Linding Wang ORCID iD: 0000-0001-6877-5791

Diagnostic Utility of Clinical Laboratory Data Determinations for

Patients with the Severe COVID-19

Yong Gao1, TuantuanLi1, Mingfeng Han1, Xiuyong Li1, Dong Wu3, Yuanhong
Accepted Article
Xu4, Yulin Zhu5, Yan Liu2, Xiaowu Wang1*, Linding Wang2*

1
Department of Clinical Laboratory, Fuyang Second People’s Hospital, Fuyang,
Anhui, China.
2
Department of Microbiology, Anhui Medical University, Hefei, Anhui, China
3
Department of pharmacy, Fuyang people's hospital, Fuyang, China.
4
Department of Clinical Laboratory, the First Affiliated Hospital of Anhui Medical
University, Hefei, China.
5
Department of Pediatrics, the First Affiliated Hospital of Anhui Medical
University, Hefei, China

* These authors equally contributed as last authors

Corresponding author

Xiaowu Wang, Department of Clinical Laboratory, Fuyang Second People’s

Hospital, Fuyang, Anhui, China.

Email: [email protected]

Linding Wang, Department of Microbiology, Anhui Medical University, Hefei,

Anhui, China.

Email: [email protected]

This article has been accepted for publication and undergone full peer review but
has not been through the copyediting, typesetting, pagination and proofreading
process, which may lead to differences between this version and the Version of
Record. Please cite this article as doi: 10.1002/jmv.25770.

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 Running Head: Clinical Laboratory Data for COVID-19 Patients

Abstract word count: 197

Text word count: 2440

Accepted Article
ABSTRACT:

The role of clinical laboratory data in the differential diagnosis of the severe forms

of COVID-19 has not been definitely established. The aim of this study was to

look for the warning index in severe COVID-19 patients. We investigated forty-

three adult patients with COVID-19. The patients were classified into mild group

(28 patients) and severe group (15 patients). Comparison of the haematological

parameters between the mild and severe groups showed significant differences in

IL-6, D-Dimer, GLU, TT, FIB and CRP (P <0.05). The optimal threshold and area

under the ROC curve of IL-6 were 24.3 pg/mL and 0.795 respectively, while those

of D-Dimer were 0.28 µg/L and 0.750, respectively. The area under the ROC

curve (AUC) of IL-6 combined with D-Dimer was 0.840. The specificity of

predicting the severity of COVID-19 during IL-6 and D-Dimer tandem testing

was up to 93.3%, while the sensitivity of IL-6 and D-Dimer by parallel test in the

severe COVID-19 was 96.4%. IL-6 and D-Dimer were closely related to the

occurrence of severe COVID-19 in the adult patients, and their combined

detection had the highest specificity and sensitivity for early prediction of the

severity of COVID-19 patients, which has important clinical value.

KEYWORDS: The severe COVID-19; IL-6; D-Dimer; diagnostic utility

INTRODUCTION

Since December 8, 2019, several cases of pneumonia of unknown etiology have

been reported in Wuhan, a city within the Hubei province of China. The disease

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 and the virus that causes it have been named as COVID-19 and SARS-COV-2

respectively. In January 2020, the outbreak spread to multiple cities in China, with

cases now confirmed in multiple countries1-3. Human to human transmission is

strongly associated with COVID-9 and SARS-COV-2. Respiratory droplets and

human to human contacts are the main routes of transmission of the virus4. In the
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early stages of this disease, symptoms of severe acute respiratory infection occur,

with some patients rapidly developing acute respiratory distress syndrome

(ARDS) and other serious complications, which are eventually followed by

multiple organ failure5. Therefore, early diagnosis and timely treatment of critical

cases is very crucial. At present, the occurrence, development, mechanism of

prognosis and immune status of patients with COVID-19 are still unclear. In this

study, we have assessed haematological characteristics of the patients. Also, we

have determined the correlation between clinical laboratory data and the severity

of COVID-19 in adult patients. Moreover, we have determined the predictive

value of clinical laboratory data for the severity of COVID-19.

