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How to analyze complement? 

For accurate complement analysis, it is important that not only the correct technique is used but also that pre-analytical sample handling is performed in an appropriate manner to avoid erroneous results.

Hycult biotech preformed a literature search using the Pubmed/MEDLINE database focusing on studies measuring the key complement components C3, C5 and/or their split products C3a/C5a and/or the soluble variant of the terminal C5b-9 complement complex (sTCC) in human blood samples that were published between February 2017 and February 2022. The identified studies were reviewed whether they had used the correct sample type and techniques for their analyses.

A total of 92 out of 376 studies were selected for full-text analysis. Forty-five studies (49%) were identified as using the correct sample type and techniques for their complement analyses, while 25 studies (27%) did not use the correct sample type or technique. For 22 studies (24%), it was not specified which sample type was used.

A substantial part of the reviewed studies did not use the appropriate sample type for assessing complement activation or did not mention which sample type was used. This deviation from the standardized procedure can lead to misinterpretation of complement biomarker levels and hampers proper comparison of complement measurements between studies. Therefore, this study underlines the necessity of general guidelines for accurate and recommended procedure.

Brandwijk RJMGE, Michels MAHM, van Rossum M, de Nooijer AH, Nilsson PH, de Bruin WCC, Toonen EJM. Pitfalls in complement analysis: A systematic literature review of assessing complement activation. Front Immunol. 2022 Oct 18;13:1007102. doi: 10.3389/fimmu.2022.1007102. PMID: 36330514; PMCID: PMC9623276.

But how can you analyze complement and overcome these pitfalls? You can find optional guidelines below or reach out to us directly to help you


This chart helps you with the selection of the appropriate sample type and technique for complement analysis.

Use the flowchart for the selection of:
(A) the appropriate sample type or
(B) the appropriate technique for the assessment of complement.

3 complement pathways

The complement system is a crucial part of the immune system, consisting of over 30 proteins that work together to help identify and destroy foreign pathogens. There are three major pathways of complement activation: the classical pathway, the lectin pathway, and the alternative pathway.

The classical pathway is activated when antibodies bind to specific antigens on the surface of pathogens. This binding triggers a cascade of complement proteins to bind and activate one another, ultimately leading to the formation of the membrane attack complex (MAC) that punctures and destroys the pathogen’s cell membrane.

The lectin pathway is activated when proteins called lectins bind to certain sugars on the surface of pathogens. This binding triggers a similar cascade of complement protein activation, leading to the formation of the MAC and pathogen destruction.

The alternative pathway is constantly active at low levels and can be triggered by the presence of certain molecules on the surface of pathogens or damaged host cells. This pathway also leads to the formation of the MAC and pathogen destruction.

All three complement pathways work together to provide a rapid and effective defense against invading pathogens. They also play a role in regulating the immune response and removing damaged host cells. Dysfunction of the complement system can lead to various diseases, including autoimmune disorders and infections.

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