	0.184	0.184	−0.034	0.126
	AST (IU/L)	p = 0.151	0.042	0.026	0.452	0.128	0.756	0.128	0.735	0.327	0.397	0.664	0.340	0.339	0.862	0.516
	ALT (IU/L)	r = 0.254	−0.119	0.116	−0.043	−0.246	0.207	0.141	−0.207	0.018	0.063	0.268	0.054	0.028	−0.019	−0.066
	ALT (IU/L)	p = 0.175	0.532	0.541	0.823	0.189	0.272	0.457	0.273	0.924	0.742	0.152	0.775	0.882	0.920	0.727
	ALP (IU/L)	r = 0.100	0.355	−0.150	0.031	−0.079	−0.084	−0.160	−0.091	0.432	−0.198	0.228	0.311	−0.197	0.050	0.140
	ALP (IU/L)	p = 0.665	0.114	0.517	0.895	0.732	0.718	0.489	0.694	0.050	0.390	0.320	0.170	0.391	0.830	0.546
	LDH (U/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	LDH (U/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	CPK (U/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	CPK (U/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	Ca (mg/dl)	r = 0.098	0.465	0.042	0.280	−0.054	−0.036	0.053	−0.670*	0.385	−0.652*	0.519	0.266	−0.424	0.512	−0.580*
	Ca (mg/dl)	p = 0.763	0.127	0.898	0.379	0.869	0.912	0.870	0.017	0.216	0.021	0.084	0.404	0.170	0.089	0.048
	P (mg/dl)	r = 0.509	0.244	0.293	−0.227	0.108	0.370	−0.028	0.383	0.258	0.076	−0.323	−0.310	−0.150	−0.058	−0.144
	P (mg/dl)	p = 0.091	0.445	0.355	0.479	0.738	0.236	0.931	0.219	0.417	0.814	0.306	0.327	0.642	0.858	0.656
	Mg (mEq/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	Mg (mEq/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	Na (mEq/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	Na (mEq/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	K (mEq/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	K (mEq/L)	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
	PMN (%)	r = −0.200	0.323	0.084	0.271	−0.361*	−0.245	0.028	0.357	0.139	0.157	−0.310	−0.214	0.146	0.320	0.144
	PMN (%)	p = 0.289	0.082	0.660	0.147	0.050	0.191	0.884	0.053	0.464	0.407	0.095	0.255	0.441	0.085	0.449
	Lymph (%)	r = 0.132	−0.308	−0.075	−0.256	0.372*	0.231	−0.075	−0.296	−0.163	−0.151	0.317	0.207	−0.121	−0.338	−0.169
	Lymph (%)	p = 0.485	0.097	0.695	0.172	0.043	0.220	0.695	0.112	0.390	0.425	0.088	0.273	0.523	0.067	0.373
	Monocyte (%)	r = −0.184	0.273	−0.042	−0.209	−0.234	−0.285	0.186	−0.249	0.014	0.223	−0.240	0.105	−0.085	0.010	0.292
	Monocyte (%)	p = 0.331	0.144	0.826	0.268	0.213	0.128	0.326	0.185	0.942	0.236	0.201	0.582	0.655	0.958	0.117
	Eos (%)	r = 10.000	−0.292	−0.123	0.381*	0.228	0.435*	0.206	−0.039	0.105	−0.265	0.226	0.013	−0.132	0.157	−0.331
	Eos (%)	<00.001	0.118	0.518	0.038	0.226	0.016	0.275	0.838	0.583	0.157	0.229	0.944	0.488	0.406	0.074
