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.

Convincing clinical and experimental data strongly suggest that B cells and donor-specific antibodies (DSA) play a significant role in inducing or accelerating acute and chronic rejection of renal allografts (¹). In a previous report (²), we have shown that DSA could be detected in more than 70% of eluates from surgical samples of renal transplants removed for chronic allograft nephropathy (CAN). It would be of interest to know what happens in still functioning grafts. Thus, the aim of this study was to assess the feasibility of detecting such antibodies (Abs) in eluates from needle core biopsies of renal transplants. Additionally, we would like to compare two methods of anti-HLA antibody screening, the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FlowPRA).

The 20 renal transplants selected for this study were removed after irreversible graft failure due to CAN. For each transplant, fresh cortical surgical samples (MacroA) (mean weight: 224.5±56.7 mg), needle core biopsies (MicroA) (mean weight: 7.52±1.04 mg) sampled at the same place as surgical samples, and needle core biopsies sampled at another randomly-chosen cortical site (MicroB) (mean weight: 8.43±0.9 mg) were frozen in liquid nitrogen.

Abs were isolated as previously described (¹) using a commercial acid elution kit (ELUKITII, Gamma biochemicals Inc., Houston, TX). Briefly, each segment was washed and minced. The 1000 g sediment was eluted with the solution of elution at room temperature for 15 min. The supernatant was removed and neutralized with the buffer solution before testing. The final volume of elution from needle core biopsies was always less than 100 μL.

Each eluate was tested in duplicate using GTI ELISA kits that detect both anti-class I (Quickscreen, GTI) and anti-class II HLA (B Screen, GTI) Abs on an automate TEKTIME ELISA reader. We used plastic trays which were precoated with class I or class II HLA antigens. After incubation with the eluates and washing, peroxidase-conjugated anti-human IgG antibodies were added to each well. A chromogenic substrate was then added; the intensity of color development which is proportional to the amount of conjugate bound to the wells was measured by the microplate reader (Optical Density). For each plate negative and positive controls and a blank were used. A positive result was defined by a ratio OD (eluate)/OD (negative control)>2.

Anti-class I and anti-class II -HLA Abs were detected in eluates with Flow PRA beads (FL12–60, One Lambda Canoga Park, CA) (³). Five microliters of class I beads together with 5 μL of class II beads were incubated with 20 μL of eluates in an Eppendorf tube for 30 min. After two washes in wash buffer, each tube was incubated with 100 μL of FITC conjugated goat anti-human IgG. They were rinsed twice and 0.5 mL of fixed solution was then added to each tube. Flow cytometric analysis was carried out with a FACSCalibur (Becton Dickinson). In each assay, negative (FL-NC, One Lambda Canoga Park, CA) and positive (FL1-PC and FL2-PC, One Lambda Canoga Park, CA) controls were used. The threshold fluorescence intensity limit was arbitrarily set at the end of the peak of the negative control. Both methods detected only IgG Abs.

Statistical analyses were performed by calculating the correlation coefficient (Spearman test) and chi-square.

In surgical samples, anti-HLA Abs were detected in 19 of the 20 eluates with FlowPRA beads and in 15 of the 20 eluates with ELISA. The results of anti-class I and anti-class II HLA Abs screening are shown in Table 1. Using flow cytometry, anti-HLA Abs were detected in 16 (80% of micro A eluates) and 12 (60% of micro B eluates) needle core biopsies. The results concerning anti-class I and anti-class II HLA Abs screening are summarized in Table 2. The correlation of the results found with flow cytometry in eluates from surgical samples and needle core biopsies are shown in Table 3. In MacroA, DSA were identified in 12 cases. Identical donor specificity was assigned using flow cytometry or cross match in 8 microA (data not shown).

TABLE 1: Anti-class I and anti-class II HLA antibodies screening in surgical samples (macroA)

TABLE 2: Screening of anti-HLA antibodies with flow cytometry (FlowPRA) in eluates from samples of different size and location

TABLE 3: Results of the statistical analysis

In this study, we have demonstrated that flow cytometry (FlowPRA) is more sensitive than ELISA (GTI) at detecting anti-HLA Abs bound to renal transplants with CAN. These results are in agreement with previous reports dealing with serum samples (^{1, 4, 5}). ELISA was initially chosen because screening for both anti-class I HLA and anti-class II HLA Abs could be run automatically in duplicate at the same time and in the same test. Nevertheless, at least 100 μL of eluate was required for each sample. Such a volume was not available for all eluates and as a result we could not test all our biopsies with ELISA. As flow cytometry was more sensitive than ELISA, the feasibility of detecting anti-HLA Abs in needle core biopsies was then conducted with flow cytometry assay. Our study shows that detection of anti-HLA Abs in needle core biopsies can be achieved with the sensitive methods of Flow PRA beads. Even if the detection rate of anti-HLA Abs was slightly higher in surgical samples, the agreement between biopsies sampled at the same place was good for anti-class I and anti-class II HLA Abs. Discrepancies noticed for anti-class I HLA Abs in needle core biopsies sampled at different places may be due to a higher sensitivity of class I anti-HLA antibodies to the acid condition of elution (⁶) or a heterogeneous distribution of these antibodies throughout the transplant. Biopsies might have involved fibrotic or severely damaged area with few cells expressing class I HLA antigens.

These results are encouraging. They indicate that anti-HLA Abs could be detected in needle core biopsies from renal transplant undertaken in the follow-up of renal transplant recipients. It might represent a complementary method for detecting anti-HLA Abs in addition of their detection in the serum. Indeed, several publications have recently underlined the role of humoral immunity in the rejection process after renal transplantation (¹). Anti-HLA Abs has been associated with acute humoral rejection (⁷) and a majority of CAN (⁸). Anti-HLA antibodies bound to renal transplants give additional information along side results obtained by detecting antibodies solely in the serum. Antibodies fixed on their target are probably harmful for the transplant and could participate to the process leading to impaired function of the graft. The detection of anti-HLA Abs bound to the transplant would be all the more relevant as diffuse deposits of C4d along peritubular capillaries could be identified (⁸). As drugs inhibiting the B-cell response are now available (^{9, 10}), it is important to detect these antibodies early after transplantation to use such therapies appropriately to prolong graft survival.