MATERIALS AND METHODS

Study Subjects

We conducted a retrospective study on COVID-9 patients from January 23, 2020

to February 2, 2020 in the Fuyang Second people's hospital. The Patients were

diagnosed according to the World Health Organization interim guidance for

COVID-19. Fluorescent RT-PCR was used to confirm each diagnosis made. 43

patients, aged 19–70 years (43.74±12.12 years), were recruited for the study.

They comprised 17 females and 26 males. Blood samples were collected from

each participant, and then used for haematological investigations. The patients

were then put into two groups in terms of the severity of the disease. Hence, there

was the mild group (consisting of 28 patients) and the severe group (consisting of

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 15 patients). Permission to conduct the study was approved by the Ethics

Committee of Fuyang Second People’s Hospital; and informed written consent

was obtained from each patient.

Clinical Laboratory Data

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Routine blood tests [White blood cell count (WBC), Lymphocyte count (LYM),

Mononuclear count (MONO), Neutrophils count (NEU)] were performed on the

blood samples. Blood biochemistry parameters [Aspartate aminotransferase

(AST), Alanine aminotransferase (ALT), Glucose (Glu), Urea, Creatinine (Cr),

Cystatin (Cys-c), Uric acid (UA) and C-reactive protein (CRP)] were measured

using HITACHI 7600-020 automated biochemistry analyzer. The reagents used

were provided by Shanghai Shenneng-DiaSys Diagnostic Technology Company.

Coagulation functions [the D-Dimer, thrombin time (TT), Prothrombin time (PT),

Fibrinogen (FIB), Activated partial thromboplastin time (APTT)] were determined

using Sysmax CS5100 hemagglutinin analyzer. Procalcitonin (PCT) was detected

by Biomerieux mini-vidas automatic fluorescence immunoanalyzer. Interleukin-6

(IL-6) was detected by Rochecobase601 on the fully automated electrochemical

luminescence immunodetector, using the corresponding reagent. Mild patients

used data from their first laboratory test on admission, while severe patients had

their most recent laboratory test before their clinical diagnosis. All the operations

were done by specially-assigned personnel and in strict accordance with the

instructions regarding the use of the reagents.

Statistical Analysis

Data on AST, Urea, Cr, Cys-c, UA, CRP, WBC, LYM, MONO, NEU, TT, FIB,

APTT, PT levels were expressed as means ± standard deviation (SD). Differences

in the levels of these parameters between the mild and severe patients of the

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 disease were determined with the student’s t-test, as the data were normally

distributed. Gender was compared using χ2 test, and ages were shown as means ±

SD. Since the data regarding ALT, GLU, PCT, IL-6 and D-Dimer levels were not

normally distributed, they were compared between the two groups using Mann

Whitney U tests. The results were presented in terms of median (IQR). The AUC
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and the 95% confidence interval of the receiver operator characteristic (ROC)

curve and logistic regression analysis were computed using the predicted

probability of the severe COVID-19. The optimal cut-off points to predict the

severity of COVID-19 were determined by Youden’s index. A two-sided P value

<0.05 was considered significant. The results of the analysis were obtained using

SPSS for windows.

RESULTS

Baseline data

The study involved forty-three patients. The mean age of the 15 patients (9 males

and 6 females) who presented with the severe form of the disease was 45.2 years

(SD: 7.68 years). The mean age of the 28 patients (17 males and 11 females) who

presented with the mild form of the disease was 42.96 years. P-values of gender in

the severe group and the mild group were 0.194 and 0.503. There were no

significant differences between the severe group and the mild group in gender and

age. Baseline Characteristics of Patients With COVID-19 were shown Table 1.

The difference between the two groups was significant in Diabetes and COPD

(chronic obstructive pulmonary disease).

