title: Antibody complement-mediated hemolytic studies with kodecytes reveal that human complement utilized in the classical pathway is more stable than generally accepted.
abstract: BACKGROUND
  Complement has significant status in the field of transfusion medicine. The accepted stability profile of complement is based on historical studies of diluted human serum hemolyzing rabbit heterophile antibody-sensitized sheep red blood cells (RBCs). Contemporary tools are available to reevaluate these historical observations using human heterophile antibodies, undiluted serum, and antigen-modified human RBCs.
  
  
  METHODS
  Human RBCs were made into "animal-like" kodecytes with heterophile Galα3Galβ4GlcNAcβ function-spacer-lipid constructs. These α-Gal-kodecytes were prepared with an antigen dilution capable of consistently producing 50% antibody-mediated hemolysis against human α1-3galactose heterophile antibodies and undiluted standardized serum. Standardized human serum aliquots from a two-donor pool stored at -85, -20, 4, 22, and 37°C for durations of up to 150 days were evaluated for loss of hemolytic activity. Where practical methodologic procedures were aligned with historical studies.
  
  
  RESULTS
  Comparison of the historical assay with the α-Gal-kodecyte assay against complement activity standards showed concordance. However, in most scenarios complement was found to be more than twice as stable as generally accepted. At least 60% of complement hemolytic activity was observed in serum stored at 22°C for 1 week or 2 months at 4°C. No loss of hemolytic activity was observed after 5 months' storage at temperatures below -20°C.
  
  
  CONCLUSIONS
  An alternative method using undiluted serum and modified human RBCs observed that classical-pathway complement hemolytic activity in stored human serum is at least twice as stable as previously accepted.
authors.affiliation: School of Science, Faculty of Health and Environmental Sciences, Auckland University of Technology, Auckland, New Zealand.|Centre for Kode Technology Innovation, School Engineering, Computer and Mathematical Sciences, Faculty of Design and Creative Technologies, Auckland University of Technology, Auckland, New Zealand.
authors.forename: Holly
authors.initials: H
authors.lastname: Perry
authors.affiliation: Centre for Kode Technology Innovation, School Engineering, Computer and Mathematical Sciences, Faculty of Design and Creative Technologies, Auckland University of Technology, Auckland, New Zealand.|Shemyakin Institute of Bioorganic Chemistry, Moscow, Russian Federation.
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bovin
authors.affiliation: Centre for Kode Technology Innovation, School Engineering, Computer and Mathematical Sciences, Faculty of Design and Creative Technologies, Auckland University of Technology, Auckland, New Zealand. kiwi@aut.ac.nz.
authors.forename: Stephen
authors.initials: S
authors.lastname: Henry
mesh_terms: D000818:Animals; D000910:Antibodies, Heterophile / Q000276:immunology*; D015478:Antigens, Heterophile / Q000276:immunology; D003167:Complement Activation; D003171:Complement Pathway, Classical; D003165:Complement System Proteins / Q000737:chemistry* / Q000276:immunology; D005690:Galactose / Q000276:immunology; D006461:Hemolysis / Q000276:immunology*; D006801:Humans; D055550:Protein Stability
chemical_list: D000910:Antibodies, Heterophile; D015478:Antigens, Heterophile; D003165:Complement System Proteins; D005690:Galactose