Noninvasive and quantitative measurement of C4d deposition for the diagnosis of antibody-mediated cardiac allograft rejection

BACKGROUND: C4d is a specific biomarker for the diagnosis of antibody-mediated rejection (AMR) after cardiac transplantation. Although strongly recommended, routine C4d surveillance is hindered by the invasive nature of endomyocardial biopsy. Targeted ultrasound (US) has high sensitivity, and C4d is abundantly expressed within the graft of patients experiencing AMR, which makes it possible to visualize C4d deposition in vivo using targeted US. METHODS: We designed a serial dilution of C4d-targeted microbubbles (MB(C4d)) using a streptavidin-biotin conjugation system. A rat model of AMR with C4d deposition was established by pre-sensitization with skin transplantation before cardiac transplantation. MB(C4d) were injected into recipients and then qualitatively and quantitatively analyzed using the destruction-replenishment method with a clinical US imaging system and analyzed by software. FINDINGS: We successfully obtained qualitative images of C4d deposition in a wide cardiac allograft section, which, for the first time, reflected real-time C4d distribution. Moreover, normal intensity difference was used for quantitative analysis and exhibited an almost nearly linear correlation with the grade of C4d deposition according to the pathologic evidence. In addition, MB(C4d) injection did not affect the survival and aggravate injury, which demonstrates its safety. INTERPRETATION: This study demonstrates a noninvasive, quantitative and safe evaluation method for C4d. As contrast-enhanced US has been widely used in clinical settings, this technology is expected to be applied quickly to clinical practice. FUND: National Natural Science Foundation of China and Guangdong Province, Leading Scientific Talents of Guangdong special support program, the Science and Technology Project of Guangdong Province and Guangzhou City.