Clinical Laboratory Data

The haematological characteristics of the patients are shown in Table 2. The

levels of WBC, LYM, MONO, NEU, AST, ALT, UR, CR, CysC, UA, APTT, PT

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 and PCT were not significantly different between the two groups. The level of

GLU in the severe group (Median: 7.73 mmol/L; IQR:5.32 mmol/L,9.91 mmol/L)

was significantly higher than in the mild group (Median: 6.00 mmol/L; IQR: 5.45

mmol/L,7.07 mmol/L) (z= -2.293, P = 0.022). The level of CRP was significantly

higher in the severe group (39.37 ± 27.68 mg/L) than in the mild group (18.76
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± 22.20 mg/L)( t = 2.660, P = 0.011). The level of IL-6 was significantly higher

in the severe group (Median:36.10 pg/mL; IQR:23.00 pg/mL,59.20 pg/mL) than

in the mild group (Median:10.60 pg/mL; IQR:5.13 pg/mL,24.18 pg/mL) (z = -

3.160, P = 0.002). TT level was significantly higher in the severe group (15.87 ±

2.11 s) than in the mild group (14.50 ± 1.71 s)(t = 2.319, P = 0.025). FIB level

was significantly higher in the severe group (3.84 ± 1.00 g/L) than in the mild

group (3.11 ± 0.83 g/L) (t = 2.553, P = 0.014). D-Dimer level was significantly

higher in the severe group (Median: 0.49µg/L; IQR:0.29µg/L,0.91 µg/L) than in

the mild group (Median: 0.21 µg/L; IQR:0.19 µg/L,0.27µg/L) (z = -2.693, P =

0.007).

Analysis by receiver operating characteristic curve (ROC)

The ROC curve was used to analyze the early-warning efficiency and the optimal

prediction threshold of COVID-19 intensification. The AUC of IL-6 which was

used to predict the severity of COVID-19 was 0.795 (P < 0.0001), which could

better predict whether COVID-19 was complicated by severe pneumonia. The

optimum critical point of IL-6 in the group was 24.3 pg/ml, which was the upper

limit of no severe pneumonia. Similarly, the AUC used by D-Dimer to predict the

severity of pneumonia was 0.750 (P = 0.0053). The optimum critical point was

0.28 ng/L, which was the upper limit of no severe pneumonia. When IL-6 and D-

Dimer were used for combined detection, the AUC for predicting the severity was

0.840 (P <0.0001), while the AUC of other indicators (GLU, TT, FIB, CRP) were

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 lower than 0.750 indicating that the combined detection increases sensitivity and

specificity. The prediction efficiency is shown in Figure 1 and Table 3.

Effects of IL-6 and D-D on the occurrence of the severe COVID-19

The severe COVID-19 was as the dependent variable (yes = 1, no = 0), and IL-6
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(> 24.3 pg/mL =1, ≤ 24.3 pg/mL= 0), D-Dimer (>0.28 µg/L = 1, ≤0.28 µg/L = 0)

were as independent variables for Logistic regression analysis. IL-6 [OR =17.304

(95%CI 2.416, 123.933), P = 0.005], D-Dimer [OR = 12.319 (95%CI) 1.716,

85.862), P = 0.012] were independent risk factors for the severity of COVID-19.

The regression equation used was: Logit (P) = -3.106+ 2.851 (IL-6) +2.496 (D-

Dimer), which was statistically significant (χ２ = 27.387, P = 0.000), and the

prediction accuracy was 86.0%, as shown in Table 4.