Recovered	WBC (μ/l)	r = 0.035	0.191	−0.255	−0.221	−0.098	0.010	−0.081	0.079	0.164	−0.297	0.126	0.299	0.244	0.065	0.582**
	WBC (μ/l)	p = 0.855	0.313	0.174	0.240	0.607	0.959	0.672	0.677	0.388	0.111	0.508	0.109	0.193	0.731	0.001
	RBC (μ/l)	r = 0.080	−0.019	0.055	−0.252	−0.152	0.069	0.068	0.283	−0.034	0.336	−0.064	0.087	−0.150	0.133	0.052
	RBC (μ/l)	p = 0.675	0.922	0.773	0.180	0.423	0.719	0.720	0.130	0.860	0.069	0.737	0.646	0.429	0.483	0.783
	Hb (mg/dl)	r = 0.184	0.191	0.063	−0.141	−0.068	0.134	0.015	0.112	−0.202	0.303	−0.074	0.000	−0.218	0.184	−0.023
	Hb (mg/dl)	p = 0.329	0.312	0.740	0.458	0.722	0.482	0.938	0.556	0.283	0.103	0.699	10.000	0.248	0.330	0.903
	HCT (%)	r = 0.246	0.097	0.052	−0.162	−0.151	0.168	0.016	0.173	−0.136	0.388*	−0.088	−0.032	−0.200	0.134	0.149
	HCT (%)	p = 0.190	0.611	0.784	0.393	0.426	0.373	0.934	0.360	0.474	0.034	0.645	0.867	0.289	0.481	0.431
	MCV (fl)	r = 0.129	0.104	−0.203	0.214	−0.104	0.204	−0.181	−0.343	−0.486**	0.173	−0.322	−0.262	−0.202	−0.253	0.230
	MCV (fl)	p = 0.496	0.586	0.283	0.256	0.585	0.279	0.339	0.063	0.006	0.361	0.082	0.161	0.284	0.178	0.221
	MCH (pg)	r = 0.150	0.237	−0.201	0.183	−0.033	0.304	−0.203	−0.354	−0.576**	0.093	−0.223	−0.176	−0.301	−0.089	0.069
	MCH (pg)	p = 0.429	0.207	0.286	0.333	0.861	0.102	0.282	0.055	0.001	0.625	0.235	0.352	0.106	0.641	0.717
	MCHC (%)	r = −0.060	0.128	0.086	−0.140	0.121	0.149	−0.183	−0.144	−0.197	0.016	0.074	0.173	−0.149	0.386*	−0.353
	MCHC (%)	p = 0.751	0.499	0.650	0.462	0.525	0.432	0.333	0.448	0.297	0.933	0.698	0.361	0.431	0.035	0.056
	PLT (mm3/μl)	r = −0.021	0.451*	0.045	0.373*	0.458*	0.139	−0.485**	−0.240	0.042	−0.371*	−0.020	0.084	−0.139	0.138	0.011
	PLT (mm3/μl)	p = 0.911	0.012	0.814	0.042	0.011	0.465	0.007	0.201	0.824	0.044	0.918	0.657	0.463	0.468	0.952
	RDW (μm)	r = −0.312	−0.246	0.005	0.004	−0.102	−0.361*	0.042	0.133	−0.050	0.229	0.134	−0.122	0.046	−0.288	0.136
	RDW (μm)	p = 0.094	0.190	0.980	0.985	0.590	0.050	0.826	0.484	0.791	0.224	0.481	0.521	0.810	0.122	0.475
	MPV (fl)	r = 0.093	−0.245	−0.030	0.024	−0.242	−0.198	0.137	0.016	0.078	0.337	−0.034	−0.199	0.303	0.088	−0.013
	MPV (fl)	p = 0.627	0.192	0.875	0.902	0.197	0.294	0.471	0.933	0.680	0.069	0.859	0.292	0.103	0.644	0.948
	PDW (%)	r = 0.154	−0.233	−0.120	−0.019	−0.349	−0.074	0.149	0.022	0.061	0.195	−0.121	−0.146	0.235	0.126	0.079
	PDW (%)	p = 0.415	0.214	0.529	0.919	0.059	0.698	0.431	0.907	0.748	0.303	0.524	0.443	0.211	0.508	0.678
	P-LCR (ng/ml)	r = 0.114	−0.313	−0.044	0.009	−0.249	−0.221	0.224	−0.010	0.087	0.245	−0.083	−0.148	0.359	0.103	0.017
