A Flow Cytometry-Based Assay for the Measurement of Total Complement Activity in the Serum and Clinical Practice

Objective. To develop a novel sensitive and accurate assay suitable for high-volume testing of the total complement activity in the serum for clinical laboratories. Methods. The total complement activity (TCA) to be measured was quantified by detecting the number of fragments produced by erythrocyte lysis and the erythrocyte fragmentation index (EFI), indicating TCA. EFI = M × M 2 / M 1 + M 2 , where M is the number of erythrocyte fragments (removed from the background), M 1 is the number of unagglutinated red cells, M 2 is the number of agglutinated red cell groups, and M 2 / M 1 + M 2 is the agglutination coefficient indicating the degree of erythrocyte agglutination. Mild changes in hemolysin and erythrocyte concentrations were made to optimize the testing conditions. The same serum samples were tested for 10 consecutive days to determine the stability of the experimental results. Serum EFI was detected in both nephrotic syndrome patients and healthy subjects. Results. There was a linear relationship between hemolysin and erythrocyte agglutination ( r = 0.999 , P < 0.001 ). A good linear relationship existed between EFI and TCA ( r = 0.991 , P < 0.001 ). The results were not affected by slight fluctuations in the concentrations of hemolysin or erythrocytes. The interbatch CV = 8.6 % of the test results showed good stability. There was a significant difference in the EFI between nephrotic syndrome patients and healthy individuals, P < 0.001 , and EFI was reduced in nephrotic syndrome patients compared to healthy individuals. Conclusion. The flow cytometry-based assay for TCA was sensitive and accurate and had potential value for clinical application.

Complement system component dysregulation is a distinctive feature of COVID-19 disease: a prospective and comparative analysis of patients admitted to the emergency department for suspected COVID-19 disease

The complement system (CS) plays a pivotal role in Coronavirus disease 2019 (COVID-19) pathophysiology. The objective of this study was to provide a comparative, prospective data analysis of CS components in an all-comers cohort and COVID-19 patients. Patients with suspected COVID-19 infection admitted to the Emergency department were grouped for definite diagnosis of COVID-19 and no COVID-19 accordingly. Clinical presentation, routine laboratory and von Willebrand factor (vWF) antigen as well as CS components 3, 4 and activated 5 (C5a) were assessed. Also, total complement activity via the classical pathway (CH50) was determined. Levels of calprotectin in serum were measured using an automated quantitative lateral flow assay. We included 80 patients in this prospective trial. Of those 19 (23.7%) were tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Patients with COVID-19 had higher levels of CS components 5a and 4 (54.79 [24.14–88.79] ng/ml vs. 35 [23.15–46.1] ng/ml; p = 0.0433 and 0.3772 [± 0.1056] g/L vs. 0.286 [0.2375–0.3748] g/L; p = 0.0168). COVID-19 patients had significantly higher levels of vWF antigen when compared to the control group (288.3 [± 80.26] % vs. 212 [151–320] %; p = 0.0469). There was a significant correlation between CS C3 and 5a with vWF antigen (rs = 0.5957 [p = 0.0131] and rs = 0.5015 [p = 0.042]) in COVID-19 patients. There was no difference in calprotectin plasma levels (4.786 [± 2.397] µg/ml vs. 4.233 [± 2.142] µg/ml; p = 0.4175) between both groups. This prospective data from a single centre all-comers cohort accentuates altered levels of CS components as a distinct feature of COVID-19 disease. Deregulation of CS component 3 and C5a are associated with increased vWF antigen possibly linking vascular damage to alternative CS activation in COVID-19.

Bacterial infections in a pediatric cohort of primary and acquired complement deficiencies

Background Acquired complement deficiency can occur in the setting of autoimmune syndromes, such as systemic lupus erythematosus (SLE), with very low or, occasionally, undetectable C3 levels. Based on inherited complement defects, patients with transiently low complement may be at similar risk for serious bacterial infection, but the degree of risk related to C3 level and temporal association is unknown. Methods We performed a retrospective study including pediatric patients with undetectable total complement activity or absent individual complement components measured at our institution from 2002 to 2018. We assessed annual rate of serious bacterial infection (SBI) defined as requiring hospitalization and/or parenteral antibiotics by manual chart review. Among included SLE patients, we assessed the 30-day probability of SBI for given C3 measurements using a logistic regression model to determine risk. Primary complement deficiency was analyzed for SBI rate as comparison. Covariates included age, level of immune suppression and history of lupus nephritis. Results Acquired complement deficiency secondary to SLE-related disease [n = 44] was the most common underlying diagnosis associated with depressed complement levels and were compared to a cohort of primary complement deficient patients [n = 18]. SBI per 100 person-years and cohort demographics were described in parallel. Our logistic regression analysis of pediatric patients with SLE showed low C3 level was temporally associated with having an SBI event. Given equivalent immunosuppression, patients with an SBI had lower C3 levels at the beginning of the observation period relative to patients without SBI. Conclusion Pediatric patients with the diagnosis of SLE can develop very low C3 levels that associate with risk of serious bacterial infection comparable to that of patients with primary complement deficiency. Patients prone to severe complement consumption may particularly be at risk.

