Neutrophil Percentage and Neutrophil-to-Monocyte Ratio as Independent Risk Factors in the Severity of COVID-19


 BackgroundInflammation plays an important role in progression of the various viral pneumonia containing COVID-19, severe inflammatory responses could lead to an imbalance of immune response. The purpose of this study was to explore the possibility of the white blood count, neutrophil percentage, neutrophil-to-monocyte ratio (NMR) and neutrophil-to-lymphocyte ratio (NLR) at admission to reflect the clinical severity in patients with COVID‐19.MethodsClinical and laboratory data of adult COVID-19 patients in Changsha, China, were collected and analyzed on admission. A logistic regression model was adopted to analyze the association between the disease severity and related risk factors. The receiver operating characteristic (ROC) curve was utilized to analyze the abilities of potential risk factors in the prediction of COVID-19 severity.ResultsCompared with non-severe patients, the severe ones had significantly higher levels of neutrophil percentage (74.9% vs. 62.1%; P < 0.001), NLR (4.1 vs. 2.1; P < 0.001) and NMR (12.4 vs. 8.0; P < 0.001). A regression analysis showed that neutrophil percentage (OR,1.113; 95% CI, 1.020-1.213; P=0.016) and NMR (OR, 1.110; 95% CI, 1.002-1.230; P = 0.046) were significantly associated with severity of COVID-19 patients. ROC curve showed that the area under the curves of neutrophil percentage, NMR and the combination of them were 0.842 (95% confidence interval (CI), 0.782-0.902), 0.790 (95% CI, 0.710-0.871) and 0.851 (95% CI, 0.790-0.911), respectively.ConclusionsNeutrophil percentage and NMR may act as independent risk factors in the severity of COVID-19.


Background
To our knowledge, coronavirus disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1], which broke out rstly in December 2019, Wuhan, China [2,3] and spread rapidly around the world. SARS-CoV-2 is the seventh member of coronaviruses family and can be transmitted from person to person [4,5]. As of July 31, 2020, nearly 88122 people were diagnosed and 4668 people died from COVID-19 in China [6], and more than seventeen million cases as well as 680000 deaths have been identi ed across the world [7].
Generally speaking, the manifestations of COVID-19 are typically mild or moderate, but it can also contribute to severe illness especially progress rapidly to dyspnea, acute respiratory failure or even death [8]. There is mounting evidence that in ammation plays an important role in progression of the various viral pneumonia containing COVID-19 [9,10]. Severe in ammatory responses could lead to an imbalance of immune response. Therefore, if early identi cations and interventions can be taken in time through recognizing the warning signals of severe COVID-19, it is possible to boost cure rate, reduce mortality and shorten length of hospital stay. White blood cell count, neutrophil percentage, neutrophil-tomonocyte ratio (NMR) and neutrophil-to-lymphocyte ratio (NLR) are good biomarkers for in ammation and immune status in vivo, which are also simple, reproducible and cost-effective [11][12][13][14][15][16][17][18]. Given this situation, we wondered if these parameters at admission could help re ect the clinical severity in patients with COVID-19.

Study Design and Participants
This case series was subjected to approval by the institutional ethics board of the Second Xiangya Hospital of Central South University (No. 2020001). Laboratory-con rmed adult COVID-19 patients using real-time polymerase chain reaction admitted to the Public Health Treatment Center of Changsha, China, by March 14th 2020, were retrospectively collected and enrolled using the following inclusion criteria: 1) ≥ 18 years old; 2) Without disease history of blood system. Additional informed consent was obtained from all patients for which identifying information is included in this article.

Data Collection
Two members of our team carefully collected and reviewed the medical records of patients individually.
The detailed information on demographic data, underlying comorbidities, medical history, symptoms, blood test parameters, and chest computed tomography (CT) scans on admission were recorded. The date of disease onset was de ned as the day when the symptoms was noticed. We used one of the following criteria to determine severe cases of COVID-19: 1) respiratory rate ≥ 30/min; 2) oxygen saturation ≤ 93%; 3) PaO2/FiO2 ≤ 300 mmHg; 4) lung lesions progressed > 50% within 24-48 hours; 5) mechanical ventilation was implemented; 6) shock; 7) intensive care unit admission.

Statistical Analysis
Because all continuous variables in our study were non-normally distributed, we used median with range and the Mann-Whitney test to depict and analyze them. The χ 2 test or Fisher exact test was utilized to compare differences for categorical variables. Univariate analyses were carried out using a logistic regression model to analyze the association between the disease severity of COVID-19 and related factors. Multivariate linear regression was used to analyze the correlation between multiple variables (gender, age, neutrophil percentage, NMR, NLR, chest CT with ground glass change, hypertension, cardiovascular disease) and the severity of COVID-19. Areas under the curve (AUCs) with 95% con dence intervals (CIs) were computed to assess the severity of COVID-19 using potential predictors; AUC > 0.70 was considered clinically relevant. All analyses were performed using IBM SPSS, version 26.
ROCs of neutrophil percentage, NMR and combination of them for the predictors of severity of COVID-19 patients.

