MO073: Ex Vivo C5B9 Deposition Test as a Diagnostic and Monitoring Tool For Primary and Transplantation Associated AHUS

Abstract BACKGROUND AND AIMS The atypical hemolytic uremic syndrome (aHUS) is a complement-mediated (CM) ultrarare disease that manifests as thrombotic microangiopathy (TMA) with preferential small kidney vessels involvement. aHUS is caused by genetic or acquired deregulation of complement (C’) system alternative pathway at the cell membrane surface which produces C’ overactivation and, eventually, membrane attack complex (MAC or C5b9) deposition, pore formation and, hence, vascular cell lysis. Currently, there is not a gold standard diagnostic test for aHUS, neither to monitor patient response to eculizumab therapy. It has been suggested that direct measurement of C5b9 deposits on cultured endothelial cells could be a plausible approach to evaluate the complement functional state in aHUS patients. Here we assess the C5b9 ex vivo deposition test applicability as a tool to diagnose aHUS and the monitoring of the Eculizumab therapy response in patients with aHUS in native, transplanted kidney and hematopoietic stem cell (HSC) transplantation (Tx) associated aHUS. METHOD A total of 14 patients with aHUS in native kidney (n = 10), aHUS recurrence after kidney Tx (n = 3) or HSC transplantation associated aHUS (n = 1), were studied during the acute phase of the disease prior to treatment, or in remission while receiving eculizumab. Chronic C3 glomerulopathy patients (n = 2) and healthy individuals were included as controls (n = 20). Serum samples from every patient to measure the C5b9 deposition on human vascular endothelial cells (HMEC-1; ATCC CRL-3234) were collected. HMEC-1 cells were platted on cover glasses in 24-well plates. Once cells reached confluence, they were treated with 10 µM adenosine diphosphate (ADP) for 10 min to activate the endothelial cells (activated conditions) or with test medium (resting conditions). Afterward, they were incubated with patient or control serum samples diluted 1:2 for 4 h. Cells were fixed with 4% paraformaldehyde solution, and C5b9 was immunodetected (1:500 rabbit polyclonal anti-complement C5b9 (Calbiochem, ref. 204 903)). Images were taken using a FV100 Fluoroview Olympus Confocal Microscope at 40×. Fifteen fields were analyzed to calculate the average staining area of each sample in pixels2 (pxl2) using the ImageJ. RESULTS C5b9 deposition on HMEC-cells using serum samples from healthy controls (n = 20) was quantified in order to establish the normal reference test range. C5b9 deposits staining area mean and standard deviation (m ± SD) on resting cells were 2018.25pxl2 ± 116.54 in healthy controls. Under ADP-activated conditions, C5b9 deposits m ± SD was 4210.51pxl2 ± 158.06 (gray boxes in Fig. 1). As expected, in the acute disease phase aHUS patients had a significant increase of C5b9 deposition (P = 0.01) as compared with controls in both resting and activated conditions (G1 in Fig. 1). The same finding was observed in a patient with aHUS associated with HSC-transplantation and in another aHUS patient who did not respond to Eculizumab (G2 and G3 in Fig. 1, respectively). All patients with aHUS in native kidneys (G4 in Fig. 1) or recurrence of the disease after kidney Tx (G5 in Fig. 1) who responded to eculizumab showed levels of C5b9 deposition comparable to the normal range except for patient 9. C3 glomerulopathy patients showed C5b9 deposition levels comparable to those found in healthy controls (G6 in Fig. 1). CONCLUSION The ex vivo C5b9 deposition test is an auspicious tool to diagnose and monitor aHUS response to eculizumab. We now demonstrated that the ex vivo C5b9 deposition test is also useful for HSC associated aHUS and for aHUS recurrence after kidney transplantation suggesting that it could be a useful diagnostic tool for any type of CM aHUS. These results expand the spectrum of patients that would benefit from a better diagnostic and treatment monitoring procedure.