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Can Anti-HLA Antibody Analyses Postcardiac Transplantation Predict Acute Allograft Rejection and Survival?
Can Anti-HLA Antibody Analyses Postcardiac Transplantation Predict Acute Allograft Rejection and Survival? G. Bhat, T.J. Schroeder, and K. Balakrishnan
I
DENTIFICATION of risk factors for acute and chronic graft rejection and allograft survival would provide guidance for immunosuppressive therapy and help in the posttransplant management of high risk patients. The panel reactive antibody (PRA) screen has been the standard to predict sensitization. Pretransplant sensitization may occur from blood transfusions, pregnancy, previous transplants, or ventricular assist devices. Cardiac transplant recipients with high PRA (usually .10%) have traditionally undergone donor specific lymphocytotoxic cross match to avoid hyperacute rejection. HLA antibody screening by PRA has been clinically utilized to predict cross match outcome and posttransplant complications, especially in renal transplant patients. Although a few studies in cardiac transplantation have correlated elevated pre- and posttransplant PRA to poor clinical outcome, other reports have been inconclusive. Pre- and posttransplant PRA is usually determined by complement-dependent cytotoxicity (CDC) or flow cytometry assays. Recently, a novel ELISA procedure for the detection of anti-HLA class I antibodies has been developed (PRA-STAT) that appears in preliminary studies to be easier to perform and more specific in detecting HLA class I IgG antibodies with higher reproducibility.1 The purpose of this study was to determine anti-HLA class I IgG antibodies by ELISA-based assay (PRA-STAT) postcardiac transplant and correlate with clinical end-points such as acute rejection and graft loss. MATERIALS AND METHODS Patients This study included 107 patients undergoing orthotopic cardiac transplantation between July 1990 and October 1995. The mean age was 52 years (range 16 to 67). Patients were predominantly male (75%) and white (89%). Pretransplant diagnosis included ischemic cardiomyopathy (49%), idiopathic dilated cardiomyopathy (49%), and other (2%). The overall graft survival was 80.4% with a mean follow-up of 45 months (range 16 to 79). All 107 patients received an OKT3-based quadruple sequential immunosuppressive protocol followed by triple therapy. Seventy-eight patients received 14 days of OKT3, and 29 patients received 10 days of OKT3. Azathioprine (4 mg/kg IV) was administered preoperatively. Methylprednisolone (500 mg IV) and OKT3 (10 mg IV) were administered intraoperatively. OKT3 (5 mg/day) was continued for 14 or 10 days in conjunction with azathioprine (1.5 mg/kg
per day), prednisone (tapered from 100 mg to 0.2 mg/kg per day), and cyclosporine (250 to 350 ng/mL tapered to 150 to 200 ng/mL), which was begun on posttransplant day 3 to 5. The endomyocardial biopsies were performed weekly for the first 2 months, then every 2 weeks for the next 2 months, and every 3 weeks for the following 2 months and then less frequently thereafter. The endomyocardial biopsies were graded by the International Society for Heart and Lung Transplantation criteria. Acute rejection episodes were defined as 3A grade or higher requiring therapy. 3A grade rejection episodes were treated with corticosteroids. Steroid-resistant rejection or 3B grade or worse rejection was treated with repeat OKT3 (5 mg/day) for 10 days. The clinical outcomes were analyzed as episodes of acute rejection within 31, 60, and 90 days, time (days) to first episode of acute rejection, graft loss within 60 days, and overall graft loss. A total of 224 serum specimens were retrospectively analyzed, 105 specimens at day 0 (transplant), 93 specimens at day 10, 55 at day 20, and 21 at day 30.
ELISA Method ELISA %PRA was determined using the PRA-STAT kit (SangStat Medical Corp., Menlo Park, Calif).1 Ninety-six–well microtiter plates were coated with monoclonal antibody TP25. This antibody reacts with the alpha 3 domain of all known HLA-A, B, C antigens. Subsequently, sHLA antigens from the culture supernatants of 46 different Epstein-Barr virus-transformed human B cells were captured on the antibody-coated plates. After the sHLA-coated plates were incubated with patient sera, bound antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Bound antibody was detected using O-phenylenediamine dihydrochloride, and absorbance was read at OD495-OD600 using an ELISA plate reader. Corrections for nonspecific binding were made for each specimen. Identification of positive reactions were accomplished by comparison to a positive reference. Assay internal cut-off values were determined using sera from nonsensitized individuals. These cut-off values were then used to identify positive reactions in each individual well.
