Transplant Immunology 11 (2003) 175–178
Analysis of AHG-PRA and ELISA-PRA in kidney transplant patients with acute rejection episodes Arnauld Kaufmana, Luciane Faria de Souza Pontesb,*, Maria Teresa Queiroz Marquesb, ´ Cristovao ´ ˜ de Moraes Sobrino Portob, Edison Regio ´ Jose´ Cavaliere Sampaioa, Luıs de Moraes Souzaa Nephrology Service, State University of Rio de Janeiro (UERJ), Av. Professor Manoel de Abreu, 444, 3o andar, DHE, 20550-170, Vila Isabel, Rio de Janeiro, Brazil b ´ ˜ Rio de Janeiro, HLA Laboratory, State University of Rio de Janeiro (UERJ), Av. Marechal Rondon, 381, Terreo, 20950-000, Maracana, Brazil
Received 1 November 2002; accepted 24 January 2003
Abstract Many centers determined a significant correlation between post-transplant anti-HLA antibodies production and clinical outcome. In order to confirm this correlation and ascertain the sequential appearance of anti-HLA antibodies, we compared the ELISA (ELISA-PRA) and the anti-human globulin enhanced complement-dependent lymphocytotoxicity panel-reactive antibodies test (AHG-PRA) with the occurrence of acute rejection episodes. Thirty patients who underwent kidney transplantation between December 1998 and October 1999 were assayed. One pre-transplant and 10 post-transplant serum samples were tested from each recipient except from one of them who lost his graft on the 1 week post-transplant. The diagnosis of acute rejection episode was based on classical criteria (fever, graft swelling and tenderness, oliguria, weight gain) and a rapid rise in serum creatinine levels, confirmed by an allograft biopsy graded by the Banff working classification. The 322 pre- and post-transplant serum specimens were tested by AHG-PRA methodology and 298 of them by the ELISA-PRA. The agreement coefficient (k) for both methodologies was 0.63. There were 27 acute rejection episodes in 19 patients. AHG-PRA results were significantly correlated (Hazard ratios10.06; Ps0.006) with this occurrence. These data were not confirmed with the ELISA-PRA娃 procedure. Our results suggest that a routine post-transplant AHG-PRA test offers an early risk assessment of acute rejection episodes and may be a useful method for monitoring the kidney transplant evolution. 䊚 2003 Elsevier Science B.V. All rights reserved. Keywords: Acute rejection; AHG; Anti-HLA antibodies; ELISA; HLA; Kidney transplantation
1. Introduction The development of acute rejection episodes is the main predictor for chronic rejection and long-term graft outcome. The precocious diagnosis and institution of anti-rejection treatment are two important hallmarks to improve graft survival w1x. The monitoring of anti-HLA class I antibodies after transplantation is still under discussion. A strong correlation between the increase of the anti-HLA class I antibodies level and an acute rejection episode was described w2x. This alloimmunization status can be effectively determined only by the humoral anti-HLA *Corresponding author. Tel.: q55-21-2587-8164; fax: q55-21587-6511. E-mail address: [email protected]
(L.F. de Souza Pontes).
panel-reactive antibody (PRA) test. Patients with pretransplantation high levels of PRA have an increased incidence of graft failure w3x. The anti-HLA antibodies production following the transplantation has been shown to be involved with the incidence and severity of acute rejection episodes and with decreased graft outcome w4x. Many authors used the lymphocyte cytotoxicity test to determine the antiHLA PRA post-transplantation in comparison with the ELISA-PRA. They concluded that the ELISA-PRA is best correlated with the occurrence of post-transplant rejections w2,5,6x. 2. Objective In the present study, we compared an ELISA methodology (ELISA-PRA) and the anti-human globulin
0966-3274/03/$ - see front matter 䊚 2003 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 6 - 3 2 7 4 Ž 0 3 . 0 0 0 0 3 - 0
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enhanced PRA (AHG-PRA) with the occurrence of acute rejection episodes, as methods for post-transplant monitoring in renal transplantation.
