Residual immune response towards decellularized homografts may be highly individual

Abstract

Objectives: Decellularized homograft valves (DHVs) have shown promising clinical results, particularly in the treatment of congenital heart disease. However, DHV appears to elicit an immune response in a subset of young patients, indicated by early valve degeneration. As the decellularization process is quality controlled for each DHV, we hypothesized that there may be residual immunogenicity within the extracellular matrix of DHV.

Methods: A semi-quantitative dot blot analysis was established to screen for preformed recipient antibodies using secondary anti-human antibodies. Fifteen DHV samples (7 aortic, 8 pulmonary) were solubilized and exposed to serum from 20 healthy controls.

Results: The sera from young controls (n = 10, 18-25 years) showed significantly stronger binding of preformed antibodies than sera from older individuals (n = 10, 48-73 years). The difference between the means of arbitrary units was 15.1 ± 6.5 (P = 0.0315). There was high intraindividual variance in the mean amounts of arbitrary units of antibody binding with some healthy controls showing >10 times higher antibody binding towards 2 different DHV. The amount of preformed antibodies bound to DHVs was higher in aortic than in pulmonary DHVs. The mean number of antibody binding (in arbitrary units) was 17.2 ± 4.5 in aortic and 14.5 ± 4.7 in pulmonary DHV (P = 0.27). The amount of preformed antibodies bound to pulmonary DHVs was statistically significantly higher in the sera of healthy males (n = 10) than in the sera of healthy females (n = 10). The mean number of arbitrary units was 17.2 ± 4.2 in male and 11.7 ± 5.3 in female sera (P = 0.036). Antibody binding to aortic DHV was also higher in males, but not significant (18.8 ± 5.0 vs 15.6 ± 4.0). Blood group (ABO) incompatibility between the serum from controls and DHV showed no impact on antibody binding, and there was no age-related impact among DHV donors.

Conclusions: Residual immunogenicity of decellularized homografts appears to exist despite almost complete cell removal. The established dot blot method allows a semi-quantitative assessment of the individual immune response towards extracellular DHV components and potentially the possibility of preoperative homograft matching.

Keywords: Decellularization; Heart valve replacement; Homograft.

Figure 1:

Overview—method to quantify the binding of preformed antibodies to decellularized homograft valve (A). Membrane loading: standards 1–6 and samples of pulmonary decellularized homograft valve (n = 8) (B). Regression of standards. Standards 1 and 2 are excluded because they were oversaturated at the chosen exposure time, which would artificially affect the regression line. A shorter exposure time would not be enough to display standards 5 and 6 (C). Amounts of arbitrary units of preformed antibodies bound to pulmonary decellularized homograft valve (n = 8) in a human serum. Negative values after baseline subtraction that arose in some cases can be explained by steric overlays between remaining donor antibodies and serum antibodies (D). DHV: decellularized homograft valve.

Figure 2:

Examples for the binding of preformed antibodies in the serum of a young female (A) and an older male (B) to pulmonary decellularized homograft valve (n = 8) as well as the binding of preformed antibodies in the sera of a young male (C) and an older female (D) to aortic decellularized homograft valve (n = 7). DHV: decellularized homograft valve.

Figure 3:

Mean amounts of arbitrary units of preformed antibodies bound to: (A) aortic and pulmonary decellularized homograft valve (n = 15) in the sera of male (n = 10) and female (n = 10) healthy subjects; (B) aortic and pulmonary decellularized homograft valve (n = 15) in the sera of young (n = 10) and old (n = 10) healthy subjects; (C) aortic (n = 7) and pulmonary (n = 8) decellularized homograft valve in the sera of 20 healthy subjects; (D) pulmonary decellularized homograft valve (n = 8) in the sera of male (n = 10) and female (n = 10) healthy subjects; and (E) aortic decellularized homograft valve (n = 7) in the sera of male (n = 10) and female (n = 10) healthy subjects. DHV: decellularized homograft valve.

Figure 4:

Graphical overview of preformed antibody binding according to age for healthy controls, showing less binding in older subjects (R₂ linear = 0.150).

Figure 5:

Preformed antibody binding differentiated by ABO compatibility between control serum and the decellularized homograft valve donor, showing no difference.

Figure 6:

Graphical overview of preformed antibody binding according to decellularized homograft valve donor age, showing no correlation. DHV: decellularized homograft valve.

Figure 7:

Graphical overview of inter-experimental variance and maximum differences in preformed antibody binding between specific decellularized homograft valve according to the age of healthy controls. DHV: decellularized homograft valve.