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

Author:

King Ruby Anne N.1ORCID,Climacosa Fresthel Monica M.12ORCID,Santos Bobbie Marie M.13,Caoili Salvador Eugenio C.1ORCID

Affiliation:

1. Biomedical Innovations Research for Translational Health Science (BIRTHS) Laboratory, Department of Biochemistry and Molecular Biology, College of Medicine, University of the Philippines Manila, Philippines

2. Department of Medical Microbiology, College of Public Health, University of the Philippines Manila, Philippines

3. Department of Ophthalmology and Visual Sciences, Philippine General Hospital, Manila, Philippines

Abstract

The complement system consists of at least 50 proteins that serve as one of the first lines of defence against foreign, or damaged, cells and invading microorganisms. Its dysregulation underlies the pathophysiology of many different diseases, which makes functional assays of complement activity crucial; they are, however, underutilised. Standard haemolysis assays for the analysis of complement function employ sensitised non-human erythrocytes (e.g. from the sheep, guinea-pig or rabbit), the use of which raises animal welfare concerns. To provide an alternative to the use of such animal-derived products for complement function assays, we developed a method that employs modified human erythrocytes to evaluate the activity of complement pathways. Human erythrocytes were subjected to various chemical and/or proteolytic treatments involving 2,4,6-trinitrobenzene sulphonate (TNBS) and pancreatin. Haemolysis assays demonstrated that sequential treatment with TNBS and pancreatin resulted in significantly greater complement-mediated haemolysis, as compared to TNBS or pancreatin treatment alone. Evidence that lysis of the modified erythrocytes was complement-mediated was provided by the chelation and subsequent restoration of calcium in the plasma. Thus, such modified human erythrocytes could be used as an alternative to animal-derived erythrocytes in haemolysis assays, in order to evaluate complement activity in human plasma during, for example, the screening of patients for complement deficiencies and other abnormalities in a clinical setting.

Funder

Philippine Council for Health Research and Development

Publisher

SAGE Publications

Subject

Medical Laboratory Technology,Toxicology,General Biochemistry, Genetics and Molecular Biology,General Medicine

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1. Editorial;Alternatives to Laboratory Animals;2020-05

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