Table 1 Correlation between PRA and rejection episodes
3. Material and methods
The study population consisted of 30 individuals who underwent kidney transplantation at the Nephrology Service, State University of Rio de Janeiro between December 1998 and October 1999. There were 12 cadaveric and 18 living kidney recipients. Seventeen recipients were men (57%) and 13 women (43%) from various ethnic groups (15 caucasians, 10 mestizos and 5 blacks). The mean patient age was 32 years (9–58). Twenty-five patients received blood transfusions and 6 women got pregnant before transplantation. All patients had received the transplant after a negative lymphocytotoxicity cross-match according to the ASHI standards w7,8x. Among the living donors 5 were identical siblings, 9 haplo-identical and 4 unrelated with poor match. All recipients received azathioprine and prednisone after transplantation and for non-identical recipients cyclosporine was included. Only 4 recipients of cadaveric kidneys received anti-CD3 monoclonal antibody (OKT3 Orthoclone Jansen-Cilag Inc.) as induction therapy. Clinical and laboratorial analysis were monitored on a 3-month period. We tested 1 pre-transplant and 10 post-transplant serum samples (from the first until the eighth week, the tenth and the twelfth weeks) from each recipient except one that lost his graft after 6 days of transplant. The diagnosis of acute rejection episodes was based on the classical criteria (fever, graft swelling and tenderness, oliguria, weight gain) and on a rapid rise in serum creatinine levels with normal or subtherapeutic levels of cyclosporine and normal graft appearance by ultrasonography. A confirmatory biopsy was performed, graded using the Banff working classification w9x. Rejections were treated with 3-g intravenous methylprednisolone boluses. Steroid-resistant cases were treated with OKT3. We tested 322 serum specimens for the detection of lymphocytotoxic antibodies using the complementdependent lymphocytotoxicity test (CDC) enhanced with AHG w8,10,11x. The PRA test was performed with 46 HLA-phenotyped donors. This panel was representative of the HLA class I antigen distribution exhibited in Brazilian population from Rio de Janeiro. The test was positive when the serum sample reacted with more than 10% of the panel with reaction graded as 6q or more, according to international standards w8x. We tested 298 serum samples using the LAT-M娃 kit (One Lambda Inc., Canoga Park, CA) w12x. The LATM娃 consists of 96 well microtiter plates covered with a purified and solubilized HLA antigens that detect IgG
Cox proportional hazard regression model (Ps0.4423)
anti-HLA class I and II antibodies. The antigens are obtained from Epstein-Barr virus transformed human cell cultures. After the plates were incubated with the patient sera, bound antibodies were detected using an alkaline phosphatase-conjugated anti-human IgG antibody. Bound antibodies were revealed with a colorimetric substrate and the absorbance was read at 630 nm using an ELISA plate reader. Identification of positive reactions was accomplished by comparison to a positive reference. Assay internal cut-off values were determined using a media between the anti-HLA positive control serum and the blanks. The test was considered positive when the serum sample had a strength reaction 4q or more. Correlation of AHG-PRA and ELISA-PRA test results with post-transplant rejection episodes were analyzed using the Cox proportional hazard regression model test and kappa agreement coefficient analysis. 4. Results The agreement coefficient (k) for the 322 serum specimens tested by AHG-PRA methodology and the 298 by ELISA-PRA was 0.63. We also analyzed the correlation of post-transplant AHG-PRA and ELISA-PRA values and the occurrence of rejection episodes. There were 27 acute rejection episodes in 19 patients, with only one graft loss. The co-variable AHG-PRA had a positive association with the rejection episodes, with a Hazard ratios10.06 and Ps0.006. These results suggest that crescent levels of anti-HLA antibodies, detected by the AHG-PRA methodology, were associated with rejection episodes. These results are presented in Table 1. 5. Discussion Besides the pre-formed anti-HLA antibodies, which are a well-characterized risk factor w13x, it has also been demonstrated that a post-transplant increase of anti-HLA antibodies correlates with acute rejections and reduced graft survival w2x, leading to a new concept on transplant monitoring, attempted by many transplant centers using different methodologies w14x. In this study, the AHG-PRA and the ELISA-PRA methodologies were compared with the occurrence of an acute rejection episode. We could determine a signif-
A. Kaufman et al. / Transplant Immunology 11 (2003) 175–178
icant correlation between the AHG-PRA and an acute rejection episode, which can indicate that the PRA posttransplant monitoring could be a complementary method for an acute rejection episode diagnosis. Different results were reported by other centers, where a correlation between the complement dependent cytotoxicity results and the clinical outcome was not found w15x. Kao et al. introduced the ELISA methodology using soluble HLA molecules in 1993 as an alternative method for testing the PRA w16x. The majority of the studies comparing the anti-HLA ELISA with lymphocytotoxicity PRA or AHG-PRA determined that the ELISA methodology is more specific and sensitive for determination of anti-HLA antibodies in the serum of transplant recipients. This is due to a reaction of the anti-HLA antibody against purified HLA molecules absorbed on the ELISA plate w13,17x. The correlation of graft loss and ELISA results of post-transplants specimens was reported by many authors w2,18x. Christiaans et al. concluded, in a recent report, that ELISA and CDC are complementary screening techniques w19x. These results could not be confirmed in our study, maybe because of the short period of follow-up and only one graft loss observed. Studying serum samples from transplanted patients at UERJ—Rio de Janeiro, we found no significant correlation between the ELISA-PRA test and an acute rejection episode. We found an agreement coefficient of 0.63 for AHGPRA and ELISA-PRA in our study, which may indicate a difference in sensitivity for the two assays that could be explained by the presence of autoantibodies or nonHLA specific antibodies detected by the AHG-PRA procedure. Technical conditions such as incubation times, washing steps and detection of bound antibodies may cause differences in results due to antibody affinities. Moreover, different laboratories reported PRA positivity considering reactivities with different panel percents, such as 10–50% or more w1,20x. Although AHG-PRA methodology can be burdened by the requirement for viable target cells, a high variability of the complement, the inability to discriminate HLA from non-HLA antibodies and more time consuming compared to ELISA-PRA, a good predictive value for a rejection episode occurring on the next week was observed. Despite all this observations, AHG enhanced lymphocytotoxicity is a very useful methodology in an HLA laboratory, mainly considering the cross-match, and recommended by the ASHI standards w7x. Our results suggest that routine post-transplant monitoring of the AHG-PRA offers an early risk assessment of acute rejection episodes. In conclusion, the AHG-PRA should be an useful method for monitoring of the kidney transplant follow-up.
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