Analysis of the effectiveness of individual and joint indicators (IL-6 and D-

Dimer) for predicting the occurrence of the severe COVID-19

When IL-6 was over 24.3 pg/mL, the severity of COVID-19 could be predicted,

with sensitivity and the specificity of 73.3% and 89.3% respectively, The severity

of COVID-19 was predicted when D-Dimer was over 0.28 µg/L, with the

sensitivity and the specificity of 86.7% and 82.1%, respectively. When IL-6 was

beyond 24.3 pg/mL or D-Dimer was beyond 0.28 µg/L, the sensitivity and the

specificity were 93.3% and 75.0%, respectively. And the corresponding AUC was

0.872. When combined IL-6 with D-Dimer by parallel testing, the sensitivity and

the specificity were 66.7% and 96.4%, respectively. The corresponding AUC was

0.815. The specificity reached the highest point at 96.4% when IL-6 and D-Dimer

were combined by tandem testing. The sensitivity was 93.3% when IL-6 and D-

Dimer were combined by parallel testing, as shown in Figure 2 and Table 5.

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 DISCUSSION

We reported here a cohort of 43 patients with laboratory confirmed COVID-19.

Patients had serious pneumonia and were admitted to the designated hospital in

Fuyang, China. All are imported cases. The clinical presentations are very similar
Accepted Article
to SARS-CoV. Coronaviruses (CoVs), a large family of single-stranded RNA

viruses, can infect a wide variety of animals, including humans, causing

respiratory, enteric, hepatic and neurological diseases6. Human coronavirus is one

of the main pathogens of respiratory infection5. Most patients have mild symptoms

and good prognosis. So far, a few patients with SARS-CoV-2 have developed

severe pneumonia, pulmonary oedema, ARDS, or multiple organ failure and have

died. Patients with severe illness developed ARDS and required ICU admission

and oxygen therapy7. So far, no specific treatment has been recommended for

coronavirus infection except for meticulous supportive care8. Currently, the source

of the infection has not yet been identified. The approach to this disease is the use

of personal precautionary measures to reduce the risk of transmission, and early

diagnosis of the disease.

This study reported the results of blood routine, blood biochemistry,

coagulation function and infection-related biomarkers of the adult patients with

COVID-19. We found that WBC, LYM, NEU, MONO counts were not

significantly different between the severe group and the mild group. However,

Huang C, et al found low lymphocytes and WBC counts in most patients 6. WBC

(the severe group: 4.26±1.64×109/L and the mild group 4.96±1.85×109/L) and

LYM (the severe group: 1.20±0.42×109/L and the mild group: 1.07±0.40×

109/L) were close to the bottom line of the normal range in many patients in our

study results. This result suggests that SARS-CoV-2 might mainly act on

lymphocytes, especially T lymphocytes, as does SARS-CoV. Virus particles

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 spread through the respiratory mucosa and infect other cells, induce a cytokine

storm in the body, generate a series of immune responses, and cause changes in

peripheral white blood cells and immune cells such as lymphocytes. Some studies

suggest that a substantial decrease in the total number of lymphocytes indicates

that coronavirus consumes many immune cells and inhibits the body’s cellular
Accepted Article

immune function. Damage to T lymphocytes might be an important factor leading

to exacerbations of patients8. The blood biochemistry indices, except for Glu,

were not different between the two groups. The median and IQR of Glu in severe

COVID-19 patients were 7.73 mmol/L and 4.59 mmol/L in the severe patients.

Chen et al. reported the Glu was 7.4 (3.4) mmol/L (median and IQR)5. It might be

because most severe patients have underlying diseases that caused high Glu level.

This study found that coagulation function was significantly different between the

severe group [0.49(0.29,0.91) µg/L)] and the mild group [0.21(0.19,0.27) µg/L].

Wang et al. found difference in laboratory findings between patients admitted to

the ICU [414(191,1324) mg/L] and those not admitted to the ICU [166(101,285)

mg/L], including higher levels of D-Dimer 9. The results showed that patients with

severe conditions would have abnormal coagulation. Coagulation activation could

have been related to sustained inflammatory response. Infection-related

biomarkers appeared to differ between the two groups (IL-6). However, the

proportion of IL-6 above normal was [36.10(23.00,59.20) pg/mL] in the severe

group, which was significantly higher than that in the mild group

[10.60(5.13,24.18) pg/mL]. This was in line with the concept of "Cytokine

Storm", which must be experienced by patients with mild illness to become

severe, emphasized by Lanjuan Li, an academician of the Chinese Academy of

Engineering. Among these risk factors, ROC curve was used to analyze the

specificity and sensitivity of different variables in the severe COVID-19 patients.