Statistical Analysis The SOFT-STAT Program (SangStat Medical) was used for data analysis. Correlation of test results with rejection episodes and/or graft loss was analyzed using chi-square analyses. From the Departments of Internal Medicine and Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio. Address reprint requests to G. Bhat, PhD, MD, Division of Cardiology, ACB, Third Floor, 550 South Jackson Street, University of Louisville, Louisville, KY 40292.
0041-1345/98/$19.00 PII S0041-1345(98)00189-4
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1154
Transplantation Proceedings, 30, 1154–1157 (1998)
ANTI-HLA ANTIBODY ROLE OF ALLOGRAFT REJECTION
Fig 1. Correlation of PRA-STAT result at day 0 and number of rejection episodes within 31 days posttransplant.
RESULTS Acute Rejection
Figure 1 shows the correlation of PRA-STAT results at day 0 and number of rejection episodes within 31 days posttransplant. The average number of acute rejection episodes in the first month after cardiac transplantation was increased in 15 patients with PRA-STAT $15% at day 0 (date of transplant) compared to 85 patients with PRASTAT ,15% (0.80 6 0.19 versus 0.45 6 0.08, P 5 .06). Figure 2 shows comparison of PRA-STAT results at day 10 posttransplant and rejection occurrence within 31 days posttransplant. Forty-one patients experiencing acute rejection within 31 days had a significantly higher PRA-STAT compared to 48 patients with no rejection (10.2 6 2.4% versus 3.3 6 2.2%, P 5 .04). PRA-STAT results at day 20 and day 30 were also compared and noted to be increased in patients who experienced acute rejection within 31 days
Fig 2. Correlation of PRA-STAT result at day 10 and rejection occurrence within 31 days posttransplant.
1155
Fig 3. Correlation of PRA-STAT results at day 10 and days to first rejection.
versus those who had no rejection, although this did not achieve statistical significance. The average number of acute rejection episodes in the first month was significantly increased in patients where PRA-STAT was $7% compared with ,7% in day 30 samples (1.25 6 0.30 versus 0.41 6 0.15, P 5 .02). Figure 3 shows that patients with elevated PRA-STAT at day 10 after transplant have earlier occurrence of first acute rejection episode. Patients with PRA-STAT values at day 10 posttransplant $15% versus ,15% had first acute rejection episodes at 22.8 6 12.4 days compared to 48.9 6 4.5 days (P 5 .05). Increasing PRA-STAT during the first month was associated with a significant increase in acute rejection episodes (Fig 4). Graft Loss
Day 10 PRA-STAT values ($10% versus ,10%) were also predictive for graft loss within the first 60 days posttrans-
Fig 4. Correlation of posttransplant changes in PRA-STAT and rejection within 31 days.
1156
Fig 5.
BHAT, SCHROEDER AND BALAKRISHNAN
Correlation of PRA-STAT results at day 10 and graft loss.