	P-LCR (ng/ml)	p = 0.547	0.092	0.817	0.960	0.184	0.240	0.235	0.957	0.649	0.192	0.663	0.434	0.051	0.588	0.928
	Urea (mg/dl)	r = −0.176	−0.124	−0.044	−0.197	−0.292	0.023	0.017	0.183	−0.118	0.139	0.245	0.008	0.068	0.045	0.247
	Urea (mg/dl)	p = 0.353	0.515	0.817	0.296	0.117	0.904	0.927	0.333	0.536	0.465	0.192	0.968	0.719	0.813	0.188
	Cr (mg/dl)	r = −0.034	0.053	0.189	0.167	−0.026	−0.313	0.240	−0.043	−0.120	0.018	−0.052	−0.061	0.284	−0.183	0.061
	Cr (mg/dl)	p = 0.860	0.779	0.318	0.377	0.893	0.092	0.202	0.823	0.529	0.924	0.786	0.749	0.129	0.334	0.748
	AST (IU/L)	r = −0.347	0.227	−0.090	−0.147	−0.045	−0.380*	−0.102	0.207	0.050	0.153	−0.024	−0.063	0.201	−0.001	0.104
	AST (IU/L)	p = 0.060	0.228	0.636	0.438	0.813	0.039	0.593	0.273	0.795	0.419	0.898	0.741	0.287	0.997	0.585
	ALT(IU/L)	r = 0.019	0.118	0.033	0.022	0.008	0.195	0.147	0.003	0.020	0.159	−0.383*	−0.106	−0.315	0.313	−0.113
	ALT(IU/L)	p = 0.922	0.533	0.863	0.906	0.966	0.301	0.437	0.987	0.915	0.401	0.037	0.576	0.090	0.093	0.554
	ALP (IU/L)	r = 0.105	−0.240	0.546**	0.158	0.249	−0.374*	0.176	−0.002	0.205	0.109	0.125	−0.214	0.065	0.050	−0.329
	ALP (IU/L)	p = 0.582	0.202	0.002	0.405	0.185	0.042	0.353	0.992	0.276	0.565	0.511	0.257	0.733	0.792	0.076
	LDH (U/L)	r = −0.203	−0.123	0.286	−0.087	0.102	−0.413*	0.191	0.020	0.144	0.124	0.021	−0.209	0.140	−0.147	−0.322
	LDH (U/L)	p = 0.281	0.517	0.125	0.647	0.593	0.023	0.311	0.917	0.449	0.514	0.913	0.269	0.461	0.438	0.083
	CPK (U/L)	r = 0.240	−0.197	0.158	0.090	−0.031	−0.248	0.467**	−0.003	0.188	−0.209	−0.259	−0.035	0.289	−0.130	0.058
	CPK (U/L)	p = 0.201	0.298	0.406	0.635	0.871	0.187	0.009	0.988	0.320	0.267	0.167	0.853	0.122	0.493	0.759
	Ca (mg/dl)	r = 0.129	0.001	0.264	−0.236	−0.077	−0.270	0.200	−0.194	0.163	−0.235	0.140	−0.017	−0.248	−0.126	−0.207
	Ca (mg/dl)	p = 0.496	0.996	0.159	0.209	0.686	0.149	0.289	0.304	0.389	0.211	0.462	0.930	0.187	0.509	0.272
	P (mg/dl)	r = −0.073	−0.029	0.006	0.124	0.280	0.209	0.059	0.198	−0.122	−0.210	−0.060	−0.375*	−0.005	0.189	0.121
	P (mg/dl)	p = 0.700	0.879	0.975	0.514	0.134	0.268	0.759	0.293	0.522	0.265	0.754	0.041	0.980	0.317	0.524
	Mg (mEq/L)	r = 0.214	−0.087	0.058	0.003	−0.214	−0.104	−0.030	0.141	0.057	−0.280	0.257	0.026	−0.101	−0.061	−0.400*
	Mg (mEq/L)	p = 0.256	0.649	0.762	0.986	0.255	0.584	0.875	0.457	0.763	0.135	0.170	0.893	0.595	0.750	0.029
	Na (mEq/L)	r = −0.325	0.212	−0.130	0.065	0.004	−0.217	−0.164	0.404*	−0.026	−0.382*	0.299	−0.313	−0.469**	0.055	−0.274
	Na (mEq/L)	p = 0.080	0.262	0.493	0.732	0.984	0.250	0.388	0.027	0.891	0.037	0.108	0.092	0.009	0.773	0.143