Discussion
To date, although the relationship between blood test parameters and COVID-19 has been repeatedly addressed, but the role of neutrophil percentage, NMR in the severity of COVID-19 has not been elaborated yet. The important nding of the study was that neutrophil percentage and NMR might act as independent risk factors in the severity of COVID-19.
Previous studies have shown that SARS-CoV-2 could contribute to hyperin ammatory response which was the main reason of the severity and death of COVID-19 patients [15,19]. Traditional systemic in ammatory is triggered by white blood cell which comprises of neutrophils, lymphocytes, monocytes, and immature cells [20]. Neutrophils are the most abundant leukocytes in blood circulation which are the rst line of host defense against pathogens [21,22]. It was reported that humans produced 1 billion neutrophils per day per kilogram of body weigh whereas increased to 10 billion when infected [21]. In vivo, the release of danger signals recruit the neutrophils to the site of tissue necrosis [23], then activated neutrophils through releasing preformed mediators, recognizing foreign nucleic acids, extruding neutrophil extracellular traps (NETs), presenting antigens to memory CD4 T cells, producing a variety of cytokines to regulate innate and adaptive immune responses, and to some degree, neutrophils could also lead to tissue necrosis [24][25][26][27][28]. Neutrophils were con rmed having in uence on not only infectious and autoimmune diseases but hematopoiesis, angiogenesis and cancer growth [25,28]. The effect of neutrophils in viral infections remains ill de ned, research has showed that neutrophils were the predominant cells in virus-induced lung infection and could control the replication and transmission of virus [29,30]. In addition, neutrophils played important role in COVID-19 [17].
Neutrophil percentage usually act as the index of in ammation and increases in infected diseases [16]. In our study, univariate regression analysis showed that neutrophil percentage was the independent risk factor in the severity of COVID-19 and the AUC was 0.842 which suggested the good predictive value in the severity of COVID-19. The optimal threshold at 49.7% for neutrophil percentage signi ed a possibility of clinical symptoms changing from non-severity to severity with high sensitivity of 92.5 percent. Zhang et al. found higher neutrophil percentage has been reported to be related with the severity of COVID-19 [31], consistent with our study, although further research hasn't been explored.
The source of monocytes is bone marrow which drives from progenitor cells. Monocytes have the ability of phagocytic activity and can differentiate into antigen-presenting cells which contribute to the participation of innate and adaptive immune responses [32]. After migrating from blood to in ammatory sites, response is different whether monocytes differentiate into macrophages or myeloid dendritic cells [33,34]. Activated monocytes release in ammatory cytokines to ght for infection, and the overexpression of cytokines is closely related with mortality, nevertheless, immunological paralysis characterized by monocyte deactivation can also explain for the poor clinical outcomes of sepsis [35,36].
Recent study suggested that bronchoalveolar uid were enriched in CCL2 and CCL7 in patients with severe COVID-19,which were the recruitment of CC-chemokine receptor 2-positive monocytes [37]. Singlecell RNA sequencing analysis also showed that mononuclear phagocyte comprising of depletion of tissue-resident alveolar macrophages and monocyte-derived macrophages was higher in severe COVID-19 than that of mild or healthy controls [38].
These peripheral blood in ammatory cells are often studied together, NLR and NMR are easily calculated and serve as systemic in ammation biomarkers [11,39]. NLR has been studied in the severity and prognosis of COVID-19 [12-14, 18, 40], in our study, NLR was also higher in severe COVID-19. According to Fang [11], segmented NMR can act as an immune dysfunction score to predict the 28-day mortality of patients with sepsis. In present study, levels of NMR were signi cantly higher in severe COVID-19 than non-severe patients, which implied that NMR might be a potential serum marker for severity of COVID-19. In addition, the logistic regression analysis showed that NMR was correlated with the severity of disease and ROC analysis also suggested that NMR was a valuable predictor.
A combination of different texts can improve the sensitivity of single test, we combined NMR with neutrophil percentage and proved a sensitivity of 72.5 percent, a speci city of 81.8 percent and AUC 0.851 in recognizing the severity of COVID-19.
This study has several limitations. First, the enrolled number of patients is small which may limit the conclusions of the study. For example, several parameters at admission are related to severity of the disease in univariate regression analysis, while only neutrophil percentage and NMR make sense in the multivariate analysis. In the future research, these indicators also need closer attention. Second, disease is a process of deterioration, the indexes at admission can't re ect the changes in diseases.

Conclusions
In summary, this retrospective cohort study revealed that the neutrophil percentage and NMR are independent risk factors for the severity of COVID-19. Further researches are needed to enlarge the sample size and assess dynamically to con rm these results. Author contributions FP and SL are joint rst authors. YZ and SW contributed to the study design, implementation, manuscript discussion and critical revision. FP, SL, CW, and BY collected and interpreted the data. All authors read and approved the nal manuscript.