A New Approach to Analyze the State of the Complement System in Patients with COVID-19. Pilot Study

One of the risk factors for infection caused by SARS-CoV-2 is hyperactivation of the complement system, which can lead to activation and damage to the endothelium, thrombosis and, in some severe cases, the development of multiple organ failure. Using quantitative immunohistochemistry, we studied the opsonization of human umbilical vein endothelial cells (HUVEC) by complement factors C3/C3b when exposed to blood plasma from patients with a confirmed diagnosis of COVID-19 in order to evaluate the possibility of using this approach to study the state of the complement system in COVID-19 ex vivo. For these purposes, FITC-labeled antibodies specific to the C3c domain of factors C3/C3b were used. The integral intensity of the recorded fluorescence and the number of C3/C3b-immunopositive structures were chosen as parameters for assessing the binding of C3/C3b to cells. We have shown that when HUVEC are incubated with plasma from patients, the total number of C3/C3b-immunopositive structures recorded on the membrane of these cells is 5.8 +/- 2.8 times (mean +/- SD, p < 0.001) higher than when exposed to plasma from healthy people. In this case, the integrated fluorescence intensity increased by 6.3 +/- 3.2 times (mean +/- SD, p <= 0.0001) compared to the control. The area of immunopositive structures recorded after exposure to plasma cells from healthy donors and patients with COVID-19 and selected for analysis ranged from 2.2 to 70 <mu>m(2) .Immunopositive particles with an area of 2.2-10.9 mu m(2) after incubation with plasma of COVID-19 patients had a more elongated shape compared to controls. The average number of particles per cell was 0.49 +/- 0.06 (mean +/- SD, n = 6) in the control, and 2.4 +/- 0.4 (mean +/- SD, n=13, p<0.001) during incubation with patient plasma. Analysis of particle area distribution showed that the most pronounced differences in the number of C3/C3b-immunopositive structures compared to the control were observed among large particles. Thus, we showed an increase in the level of opsonization of endothelial cells by complement factors C3/C3b in the presence of plasma from COVID-19 patients in comparison with control plasma. The increase is due to an increase in the number of C3/C3b-immunopositive structures, mostly large ones. We assume that the proposed approach will allow us to study the role of the complement system in damage to vascular endothelial cells in patients with COVID-19 using an ex vivo model, as well as to evaluate the level of complement activation in the plasma of patients and the effectiveness of their treatment.