	K (mEq/L)	r = 0.128	0.105	−0.447*	0.288	0.006	−0.317	0.043	−0.087	0.087	0.010	0.142	0.029	−0.116	−0.249	−0.095
	K (mEq/L)	p = 0.500	0.579	0.013	0.122	0.976	0.088	0.822	0.648	0.648	0.958	0.454	0.879	0.542	0.185	0.619
	PMN (%)	r = −0.491**	−0.039	−0.034	−0.105	−0.307	−0.125	−0.039	0.187	0.082	0.005	−0.182	0.079	0.220	0.110	0.129
	PMN (%)	p = 0.006	0.837	0.860	0.580	0.099	0.510	0.838	0.324	0.666	0.980	0.335	0.677	0.243	0.563	0.497
	Lymph (%)	r = 0.261	0.203	−0.042	0.276	0.407*	0.183	−0.022	0.071	−0.203	0.005	0.089	−0.181	−0.151	−0.089	−0.032
	Lymph (%)	p = 0.164	0.281	0.825	0.139	0.026	0.334	0.908	0.709	0.281	0.979	0.642	0.339	0.424	0.642	0.868
	Monocyte (%)	r = 0.340	−0.215	0.221	−0.011	0.083	0.088	−0.263	−0.071	0.091	−0.016	−0.141	−0.047	0.009	0.246	−0.003
	Monocyte (%)	p = 0.066	0.253	0.242	0.955	0.662	0.643	0.161	0.708	0.634	0.935	0.458	0.805	0.963	0.189	0.989
	Eos (%)	r = 10.000	0.105	0.176	0.138	0.233	0.283	0.113	−0.074	−0.229	0.126	−0.038	0.004	−0.009	−0.310	0.063
	Eos (%)	p = <00.001	0.580	0.353	0.468	0.216	0.130	0.552	0.697	0.223	0.506	0.843	0.983	0.962	0.096	0.740
Total	WBC (μ/l)	r = 0.345**	0.354**	−0.048	0.106	0.168	0.277**	0.086	0.179	0.257*	−0.024	0.222*	0.253*	0.354**	0.284**	0.357**
	WBC (μ/l)	p = 0.001	0.001	0.656	0.321	0.113	0.008	0.420	0.092	0.014	0.821	0.035	0.016	0.001	0.007	0.001
	RBC (μ/l)	r = −0.317**	−0.172	−0.127	−0.345**	−0.322**	−0.386**	−0.094	0.108	−0.137	−0.114	−0.171	−0.101	−0.040	−0.291**	−0.180
	RBC (μ/l)	p = 0.002	0.104	0.233	0.001	0.002	0.000	0.376	0.312	0.197	0.285	0.107	0.342	0.709	0.005	0.090
	Hb (mg/dl)	r = 0.030	0.090	−0.090	−0.119	−0.086	−0.031	−0.080	0.073	−0.056	0.086	0.069	−0.089	−0.012	0.059	−0.220*
	Hb (mg/dl)	p = 0.776	0.398	0.399	0.263	0.422	0.768	0.455	0.492	0.598	0.421	0.520	0.405	0.910	0.579	0.037
	HCT (%)	r = −0.049	−0.013	−0.106	−0.166	−0.185	−0.105	−0.065	0.051	−0.098	0.026	0.035	−0.086	−0.033	−0.039	−0.229*
	HCT (%)	p = 0.647	0.904	0.321	0.117	0.082	0.326	0.540	0.630	0.359	0.807	0.741	0.420	0.760	0.717	0.030
	MCV (fl)	r = 0.472**	0.210*	0.218*	0.367**	0.313**	0.452**	0.210*	−0.066	0.119	0.321**	0.063	0.188	0.131	0.293**	0.374**
	MCV (fl)	p = 0.000	0.047	0.039	0.000	0.003	0.000	0.046	0.536	0.264	0.002	0.555	0.077	0.220	0.005	0.000
	MCH (pg)	r = 0.427**	0.208*	0.123	0.268*	0.255*	0.356**	0.171	−0.039	0.107	0.229*	0.167	0.029	0.047	0.255*	0.160
	MCH (pg)	p = <00.001	0.050	0.248	0.011	0.015	0.001	0.107	0.714	0.315	0.030	0.116	0.786	0.660	0.015	0.132
