INVESTIGATION OF HEMOLYSIS ASSOCIATED WITH LONG-TERM PERFUSION OF hDAF AND NORMAL PORCINE LIVERS WITH HUMAN BLOOD

Abstract

479 An extra-corporeal perfusion model has been established in order to test the viability of hDAF transgenic pig livers when perfused with fresh, whole, human blood. Three groups have been studied: alloperfusions (pig blood), xenoperfusions of unmodified pig livers and xenoperfusions of hDAF transgenic pig livers. Liver function was measured using pH, base excess, potassium, bile production, oxygen consumption, ALT, bilirubin, urea synthesis, factor V synthesis and complement production. Hemolysis was assessed by serial hematocrit and free hemoglobin assessment. Histological appearances were assessed using light microscopy and immunohistochemistry staining for IgM, IgG, and complement deposition. In the alloperfusion control group function and structural integrity was demonstrated for at least 72 hours. Xenoperfusions were carried out for periods of up to 72 hours and, unlike alloperfusion, were limited by progressive hemolysis; histological appearances were of patchy necrosis with most lobules remaining normal. The cause of hemolysis has been investigated; porcine antibodies were demonstrated in the human blood perfusing both normal and transgenic pig livers. Peak antibody levels were reached within one minute of commencing perfusion and did not significantly change throughout the duration of perfusion. This implies elution of porcine antibodies rather than production of new antibodies by the porcine livers. We have further demonstrated by FACS analysis that a proportion of these antibodies are pig anti-human specific. In addition, porcine livers perfused with human blood have been shown to produce supraphysiologic levels of complement activity as assessed by CH50 analysis on sheep red blood cells (RBC). Interestingly, however, when serum from these xenoperfusions was incubated with fresh human RBC, it was not possible to demonstrate lysis. Thus, while there appear to be both pig anti-human antibodies and complement present, classical pathway complement hemolysis could not be demonstrated. Investigations of the hemolysis continues, in order to assess whether this would be a limitation in clinical studies. This research was funded by the Wellcome Trust and carried out in collaboration with Imutran (Novartis).