plant (16.6% versus 2.6%, P 5 .05) and over the entire follow-up period (39% versus 14.6%, P 5 .04) (Fig 5). Overall, day 10 PRA-STAT values were significantly higher in patients with early graft loss compared with no graft loss (21.4% 6 7.5 versus 6.3% 6 1.8, P 5 .05) (Fig 6). Similarly, overall graft loss (P 5 .003) and graft loss within 60 days (P 5 .021) was significantly increased in the higher PRASTAT group at day 20 posttransplant. DISCUSSION
In this study, anti-HLA antibodies postcardiac transplantation detected by the ELISA-based assay, PRA-STAT, correlated with early and late graft loss and incidence of acute allograft rejection episodes. PRA has been traditionally determined by the lymphocytotoxicity method (CDC-PRA) in a small number of
studies dealing with posttransplant monitoring of anti-HLA antibodies, and the results have been inconsistent. There was no correlation between CDC-PRA and the incidence and severity of rejection in 82 patients in whom lymphocytoxic antibodies were determined 6 months following cardiac transplantation, although donor-specific reactive antibodies strongly correlated with rejection.2 A study of 238 renal and 199 heart transplant recipients showed significant association between development of anti-HLA antibodies and that of chronic allograft rejection and survival.3 In a study involving the role of anti-HLA antibodies in 107 cardiac transplant patients, the long-term cardiac transplant survivors showed a significantly lower frequency of anti-HLA antibodies after transplantation.4 PRA . 25% was shown to be a risk factor for long-term survival in a group of cardiac transplant recipients, although increased PRA did not correlate with acute rejection.5 In a review of 463 heart transplants where the relationship of CDC-PRA level and the donor-specific lymphocytotoxic crossmatch was examined, CDC-PRA .10% was shown to be a risk factor for rejection-related events.6 A negative crossmatch in patients with an elevated CDC-PRA did not reduce the risk of death from acute or chronic rejection. This study concluded that the effect of elevated PRA on survival from rejection appeared to override the effect of a negative or positive crossmatch. Heart transplant recipients with CDC-PRA levels .10% compared to ,10% during the first 6 months after transplantation showed decreased long-term survival and increased graft atherosclerosis but no correlation to acute cellular rejection episodes.7 A previous study involving posttransplant monitoring by PRA-STAT has associated high PRA-STAT with graft survival and acute rejection in kidney transplant recipients.8 Although preliminary reports have described the pretransplant evaluation of PRA-STAT in small groups of cardiac transplant recipients,9 –11 our study is unique in correlating clinical outcome with PRA-STAT elevation after cardiac transplantation. The majority of cardiac transplant patients in previous studies have been treated with cyclosporine-based triple immunotherapy, with one study using CDC-PRA excluding patient’s sera obtained during treatment with OKT3 or ATG.2 PRA-STAT testing should overcome the difficulty in differentiating HLA-specific antibodies from other antibodies and, as shown in our study, could be used for monitoring during antilymphocyte induction therapy. CONCLUSION
Fig 6. Correlation of PRA-STAT results at day 10 and graft loss within 60 days posttransplant.
In this study, PRA-STAT results posttransplant correlated significantly with early and late graft loss. Patients at high risk for increased acute rejection within 1 month posttransplant as well as a shorter duration to first rejection episode were identified by PRA-STAT evaluation. Increasing PRASTAT after transplant correlated with increased acute rejection episodes compared to decreasing PRA-STAT value posttransplant.
ANTI-HLA ANTIBODY ROLE OF ALLOGRAFT REJECTION
The study of anti-HLA antibodies by PRA-STAT in sequential samples of sera after cardiac transplantation appears to be a useful prognostic test to identify high risk recipients, and further testing is warranted in larger number of patients. Currently, prospective studies are ongoing to noninvasively identify by PRA-STAT those patients at high risk for acute rejection and graft loss to guide immunosuppressive therapy and optimal outcome posttransplant. REFERENCES 1. Buelow R, Mercier I, Glanville L, et al: Hum Immunol 44:1, 1995 2. Smith JD, Danskine AJ, Rose ML, et al: Transplantation 53:1358, 1992 3. Barr ML, Cohen DJ, Benvenisty AI, et al: Transplant Proc 25:262, 1993