	MCHC (%)	r = 0.384**	0.406**	0.120	0.154	0.311**	0.446**	0.074	0.131	0.296**	0.266*	0.248*	0.293**	0.081	0.542**	0.168
	MCHC (%)	p = <00.001	<00.001	0.261	0.146	0.003	<00.001	0.489	0.219	0.005	0.011	0.019	0.005	0.451	0.000	0.113
	PLT (mm3/μl)	r = −0.240*	−0.020	−0.112	−0.169	−0.126	−0.200	−0.302**	−0.193	−0.127	−0.257*	−0.104	−0.164	−0.085	−0.237*	−0.175
	PLT (mm3/μl)	p = 0.023	0.855	0.292	0.111	0.235	0.058	0.004	0.068	0.232	0.014	0.330	0.123	0.424	0.024	0.099
	RDW (μm)	r = 0.023	0.855	0.292	0.111	0.235	0.058	0.004	0.068	0.232	0.014	0.330	0.123	0.424	0.024	0.099
	RDW (μm)	p = 0.198	0.908	0.212	0.007	0.178	0.870	0.003	0.181	0.077	0.110	0.054	0.676	0.066	0.902	0.006
	MPV (fl)	r = 0.229	−0.100	−0.101	−0.074	−0.260*	−0.092	0.257*	0.012	0.266*	0.326*	0.027	0.017	0.451**	0.026	0.166
	MPV (fl)	p = 0.078	0.446	0.442	0.574	0.045	0.484	0.047	0.928	0.040	0.011	0.837	0.898	0.000	0.842	0.206
	PDW (%)	r = 0.290*	−0.126	−0.078	−0.052	−0.227	−0.050	0.211	−0.013	0.278*	0.199	−0.008	0.047	0.385**	0.086	0.179
	PDW (%)	p = 0.290*	−0.126	−0.078	−0.052	−0.227	−0.050	0.211	−0.013	0.278*	0.199	−0.008	0.047	0.385**	0.086	0.179
	P-LCR (ng/ml)	r = 0.242	−0.190	−0.046	−0.023	−0.192	−0.166	0.270*	−0.012	0.295*	0.239	0.015	0.029	0.484**	0.037	0.155
	P-LCR (ng/ml)	p = 0.063	0.146	0.728	0.860	0.141	0.204	0.037	0.925	0.022	0.066	0.909	0.825	0.000	0.780	0.238
	Urea (mg/dl)	r = 0.375**	0.163	0.201	0.324**	0.294**	0.198	0.185	0.031	0.046	0.121	0.173	−0.028	0.154	0.182	0.135
	Urea (mg/dl)	p = <00.001	0.125	0.058	0.002	0.005	0.061	0.082	0.773	0.667	0.257	0.102	0.795	0.147	0.087	0.205
	Cr (mg/dl)	r = 0.314**	0.240*	0.272**	0.336**	0.311**	0.118	0.311**	0.066	0.080	0.104	0.071	0.184	0.232*	0.207*	0.087
	Cr (mg/dl)	p = 0.003	0.023	0.009	0.001	0.003	0.268	0.003	0.535	0.451	0.327	0.503	0.083	0.027	0.050	0.413
	AST (IU/L)	r = 0.416**	0.474**	0.157	0.469**	0.433**	0.359**	0.109	0.226*	0.272**	0.311**	0.253*	0.279**	0.241*	0.426**	0.358**
	AST (IU/L)	p = <0.001	<00.001	0.142	<0.001	<0.001	0.001	0.310	0.033	0.010	0.003	0.017	0.008	0.023	<0.001	0.001
	ALT (IU/L)	r = 0.543**	0.392**	0.256*	0.400**	0.352**	0.526**	0.259*	0.102	0.360**	0.246*	0.201	0.280**	0.144	0.514**	0.343**
	ALT (IU/L)	p = <0.001	<0.001	0.015	<0.001	0.001	<0.001	0.014	0.340	<0.001	0.019	0.058	0.008	0.176	<00.001	0.001
	ALP (IU/L)	r = 0.216	0.131	0.283*	0.223*	0.219*	0.132	0.173	0.042	0.250*	0.105	0.128	0.180	0.085	0.242*	0.159
	ALP (IU/L)	p = 0.053	0.245	0.011	0.045	0.050	0.241	0.123	0.708	0.024	0.350	0.253	0.107	0.451	0.030	0.155
