key: cord-0790819-4j0cqkuo authors: Bagherimoghaddam, Ahmad; Rafatpanah, Houshang; Mansouritorghabeh, Hassan title: Elevated levels of C3, C4, and CH50 of the complement system in ICU and non‐ICU patients with COVID‐19 date: 2022-02-16 journal: Health Sci Rep DOI: 10.1002/hsr2.519 sha: ed0e40359a6e2a68c96bb75c9be704e45b0aa083 doc_id: 790819 cord_uid: 4j0cqkuo PURPOSE: SARS‐CoV‐2 infection has spread in each corner of the world. Many health systems have dealt with it intensively. The complement system is an instrumental component in the inflammatory immune response and plays a role in the activation of blood coagulation. Our understanding of the pathophysiology of SARS‐CoV‐2 is still limited but is constantly expanding. This study aimed to determine changes in the complement system in intensive care unit (ICU) and non‐ICU patients with COVID‐19. METHODS: In a cross‐sectional study, plasma levels of C3, C4, and CH50 were determined in two groups of ICU and non‐ICU patients with COVID‐19 to understand the potential effects of SARS‐CoV‐2 on the innate immune system. The assays of C3 and C4 were conducted using turbidimetry method. The CH50 test was conducted using the functional method. RESULTS: The present study revealed that the C3, C4, and CH50 plasma levels were 142.48 ± 30.38 mg/dL, 32.58 ± 8.78 mg/dL, and 61.74 ± 19.54%, respectively. These results indicate high levels of complement components C3 and C4 and complement function (CH50) in patients with COVID‐19 than normal ranges. Plasma levels of C3, C4, and CH50 were higher in ICU patients than in non‐ICU COVID‐19 groups. CONCLUSION: These results indicate that the innate immune system was activated in both ICU and non‐ICU patients in response to SARS‐CoV‐2 infection. Further studies with a larger number of COVID‐19 patients and additional testing of complement components (C3a and C5a) may reveal the role of COVID‐19 infection in the activation of the complement system. In March 2020, the World Health Organization (WHO) declared a COVID-19 pandemic. 1 According to WHO, 259502031 people have been definitively infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as of November 26, 2021 . In addition, more than 5 183 003 deaths have been recorded due to COVID-19 spreads 1000 times faster in the body than most related viruses. 3 Mild-to-moderate symptoms are predicted in the majority of COVID-19 patients. COVID-19 causes multiple organ dysfunction or acute respiratory distress syndrome in approximately 5% of cases. 4, 5 In addition, symptoms in children are usually milder than in adults. Pneumonia is a more serious condition in adults than in children. 6 Multiple organ dysfunction in COVID-19 has been associated with a high risk of death due to advanced respiratory failure and systemic coagulopathy. 7, 8 The complement system, a core component of innate immunity, elicits proinflammatory responses during viral infections. 9 It has been hypothesized that activation of the complement system begins with direct lectin activation, followed by immune complex activation in the classical pathway, and finally by activation of the Toll-like receptor by the alternative pathway. 10 Activation of the C3 component of the complement system exacerbates acute respiratory distress syndrome, according to a report on SARS-CoV, which is closely related to SARS-CoV-2. 11 In five COVID-19 patients, skin and lung biopsies revealed deposits of C4d and C5b-9 in both skin and lung tissues. In addition, one patient with COVID-19 had an increased number of activated plasma cells in his bronchoalveolar lavage. 12 The relationship between complement activation and significant thrombophilia has been demonstrated previously. [13] [14] [15] In addition, activation of the coagulation system after inflammation has been demonstrated to be caused by downregulation of physiological anticoagulants, tissue factor-mediated thrombin generation, and inhibition of fibrinolysis. 16 Recent studies emphasize the prognostic value of D-dimer in COVID-19 infections. 17 The role of microvascular damage caused by complement activation and subsequent procoagulant conditions in the pathogenesis of SARS-CoV-2 in tissues is supported by these findings. 18 There is an urgent need to shed more light on this topic to understand the role of the complement system in the pathophysiology of COVID-19. In patients with COVID-19, the deposition of C4d and C5b-9 could be an indicator of strong activation of the alternative and lectin-based pathways of the complement system. If the disruptive role of the complement system in the deterioration of COVID-19 is confirmed, scientists may consider the use of complement inhibitors as a potential therapeutic option in patients with CIVID-19. Here, a cross-sectional study was conducted to examine C3, C4, and CH50 levels in two groups of patients with COVID-19 admitted to the intensive care unit (ICU) and outside the ICU. It is assumed that patients with COVID-19 admitted to the ICU completely are affected by SARS-CoV-2. Therefore, here, we tried to compare them in two groups of patients hospitalized for COVID-19. Under these circumstances, merely the influence of COVID-19 on the complement system would be measurable. Exclusion criteria included: 1. Infestation with any of the above diseases after entering into the study. 