Bacterial infections in a pediatric cohort of primary and acquired complement deficiencies.

Background: Acquired complement deficiency can occur in the setting of autoimmune syndromes, such as systemic lupus erythematosus (SLE), with very low or, occasionally, undetectable C3 levels. Based on inherited complement defects, patients with transiently low complement may be at similar risk for serious bacterial infection, but the degree of risk related to C3 level and temporal association is unknown. Methods: We performed a retrospective study including pediatric patients with undetectable total complement activity or absent individual complement components measured at our institution from 2002 to 2018. We assessed annual rate of serious bacterial infection (SBI) defined as requiring hospitalization and/or parenteral antibiotics by manual chart review. Among included SLE patients, we assessed the 30-day probability of SBI for given C3 measurements using a logistic regression model to determine risk. Primary complement deficiency was analyzed for SBI rate as comparison. Covariates included age, level of immune suppression and history of lupus nephritis.Results: Acquired complement deficiency secondary to SLE-related disease [n=44] was the most common underlying diagnosis associated with depressed complement levels and were compared to a cohort of primary complement deficient patients [n=18]. SBI per 100 person-years and cohort demographics were described in parallel. Our logistic regression analysis of pediatric patients with SLE showed low C3 level was temporally associated with having an SBI event. Given equivalent immunosuppression, patients with an SBI had lower C3 levels at the beginning of the observation period relative to patients without SBI. Conclusion: Pediatric patients with the diagnosis of SLE can develop very low C3 levels that associate with risk of serious bacterial infection comparable to that of patients with primary complement deficiency. Patients prone to severe complement consumption may particularly be at risk.

Risk of Bacterial Infection in Acquired Complement Deficiencies

Background: Acquired complement deficiency can occur in the setting of autoimmune syndromes, such as systemic lupus erythematosus (SLE), with very low or, occasionally, undetectable C3 levels. Based on data from patients with inherited complement defects, a perceived risk for serious bacterial infection exists amongst patients with transiently low complement, but the degree of risk related to C3 level is unknown. Methods: We performed a retrospective study of all pediatric patients with an undetectable total complement activity or absent individual complement components measured at our institution from 2002 to 2018. We assessed annual rate of serious bacterial infection (SBI) defined as requiring hospitalization and/or parenteral antibiotics. Among included SLE patients, we assessed the 30-day probability of SBI for given C3 measurements using a logistic regression model to determine risk. Results: Acquired complement deficiency secondary to SLE-related disease [n=44] was the most common underlying diagnosis associated with depressed complement levels. While controlling for immunosuppression level and lupus nephritis diagnosis, our logistic regression analysis of pediatric patients with SLE showed low C3 level was temporally associated with having an SBI event. Even in patients with equivalent immunosuppression, patients with an SBI were found to have lower C3 levels preceding the infection relative to patients without SBI. Conclusion: Pediatric patients with the diagnosis of SLE can develop very low C3 levels that are independently associated with risk of serious bacterial infection. Patients prone to complement consumption may particularly be at risk.

Differences of Total Activity and Heat Tolerance of Complement in Plasma Between Black and Yellow Populations

Background The goal of this paper is to compare and evaluate the differences in total activity and Heat Tolerance of complement distribution Between Black and Yellow populations.Methods Blood samples of Black and Yellow healthy individuals were randomly collected, and plasma was obtained. The test plasma was diluted in a five series concentration, following with standard reaction and experimental reaction measurements. The basic principle of heat tolerance temperature calculation is to consider the ratio value (ratio method) in which heat tolerance of TCA calculated and plotted according to the area of trapezoidal and triangle as follows: C x°C=OD1-OD5+(OD2-OD5+OD3-OD5+OD4-OD5)*2. Where Cx°C is the total complement activity of the specimen measured in the two reaction conditions.Results The total activity of complement in Black individuals was statistically significantly lower than Yellow individuals (P< 0.05). The heat tolerance of total complement activity was higher among blacks than yellow individuals with no differences between the two groups (P> 0.05). The frequency values of total activity of complement distributed in the yellow individuals was statistically higher than Blacks (P<0.05) and The heat tolerance of total complement activity was statistically higher significant among Blacks than Yellow individuals (P<0.05).Conclusions There is a high differences in total complement activity and heat Tolerance distribution between Black and Yellow populations, may predict health status and innate immunity level differences between African race and Asian race.