1157 4. Suciu-Foca N, Reed E, Marboe C, et al: Transplantation 51:716, 1991 5. Loh E, Bergin JD, Couper GS, et al: J Heart Lung Transplant 13:194, 1994 6. Lavee J, Kormos RL, Duquesnoy RJ, et al: J Heart Lung Transplant 10:921, 1991 7. Rose EA, Pepino P, Barr ML, et al: J Heart Lung Transplant 11:5120, 1992 8. Kerman RH, Susskind B, Buelow R, et al: Transplantation 62:201, 1996 9. Kerman R, Susskind B, Katz S, et al: Hum Immunol 49:97, 1996 10. Ting A, Vayntrub T, Mercier I, et al: Hum Immunol 49:93, 1996 11. Sundt TM, Ewald GA, Moorhead SL, et al: J Heart Lung Transplant 16:78, 1997
I
DENTIFICATION of risk factors for acute and chronic graft rejection and allograft survival would provide guidance for immunosuppressive therapy and help in the posttransplant management of high risk patients. The panel reactive antibody (PRA) screen has been the standard to predict sensitization. Pretransplant sensitization may occur from blood transfusions, pregnancy, previous transplants, or ventricular assist devices. Cardiac transplant recipients with high PRA (usually .10%) have traditionally undergone donor specific lymphocytotoxic cross match to avoid hyperacute rejection. HLA antibody screening by PRA has been clinically utilized to predict cross match outcome and posttransplant complications, especially in renal transplant patients. Although a few studies in cardiac transplantation have correlated elevated pre- and posttransplant PRA to poor clinical outcome, other reports have been inconclusive. Pre- and posttransplant PRA is usually determined by complement-dependent cytotoxicity (CDC) or flow cytometry assays. Recently, a novel ELISA procedure for the detection of anti-HLA class I antibodies has been developed (PRA-STAT) that appears in preliminary studies to be easier to perform and more specific in detecting HLA class I IgG antibodies with higher reproducibility.1 The purpose of this study was to determine anti-HLA class I IgG antibodies by ELISA-based assay (PRA-STAT) postcardiac transplant and correlate with clinical end-points such as acute rejection and graft loss. MATERIALS AND METHODS Patients This study included 107 patients undergoing orthotopic cardiac transplantation between July 1990 and October 1995. The mean age was 52 years (range 16 to 67). Patients were predominantly male (75%) and white (89%). Pretransplant diagnosis included ischemic cardiomyopathy (49%), idiopathic dilated cardiomyopathy (49%), and other (2%). The overall graft survival was 80.4% with a mean follow-up of 45 months (range 16 to 79). All 107 patients received an OKT3-based quadruple sequential immunosuppressive protocol followed by triple therapy. Seventy-eight patients received 14 days of OKT3, and 29 patients received 10 days of OKT3. Azathioprine (4 mg/kg IV) was administered preoperatively. Methylprednisolone (500 mg IV) and OKT3 (10 mg IV) were administered intraoperatively. OKT3 (5 mg/day) was continued for 14 or 10 days in conjunction with azathioprine (1.5 mg/kg
per day), prednisone (tapered from 100 mg to 0.2 mg/kg per day), and cyclosporine (250 to 350 ng/mL tapered to 150 to 200 ng/mL), which was begun on posttransplant day 3 to 5. The endomyocardial biopsies were performed weekly for the first 2 months, then every 2 weeks for the next 2 months, and every 3 weeks for the following 2 months and then less frequently thereafter. The endomyocardial biopsies were graded by the International Society for Heart and Lung Transplantation criteria. Acute rejection episodes were defined as 3A grade or higher requiring therapy. 3A grade rejection episodes were treated with corticosteroids. Steroid-resistant rejection or 3B grade or worse rejection was treated with repeat OKT3 (5 mg/day) for 10 days. The clinical outcomes were analyzed as episodes of acute rejection within 31, 60, and 90 days, time (days) to first episode of acute rejection, graft loss within 60 days, and overall graft loss. A total of 224 serum specimens were retrospectively analyzed, 105 specimens at day 0 (transplant), 93 specimens at day 10, 55 at day 20, and 21 at day 30.
ELISA Method ELISA %PRA was determined using the PRA-STAT kit (SangStat Medical Corp., Menlo Park, Calif).1 Ninety-six–well microtiter plates were coated with monoclonal antibody TP25. This antibody reacts with the alpha 3 domain of all known HLA-A, B, C antigens. Subsequently, sHLA antigens from the culture supernatants of 46 different Epstein-Barr virus-transformed human B cells were captured on the antibody-coated plates. After the sHLA-coated plates were incubated with patient sera, bound antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Bound antibody was detected using O-phenylenediamine dihydrochloride, and absorbance was read at OD495-OD600 using an ELISA plate reader. Corrections for nonspecific binding were made for each specimen. Identification of positive reactions were accomplished by comparison to a positive reference. Assay internal cut-off values were determined using sera from nonsensitized individuals. These cut-off values were then used to identify positive reactions in each individual well.