The AUC of IL-6 and D-Dimer were 0.795 and 0.750, respectively, while those of

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 Glu, TT, CRP and FIB were below 0.750, thus leading to poor predictive value.

When IL-6 and D-D were jointly predicted, the ROC curve integral of severe

COVID-19 was 0.840 (p< 0.01) as good predictors of severe COVID-19 under the

ROC curve, and the combined detection effect was better. Combined detection

was more efficient than independent detection. Logistic regression analysis

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showed that IL-6 and D-Dimer could predict severe COVID-19. The combined

detection of IL-6 and D-Dimer plays a complementary role. The combination of

the severe COVID-19 can be greatly improved by selecting different combinations

according to different situations. Early prediction plays an important role. When

IL-6 was over 24.3 pg/mL and D-Dimer was over 0.28 µg/L by series test and

parallel test, the AUC of the COVID-19 with or without the severe was over

0.750, which also confirmed the high prediction efficiency.

In conclusion, our findings suggest that IL-6 and D-Dimer level can be used to

estimate the severity of COVID-19. If necessary, the levels of IL-6 and D-Dimer

should be measured, as they can help diagnose the severity of adult COVID-19

patient.

This study has several limitations. Firstly, the sample size was relatively small

compared with Wuhan, where the disease originated, which may have some

impact on the statistical results. Secondly, due to the large-scale outbreak of the

epidemic restricting the flow of people, data on healthy patients are lacking as

blank controls. Since this study was a retrospective study, not every patient was

continuously monitored for all indicators in the blood including IL-6 and d-dimer

levels. In future studies, data will be collected from healthy patients as blank

controls to further explore the predictive value of IL-6 and D-dimer for patients

with SARS-COV-2 infection.

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 ACKNOWLEDGMENTS

This study was supported by National Science and Technology Major Project of

the 13th five-year Plan (Project No:2018ZX10711001-005-002), Natural Science

Foundation of Anhui Province (Project No:1708085MH193), and the Basic and

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Clinical Cooperative Research and Promotion Program of Anhui Medical

University（2019xkjT024）.

AUTHOR CONTRIBUTIONS

All authors participated in the research design. Yong Gao, Xiaowu Wang and

Linding Wang performed the data management and statistical analyses after

discussion with all authors. All authors participated in data interpretation and in

writing the manuscript. All authors took responsibility for the decision to submit

for publication.

CONFLICT OF INTERESTS

The authors declare that there is no conflict of interests.

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 blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP).
medRxiv 2020.

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Accepted Article
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FIGURE 1 ROC curves comparing the potential of different variables to predict

the severe COVID-19. (a) The prediction of the severe COVID-19 variables for

Individual indicators. (b) The prediction of the severe COVID-19 variables for IL-

6 combine with D-Dimer.

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 FIGURE 2 ROC curves of independent and joint detection were obtained when

IL-6 and D-Dimer both took the best critical values. D-Dimer or IL-6 represented

serial detection. D-Dimer and IL-6 represented parallel detection.

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TABLE 1 Baseline Characteristics of Patients With COVID-19

No. (%)

P value
Total (N = 43) Severe Mild

group(15) group(28)

Age,y 45.20±7.68 42.96±14.00 0.503

Sex 0.194

Female 6 11

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 Male 9 17

Comorbidities
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Diabetes 7（16.28） 6（40.00） 1（3.57） 0.005

Hypertension 13（30.23） 6（40.00） 7（25.00） 0.501

Cardiovascular
3（69.77） 1（6.67） 2（8.00） 0.725
disease

COPD 8（18.60） 3（20.00） 0 0.037

obesity 4（9.30） 3（20.00） 1（3.57） 0.114

Data are mean ± SD； P values indicate differences between severe group and

mild group patients. P < .05 was considered statistically

significant.

