Novel Mechanisms for Controlled Antigen Expression (KODE™ CAE) on Erythrocyte Membranes

Gilliver, Lissa
Henry, Stephen
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Doctor of Philosophy
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Auckland University of Technology

Background: A major function of blood banking laboratories around the world is the ABO typing of blood from donors and recipients for the purposes of transfusion. Incorrect determination of donor and to a lesser degree recipients ABO blood types can result in a fatal outcome. Figures indicate that about 1:10,000 units of blood transfused is of incompatible group and death has been reported to ensue in 10% of cases. Some 33% of the mistakes that lead to these transfusion reactions have been attributed to breakdowns in blood banking procedure. Astoundingly, there is no standardised positive control in this field. A cell that weakly expressed blood group A and/or B antigens would be ideal for this purpose, but no natural weak subgroup cell is suitable as a control. The natural phenomenon by which glycolipids are able to be taken up by the RBC membrane was exploited to add blood group active A or B antigens and synthetic analogues to group O RBCs, thereby making them blood group A or B respectively. Control over the amount of antigen expressed is crucial.

Methods: Several methods for the transformation of cells to express low amounts of A and/or B antigen by insertion were evaluated; including insertion of natural, modified-natural, and specifically engineered synthetic glycolipids into group O cells. Alternative methods using enzymes were also investigated - using glycosidases to strip antigen from cells and glycosyltransferases to synthesise A and/or B antigens on group O cells.

Results: All the methods trialled were able to produce cells that showed weak expression of antigen against ABO blood grouping antisera. However, the synthetic glycolipid was found to offer significant advantages over the other methods. These features include: aqueous solubility, reactivity with all relevant active antibodies due to the generic presentation of the trisaccharide antigenic determinant, excellent batch to batch reproducibility, purity and homogeneity, capacity for production of unlimited quantity, superior insertion (compared to natural glycolipids both in efficiency and in low temperature insertion), and unmatched stability of transformed cells.

Conclusion: Following a successful field trial and further validation trials, synthetic glycolipids were used to prepare an ABO analytical control in the product known as Securacell®, which was launched onto the Australian market by CSL Biosciences. This thesis details the successful development of the world’s first ABO blood grouping analytical sensitivity control system.

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