A Human Erythrocyte-based Haemolysis Assay for the Evaluation of Human Complement Activity

Area of Science:

• Immunology
• Hematology

Background:

• The complement system is vital for innate immunity, acting as a first defense against pathogens and damaged cells.
• Complement system dysregulation is implicated in numerous diseases, necessitating reliable functional assays.
• Current complement assays use animal-derived red blood cells, raising ethical concerns and limiting clinical utility.

Purpose of the Study:

• To develop an alternative method for assessing complement system activity using modified human erythrocytes.
• To provide a more ethical and potentially more clinically relevant approach to complement functional assays.

Main Methods:

• Human erythrocytes were chemically and proteolytically modified using 2,4,6-trinitrobenzene sulphonate (TNBS) and pancreatin.
• Complement-mediated haemolysis assays were performed using these modified erythrocytes.
• Calcium chelation and restoration experiments confirmed the complement-dependent nature of the lysis.

Main Results:

• Sequential treatment of human erythrocytes with TNBS and pancreatin significantly enhanced complement-mediated haemolysis compared to single treatments.
• The lysis of modified erythrocytes was confirmed to be complement-dependent.
• Modified human erythrocytes show promise as a substitute for animal-derived erythrocytes in complement assays.

Conclusions:

• Modified human erythrocytes provide a viable, animal-free alternative for evaluating complement pathway activity.
• This method can be applied in clinical settings for diagnosing complement deficiencies and other related abnormalities.
• The developed assay addresses animal welfare concerns associated with traditional haemolysis assays.