	LDH (U/L)	r = −0.083	0.184	0.102	0.063	0.002	−0.120	−0.127	−0.018	0.074	0.130	0.019	−0.054	−0.054	−0.097	−0.128
	LDH (U/L)	p = 0.526	0.160	0.436	0.632	0.987	0.361	0.332	0.889	0.576	0.321	0.888	0.684	0.683	0.461	0.331
	CPK (U/L)	r = 0.131	0.007	0.198	0.202	0.138	0.042	0.193	0.003	0.152	0.001	−0.091	0.149	0.157	0.123	0.118
	CPK (U/L)	p = 0.318	0.958	0.130	0.121	0.293	0.751	0.139	0.985	0.247	0.995	0.489	0.256	0.230	0.348	0.368
	Ca (mg/dl)	r = 0.148	−0.321**	−0.227	−0.225	−0.252*	−0.375**	−0.218	−0.170	0.136	−0.378**	−0.036	−0.190	−0.397**	−0.122	−0.389**
	Ca (mg/dl)	p = 0.218	0.006	0.056	0.059	0.034	0.001	0.068	0.156	0.258	0.001	0.763	0.113	0.001	0.309	0.001
	P (mg/dl)	r = −0.128	0.133	0.056	0.151	0.231	0.155	0.137	0.217	0.002	−0.011	0.027	−0.236*	−0.005	0.152	0.051
	P (mg/dl)	p = 0.283	0.264	0.641	0.205	0.051	0.193	0.250	0.067	0.984	0.930	0.822	0.046	0.965	0.202	0.668
	Mg (mEq/L)	r = −0.058	0.158	0.375**	−0.165	−0.407**	−0.082	0.050	0.068	0.062	−0.034	0.303*	0.139	0.071	−0.073	−0.028
	Mg (mEq/L)	p = 0.660	0.228	0.003	0.208	0.001	0.533	0.707	0.604	0.639	0.795	0.019	0.289	0.589	0.577	0.831
	Na (mEq/L)	r = −0.293*	0.237	0.019	0.071	0.006	−0.298*	−0.050	0.330*	0.121	−0.071	0.300*	−0.098	−0.181	0.126	−0.226
	Na (mEq/L)	p = 0.023	0.069	0.888	0.588	0.961	0.021	0.704	0.010	0.357	0.589	0.020	0.456	0.167	0.337	0.083
	K (mEq/L)	r = 0.143	0.123	−0.394**	0.319*	0.034	−0.146	0.002	0.038	0.097	0.138	0.197	0.086	−0.253	−0.146	−0.183
	K (mEq/L)	p = 0.275	0.348	0.002	0.013	0.797	0.265	0.986	0.770	0.460	0.293	0.132	0.513	0.051	0.267	0.163
	PMN (%)	r = −0.025	0.713**	0.482**	0.305**	0.459**	0.497**	0.605**	0.336**	0.286**	0.481**	0.299**	0.355**	0.396**	0.340**	0.594**
	PMN (%)	p = 0.813	<0.001	<0.001	0.003	<0.001	<0.001	<0.001	0.001	0.006	0.000	0.004	0.001	<0.001	0.001	<0.001
	Lymph (%)	r = −0.061	−0.679**	−0.478**	−0.249*	−0.430**	−0.469**	−0.604**	−0.272**	−0.321**	−0.458**	−0.305**	−0.372**	−0.343**	−0.332**	−0.564**
	Lymph (%)	p = 0.568	<0.001	<0.001	0.018	<0.001	<0.001	<0.001	0.009	0.002	<0.001	0.003	<0.001	0.001	0.001	<0.001
	Monocyte (%)	r = 0.088	−0.279**	−0.155	−0.196	−0.251*	−0.255*	−0.323**	−0.198	−0.028	−0.189	−0.264*	−0.182	−0.281**	−0.111	−0.193
	Monocyte (%)	p = 0.408	0.008	0.144	0.064	0.017	0.015	0.002	0.062	0.791	0.074	0.012	0.086	0.007	0.296	0.068
	Eos (%)	r = 1.000	0.099	0.020	0.182	0.196	0.227*	0.201	−0.031	−0.093	0.076	0.108	0.017	−0.014	−0.052	0.074
	Eos (%)	p = <0.001	0.355	0.848	0.087	0.065	0.031	0.058	0.770	0.385	0.476	0.310	0.874	0.898	0.627	0.488