2. Refusal to participate in the study. The 60 patients with COVID-19 were divided into two groups: ICU patients and non-ICU patients with COVID-19 (30 patients in each group). The non-ICU patients with COVID-19 were patients who did not require admission to the ICU and did not require supplemental oxygen. These patients were selected from a general acute respiratory care unit. Admission to an ICU and the need for intubation or ventilation were the prerequisites for admission in the intensive care category. For the performance of the upcoming laboratory tests, the obtained sera were aliquoted and frozen after centrifugation at 1500g for 10 min. The CH50 assay was performed for all patients in one run after thawing the serum samples. The CH50 assay was conducted using the functional power of the complement system to lyse 50% of sheep red blood cells. The sheep red cells were precoated with rabbit antibodies against sheep red cells (hemolysin). The details of the procedure are described in the article by Dong and Liu. 19 The amboceptor used was purchased commercially from the Behring Company (Dade Behring, Marburg, Germany). A qualified laboratory technician experimented and read the results. Turbidimetry assay was used to determine C3 and C4 concentrations. C3 and C4 concentrations were measured using commercial biochemistry kits from Bionik Company (Tehran, Iran). Data were read using a Diatron instrument (Pictus 700). Normal values for C3 and C4 in adults were 90 to 180 and 10 to 40 mg/dL, respectively. All methods were performed in accordance with current guidelines and regulations. D-dimer, taken from medical records, was determined using commercial biochemistry kits from Bionik Company (Tehran, Iran). Among the 60 patients with COVID-19, there were 29 females (48.4%) and 31 males (51.6%). The youngest affected patient who participated in the study was 20 years old, while the oldest was 103 years old. The mean age of the patients was 57.70 ± 19.79 years. Table 1 showed the minimum, maximum, and mean ± SD of some of the major laboratory findings in these patients, including C3, C4, and CH50 levels. Table 2 listed these findings separately for the two groups of ICU and non-ICU patients with COVID-19. Overall, patients with COVID-19 had increased plasma levels of C3, C4, and CH50 than normal ranges (Figure 1 ). All three markers were elevated in both ICU and non-ICU patients with COVID-19. Moreover, complement system activity, which measured as CH50 was increased in ICU and non-ICU groups ( Table 2) . Although there was no statistical difference in plasma levels of C3, C4, and CH50 between ICU and non-ICU patients with COVID-19, the plasma levels of the current markers in two groups of ICU and non-ICU patients with COVID-19 were higher than normal ranges. The minimum, maximum, and mean of D-dimer in all patients were 100, 10 000, and 2484.75 ± 2184.86 ng/mL, respectively. The mean level of D-dimer was higher in ICU patients with COVID-19 than non-ICU patients with COVID-19 (2606.33 ± 1722.32 vs 2317.58 ± 2728.60 ng/mL respectively). The levels of D-dimer were elevated in both groups without a statistical difference, but were higher than the normal range (0-500 ng/mL). The complement system consists of several proteins that can lyse cells after activation. 20 The CH50 assay measures the ability of complement components to bind antibody-sensitized sheep erythrocytes. In other words, the CH50 assay is a screening tool to determine whether or not the classical pathway of the complement system has been activated. This process is mediated by assembling a membrane attack complex. In the case of SARS-CoV-2 infection, it is assumed that the cells infected by the virus are recognized by the body's immune There are limited data on the levels of C3 and C4 in COVID-19. Laurence et al. 21 reported elevated C3 levels in a patient with COVID-19 and found that anti-C5 treatment with Eculizumab produced the best results. There is increasing evidence of successful C3 inhibitor therapy in COVID-19 patients. 22, 23 On the other hand, two papers reported normal C3 and C4 levels in patients with COVID-19. 24, 25 Compared with the standard C3 and C4 assays, the C3a and C5a assays will depict complement-driven disease activation. 26 expected that many more studies will uncover the mysteries behind SARS-CoV-2 infection. This study was approved by The Regional Ethics Committee in Mashhad University of Medical Sciences (IR.MUMS.REC.1399.153). This study was approved by The Vice Chancellor of the Research in Mashhad University of Medical Sciences (Approval number 990243). The regional Ethics Committee in Mashhad University of Medical Sciences issued an approval letter for this manuscript. There is no conflict of interest regarding this manuscript. The authors affirm that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained. The raw data are available on request from the corresponding author. 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SLE and serum complement: causative, concomitant or coincidental? Elevated levels of C3, C4, and CH50 of the complement system in ICU and non-ICU patients with COVID-19 The authors would like to express their gratitude to the Regional Ethics Committee at Mashhad University of Medical Sciences for approvals of the manuscript.