TABLE 2 Clinical Laboratory Data of 43 patients with COVID-19

Severe Mild group t/z P

Variable group(n=15) (n=28) values value

AST(U/L) 27.80±11.42 33.21±18.24 -1.042 0.300

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 24.50(15.75,37.75

ALT(U/L) 27.00(21.00,41.00) ) -0.446 0.655

Glu(mmol/L) 7.73(5.32,9.91) 6.00(5.45,7.07) -2.293 0.022▲

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Urea(mmol/L) 4.51±1.76 4.09±1.29 0.913 0.367

Cr(µmol/L) 65.33±15.55 66.96±13.38 -0.393 0.696

Cys-c(mg/L) 0.862±0.21 0.820±0.130 0.760 0.452

UA (µmol/L) 201.60±90.59 256.54±85.86 -1.963 0.056

CRP (mg/L) 39.37±27.68 18.76±22.20 2.660 0.011▲

PCT (ng/mL) 0.04(0.02,0.09) 0.02(0.01,0.04) -1.719 0.086

IL-6 (pg/mL) 36.10(23.00,59.20) 10.60(5.13,24.18) -3.160 0.002▲

WBC (×109/L) 4.26±1.64 4.96±1.85 -1.229 0.220

LYM (×109/L) 1.20±0.42 1.07±0.40 1.031 0.309

MONO (×

109/L) 0.37±0.16 0.43±0.19 -1.104 0.276

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 NEU (×109/L) 2.65±1.49 3.43±1.63 -1.556 0.127

TT(s) 15.87±2.11 14.50±1.71 2.319 0.025▲

0.014▲
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FIB(g/L) 3.84±1.00 3.11±0.83 2.553

APTT(s) 27.29±6.09 30.41±5.31 -1.745 0.089

PT (s) 11.26±1.42 12.03±1.21 -1.872 0.068

D-D(µg/L) 0.49(0.29,0.91) 0.21(0.19,0.27) -2.693 0.007▲

Data are mean ± SD and median (IQR); P values for differences between two
▲
groups were obtained by a Student’s t-test or Mann Whitney U test. is

statistically significant (P＜0.05).

TABLE 3 ROC curve analysis of Clinical Laboratory Data

Variables AUC 95%CI P value

IL-6 (pg/mL) 0.795 0.645~0.903 <0.0001

D-D (µg/L) 0.750 0.595~0.869 0.0053

Glu(mmol/L) 0.714 0.556~0.841 0.0464

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 TT(s) 0.711 0.552~0.826 0.0092

FIB(s) 0.695 0.536~0.789 0.0229

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CRP (mg/L) 0.600 0.440~0.746 0.3688

D-D_IL-6 0.840 0.697~0.934 <0.0001

TABLE 4 Analysis on the occurrence of severe COVID-19 in IL-6 and D-Dimer

B SE Wald P value OR (95%CI)

IL-6 2.851 1.005 8.055 0.005 17.304(2.416,123.933)

D-D 2.496 0.998 6.255 0.012 12.139(1.716,85.862)

constant -3.106 0.903 11.830 0.001

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 TABLE 5 Analysis of the effectiveness of individual and joint indicators (IL-6

and D-Dimer) for predicting the occurrence of the severe COVID-19

Sensitivity (%) Specificity (%) AUC (95%CI)

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IL-6 73.3 89.3 0.813(0.665,0.915)

D-D 86.7 82.1 0.844(0.701,0.936)

IL-6 or D-D 93.3 75.0 0.872(0.698,0935)

IL-6 and D-D 66.7 96.4 0.815(0.668,0.917)

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