WBC: White Blood Cells, RBC: Red Blood Cell, Hb: Hemoglobin, HCT: Hematocrit, MCV: Mean Corpuscular Volume, MCH: Mean Corpuscular Hemoglobin, MCHC: Mean Corpuscular Hemoglobin Concentration, PLT: Platelet Cells, RDW: Red Cell Distribution Width, MPV: Mean Platelet Volume, PDW: Platelet Distribution Width, P-LCR: Platelet-large cell ratio, Cr: Creatinine, AST: Aspartate Aminotransferase, ALT: Alanine Aminotransferase, ALP: Alkaline Phosphatase, CPK: Creatinine Phosphokinase, LDH: Lactate Dehydrogenase, Mg: Magnesium, Ca: Calcium, P: Phosphorus, Na: Sodium, K: Potassium, PMN: Polymorph nuclear leukocytes, Lymph: Lymphocyte, NA: Not Available.

*Correlation is significant at the 0.05 level (2-tailed).

**Correlation is significant at the 0.01 level (2-tailed).

r = Correlation coefficient.

p = p-value.

Discussion

The present study confirms previous studies where fever, cough and dyspnea are the most common clinical symptoms in COVID-19 disease.^19,20 We found that some laboratory findings (ALP, Calcium, Phosphorus, PMN, monocyte counts, and PLT) are associated with an increased risk of death and may be considered predictors of disease severity. Also, significant differences were found between COVID-19 patients and healthy. Association analyses between cytokine/chemokine levels and clinical data revealed that cytokine/chemokine levels (except IL-10) were significantly associated with symptoms in COVID-19 patients. Previous studies have demonstrated that fever, cough, and sputum are the most common clinical symptoms and findings, whereas myalgia, diarrhea, and vomiting have been reported less common,^22,23 similar to reports on infections like seasonal influenza, SARS and MERS.^24,25 Reports on epidemiological characteristics of COVID-19 revealed that nearly 80% of patients are asymptomatic or have a mild disease.^26,27 In contrast, all individuals in this study had severe disease, and fever, cough, and dyspnea are the most frequently reported clinical findings in COVID-19 patients.²⁸

Our data revealed early elevated LDH and PMN in COVID-19 patients compared to healthy, as well as low platelet counts. This implies that assessing inflammation markers may be critical for early detection of suspected cases and may help in identifying patients at risk of developing severe disease.²⁹ Previous studies revealed that lymphopenia and albuminuria are other common findings,^28,30 in accordance with this study that found lymphopenia and/or an elevation in WBC as prognostic factors in COVID-19 patients. The marked lymphopenia may indicate that the virus directly or indirectly affect lymphocytes.²⁸ SARS-CoV-2 epidemiological studies showed lymphopenia, thrombocytopenia and leukocytosis, and increased levels of LDH, AST, ALT and creatinine, to be the frequently reported laboratory abnormalities.³¹ This indicates that COVID-19 infection indirectly affects the liver and other organs.³² This seems important as abnormal liver function and kidney tests are associated with increased mortality rates and poor prognosis^33,34 in line with our findings. Also, coagulation measures like PT and PTT seem to be important when evaluating prognosis in a COVID-19 patient, as well as decreased platelet counts.³⁵ Moreover, acute phase factors including CRP, LDH and ferritin are all associated with disease severity in COVID-19.³³ these elements could be considered to evaluate the patient’s disease condition and prognosis.

SARS-CoV-2, crossing the respiratory barriers and invading host cells, lead to elevation of proinflammatory cytokine/chemokine and stimulation of the cytokine storm. Some of the biomarkers analyzed here, are potent anti-inflammatory cytokines that inhibits production of proinflammatory cytokines, supposed to result in a diminution of pathological inflammation, and these are activated in parallel with proinflammatory cytokines.⁵ The complex network of cytokine/chemokine interactions in COVID-19 disease is therefore challenging to explore and understand and modulation of immune cell activation, recruitment and involvement in the inflammatory response. We examined expression levels of 15 cytokines/chemokines in deceased and recovered COVID-19 patients and healthy subjects. We detected over-expression of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IP-10, MIP-1, MCP1, RANTES, and TNF-α in COVID-19 patients compared to healthy subjects of both genders. However, none of these cytokines/chemokines were significantly different between recovered and deceased patients, although they tended to be higher in the deceased group (1.15-1.86-fold). These mediators have also been studied by Chen et al. who observed increased expression of IL-2R and IL-6, proposed to predict the severity of COVID-19 pneumonia and the prognosis of their patients.³⁶ While many studies approved the association between cytokine/chemokine profiles and COVID-19 clinical course and outcomes, there are no comprehensive studies in this area.

The elevation in IL-5 was correlated with lymphopenia and elevated IFN-γ level, one of the main acute phase cytokines, but with no difference between deceased and recovered patients. We found no difference in cytokine/chemokine patterns between deceased and recovered COVID-19 patients, in contrast to previously reported higher levels of IL-6 in ICU-admitted patients, compared to milder cases.³⁷ Chen et al. studied critical, severe and mild COVID-19 patients,³⁶ and in accordance with us found no differences in IL-1, IL-8 and TNF-α. In a study from China, the chemokine RANTES was significantly elevated in patients with mild but not severe disease, even in an early stage of infection.²⁹ In contrast, we found similar levels of RANTES in deceased and recovered patients, above levels in healthy individuals. We proved elevated IFN-γ levels compared to healthy, in accordance with a recent report on nucleoprotein-related IFN-γ secretion in COVID-19 patients.³⁸ Hu et al. studied recovered COVID-19 patients and found elevated levels of IFN-γ to protect against development of lung fibroses.³⁹ While the study did not evaluated outcomes such as lung fibrosis in this study, we noticed similar levels of IFN-γ in recovered and deceased patients. Previous studies have suggested that IL-1β and IL-6 are key pro-inflammatory biomarkers in initiation of the acute phase response, resulting in a broad range of local and systemic events such as fever and recruitment of leukocytes.^18,19 Increased IL-17 in patients with COVID-19 pneumonia has been observed in other studies,⁴⁰ and Th-17 cells contribute in the cytokine storm triggered by SARS-CoV-2.⁴¹ Further, elevations of IL-2, IL-4 and IL-17 levels are observed in COVID-19 patients’ serums with prominent lung damage.⁴⁰ Studies on TNF-α highlight differences in immunological responses during COVID-19 infection, dependent on disease severity.⁵ Due to several studies reporting an increase in TNF-α, it has been proposed that TNF-α should be the target for immunoregulatory therapies in COVID-19 disease.^19,20

