Detection of Anti-HLA Antibodies with Flow Cytometry in Needle Core Biopsies of Renal Transplants Recipients with Chronic Allograft Nephropathy

Abstract

The aim of this study was to assess the feasibility of detecting anti-HLA antibodies in eluates from needle core biopsies of renal transplants with chronic allograft nephropathy. Two methods of screening, the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FlowPRA) were compared. Twenty renal transplants with CAN were removed after irreversible graft failure. To assess the feasibility of detecting anti-HLA antibodies in small samples, needle core biopsies were sampled at the same place as surgical samples and at a second cortical area. Antibodies were eluted with an acid elution kit and anti-class I and class II IgG HLA antibodies detected using ELISA and flow cytometry. Flow cytometry was found to be more sensitive than ELISA for detecting anti-HLA antibodies in eluates from renal transplants with CAN (95% vs. 75% of positive cases). Detection of anti-HLA antibodies showed good agreement between surgical samples and needle core biopsies performed at the same place for anti-class I (80% vs. 65%, r=0.724 P<0.01) and anti-class II HLA antibodies (70% vs. 55%, r=0.827 P<0.01). In addition, differences in the detection of anti-class I HLA antibodies in needle core biopsies sampled at different sites suggests that immunization to class I donor antigen could be underestimated in needle core biopsy samples. These data indicate that anti-HLA antibodies can be detected in needle core biopsies from renal transplants. Provided further evaluation is done, elution might be a complementary method to detect anti-HLA antibodies when they are bound to the transplant.

Antibody-Mediated Renal Allograft Rejection

“…Antibodies to donor ABO antigens show a similar association. C4d deposition without detectable circulating antibody can be due to absorption by the graft, as demonstrated by elution of anti-HLA antibodies from rejected grafts in patients who had no detectable circulating antibody at the time, even from needle biopsies (20). Non-HLA, non-ABO antigens are the target in a minority of cases, probably accounting for the rare C4d ϩ acute rejection in HLA-identical grafts (Ͻ2% of patients) (21).…”

Mechanisms and Role of HLA and non-HLA Alloantibodies

“…Anti-HLA antibody also has been eluted from needle biopsies of functional grafts with chronic allograft nephropathy (89), and a significant correlation between their presence and the presence of C4d staining and plasma cell infiltrate was found (90). Furthermore, anti-HLA antibodies were not found in the biopsy eluates of well-tolerated transplants, strongly supporting a pathogenic role of donorspecific anti-HLA antibody in chronic rejection and chronic allograft nephropathy.…”

Comparison of Different Methods of Anti-Hla Antibodies Detection in Diagnosis of Antibody-Mediated Renal Allograft Rejection

“…2. Отсутствие АТ к HLA при имеющейся экспрес-сии C4d на ПТК (АТ-/С4d+), что может объ-ясняться абсорбцией имеющихся антител на трансплантате [31], а также действием не-HLA-антител, таких как антитела к рецептору 1-го типа ангиотензина II (AT1R), в развитии острого повреждения трансплантата [32]. Помимо этого, отсутствие циркулирующих антител возмож-но вследствие недостаточной чувствительнос-ти методов, используемых для определения АТ к HLA.…”