The significance of surface neutrophilic MPO expression level in NETosis and NETosis-associated coagulopathies in covid-19 infected patients

Abstract

Introduction: Inflammatory response-induced coagulopathy is a common complication associated with severe form of covid-19 infection. Evidences suggest that neutrophil extracellular traps (NETs) play a significant role in triggering the immunothrombosis in this condition. We aimed to evaluate the diagnostic value of surface neutrophilic myeloperoxidase (MPO) as NETosis biomarker for predicting the risk of covid-19-associated coagulopathies.

Methods: Covid-19 infection was assessed by real-time-PCR and plasma d-dimer levels were measured by ELFA. Based on the covid-19 infection and d-dimer level outcomes, patients were categorized into four groups. Any alteration in the serum level of IL-6, H3Cit and neutrophilic surface MPO were analyzed by CLIA, ELISA, and flow cytometry, respectively.

Results: H3Cit variations and different d-dimer values confirmed the association between NETosis and coagulopathies. Findings showed that the expression of neutrophilic MPO reduced in cases with NETosis, which was correlated with increased levels of H3Cit. ANC/MPO ratio was signified as a valuable marker to discriminate the covid-19 and non covid-19-associated coagulopathies and could be considered as a prognostic factor due to its noteworthy correlation with serum IL-6 concentration.

Conclusion: Declined levels of surface neutrophilic MPO in NETosis correlate with covid-19-associated coagulopathies and increased IL-6 levels, as a potential biomarker of covid-19 disease severity.

Keywords: Coagulopathies; NETosis; Surface myeloperoxidase.

Fig. 1

Representative gating strategy of neutrophil population (Dot-plot), IgG1 FITC isotype control (Histogram, Left), and FITC-anti-MPO treated sample (Histogram, Right). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Bar chart demonstrates the significant reduction in MPO expression in the surface of neutrophils in cases with higher d-dimer level (*P value < 0.05) (A). Pearson correlation analysis of MFI obtained by flow cytometry and d-dimer levels illustrated a significant reverse correlation between these two variables (Pearson r: −0.3, *P value < 0.05) (95% confidence interval: −0.4338 to −0.05469) (B).

Fig. 3

Comparison of neutrophilic surface MPO expression in different groups by flow cytometry. Results evidenced a significant decrease in neutrophilic MPO expression in all studied groups when compared with healthy controls. Meanwhile, this parameter did not reveal any significant change among different patient groups.

Fig. 4

Bar chart compares the serum H3Cit level among the different groups. The level of H3Cit in group A was expressively higher than those of the other three groups. The difference was not significant between groups B and C, while their related H3Cit levels were meaningfully greater than the control group.

Fig. 5

The Pearson correlation analysis of neutrophilic surface MPO and serum H3Cit in all studied cases revealed a noteworthy reverse association between these two variables (**P value < 0.01) with a 95% confidence interval of −0.4915 to −0.1025 (A). Evaluating the correlation between these two variables within different groups showed significant association in group A (B) and D (E), while this association was not evident in group B (C) and C (D).

Fig. 6

The bar chart shows One-way analysis of variance for ANC/MPO index among cases who were already grouped based on their coagulopathy status and covid-19 results. Results represent a significant increase in this index in group A. The differences are not significant when compared among other groups (A). ROC curve analysis of this index in group A and B (covid-19 positive case) suggest a cutoff value of <0.13 for predicting the coagulopathy in covid-19 positive cases with a sensitivity of 76.9% and specificity of 92.3% (***P value < 0.001) (B).

Fig. 7

Representative of Pearson correlation analysis for ANC/MPO ratio and serum IL-6 level. Assessments confirmed a direct and considerable association between these two parameters (***P value < 0.001).