SARS-CoV-2 has been reported to stimulate IL-1β increase that sequentially triggers elevation of other pro-inflammatory cytokines, including IL-6 and TNF-α.¹⁶ Although, we detected elevation of such cytokines in COVID-19 patients, we could not show different levels in deceased compared to recovered patients. We assessed correlations and associations between different cytokines, between cytokines and outcomes (healthy, recovered, deceased), and routine laboratory tests. In contrast to previous studies,^16,40 we did not find IL-6 to be correlated to WBC counts, PMN percentage or fever. However, IFN-γ was correlated to increased numbers of PMN, and suggested to be the major trigger of early inflammatory response in COVID-19 disease. IL-1β, IL-4, IL-5, IL-17, and MIP-1α were all correlated with decreased PLT counts. Levels of IL-4 and IL-5 were correlated with increased urea and creatinine, indicative of kidney organ failure. Liver failure is suspected with raised liver enzymes and these were correlated to levels of IFN-γ, IL-2, IL-6 and IP-10.

Pro-inflammatory cytokine/chemokine have a key role in viral infections through activating the adaptive immune cells; whereas an unbalanced pro- versus anti-inflammatory response can result in damage of lung tissue in the course of the infection.⁴² Recent studies showed that key pro-inflammatory cytokines and chemokines, including IFN-γ, IL-2, CCL2, and CCL3, can be anti-inflammatory mediators.^43,44 Similarly, anti-inflammatory effectors such as IL-10, under certain conditions and in combination with other cytokines, may induce a pro-inflammatory response.⁴⁵ We found similar cytokine patterns in recovered and deceased COVID-19 patients, possibly suggesting a regulatory mechanism of cytokine secretion in severe COVID-19 disease. A limitation in this study was the lower age in COVID-19 patients compared to controls. However, differences between patients with COVID-19 disease and healthy were substantial and highly significant for all measures. Therefore, the age differences should be acceptable for conclusions. There are discrepancies between our and other studies as discussed above, maybe because of differences in sample size, ethnicity, age, comorbidities, time of sampling, as well as season and climate differences. The limited number of cases when performing the study may be led to a reduced study’s power in showing statistically significant differences in different parameters.

Conclusion

This study provides more evidence for the association of cytokine/chemokine levels with the clinical course and outcome of COVID-19 disease. More studies are needed to explore if this measures could be an indicator of disease stage, help in strategy for treatment and/or prognosis for outcome.

Supplemental Material

Supplemental material - Serum cytokine/chemokine profile and clinical/paraclinical data in COVID-19 deceased and recovered patients

Supplemental material for Serum cytokine/chemokine profile and clinical/paraclinical data in COVID-19 deceased and recovered patients by Bahman Aghcheli, Emad Behboudi, Alijan Tabarraei, Hadi Razavi Nikoo, Abdolhalim Rajabi, Abdolvahab Moradi, Britt Nakstad and Alireza Tahamtan in European Journal of Inflammation

Footnotes

Acknowledgements

The authors would like to thank Golestan University of Medical Sciences and SARS-CoV-2 laboratory and nursing team that are fighting against the illness. The authors appreciate the financial support of the Research Deputy at Golestan Medical University (111504). This project was extracted from a PhD thesis.

Author contributions

AT conceptualized and designed the study. BA and EB did the experiments and collected data. AR carried out the initial data analyses. BA and EB drafted the initial manuscript. AT and BN coordinated and supervised data collection, and critically reviewed the manuscript. ATb, HRN, BN, and AM reviewed and revised the manuscript. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethics approval

The study was approved by the Ethics Committee of Golestan University of Medical Sciences (IR.GOUMS.REC.1399.007).

Informed consent

Written informed consent was obtained from all subjects before the study.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding authors.

ORCID iDs

Emad Behboudi

Alireza Tahamtan

Supplemental Material

Supplemental material for this article is available online.

Appendix

Abbreviations

References

https://covid19.who.int/.

Samadizadeh

Masoudi

Rastegar

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