Statistical Analysis The SOFT-STAT Program (SangStat Medical) was used for data analysis. Correlation of test results with rejection episodes and/or graft loss was analyzed using chi-square analyses. From the Departments of Internal Medicine and Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio. Address reprint requests to G. Bhat, PhD, MD, Division of Cardiology, ACB, Third Floor, 550 South Jackson Street, University of Louisville, Louisville, KY 40292.
0041-1345/98/$19.00 PII S0041-1345(98)00189-4
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1154
Transplantation Proceedings, 30, 1154–1157 (1998)
ANTI-HLA ANTIBODY ROLE OF ALLOGRAFT REJECTION
Fig 1. Correlation of PRA-STAT result at day 0 and number of rejection episodes within 31 days posttransplant.
RESULTS Acute Rejection
Figure 1 shows the correlation of PRA-STAT results at day 0 and number of rejection episodes within 31 days posttransplant. The average number of acute rejection episodes in the first month after cardiac transplantation was increased in 15 patients with PRA-STAT $15% at day 0 (date of transplant) compared to 85 patients with PRASTAT ,15% (0.80 6 0.19 versus 0.45 6 0.08, P 5 .06). Figure 2 shows comparison of PRA-STAT results at day 10 posttransplant and rejection occurrence within 31 days posttransplant. Forty-one patients experiencing acute rejection within 31 days had a significantly higher PRA-STAT compared to 48 patients with no rejection (10.2 6 2.4% versus 3.3 6 2.2%, P 5 .04). PRA-STAT results at day 20 and day 30 were also compared and noted to be increased in patients who experienced acute rejection within 31 days
Fig 2. Correlation of PRA-STAT result at day 10 and rejection occurrence within 31 days posttransplant.
1155
Fig 3. Correlation of PRA-STAT results at day 10 and days to first rejection.
versus those who had no rejection, although this did not achieve statistical significance. The average number of acute rejection episodes in the first month was significantly increased in patients where PRA-STAT was $7% compared with ,7% in day 30 samples (1.25 6 0.30 versus 0.41 6 0.15, P 5 .02). Figure 3 shows that patients with elevated PRA-STAT at day 10 after transplant have earlier occurrence of first acute rejection episode. Patients with PRA-STAT values at day 10 posttransplant $15% versus ,15% had first acute rejection episodes at 22.8 6 12.4 days compared to 48.9 6 4.5 days (P 5 .05). Increasing PRA-STAT during the first month was associated with a significant increase in acute rejection episodes (Fig 4). Graft Loss
Day 10 PRA-STAT values ($10% versus ,10%) were also predictive for graft loss within the first 60 days posttrans-
Fig 4. Correlation of posttransplant changes in PRA-STAT and rejection within 31 days.
1156
Fig 5.
BHAT, SCHROEDER AND BALAKRISHNAN
Correlation of PRA-STAT results at day 10 and graft loss.
plant (16.6% versus 2.6%, P 5 .05) and over the entire follow-up period (39% versus 14.6%, P 5 .04) (Fig 5). Overall, day 10 PRA-STAT values were significantly higher in patients with early graft loss compared with no graft loss (21.4% 6 7.5 versus 6.3% 6 1.8, P 5 .05) (Fig 6). Similarly, overall graft loss (P 5 .003) and graft loss within 60 days (P 5 .021) was significantly increased in the higher PRASTAT group at day 20 posttransplant. DISCUSSION
In this study, anti-HLA antibodies postcardiac transplantation detected by the ELISA-based assay, PRA-STAT, correlated with early and late graft loss and incidence of acute allograft rejection episodes. PRA has been traditionally determined by the lymphocytotoxicity method (CDC-PRA) in a small number of
studies dealing with posttransplant monitoring of anti-HLA antibodies, and the results have been inconsistent. There was no correlation between CDC-PRA and the incidence and severity of rejection in 82 patients in whom lymphocytoxic antibodies were determined 6 months following cardiac transplantation, although donor-specific reactive antibodies strongly correlated with rejection.2 A study of 238 renal and 199 heart transplant recipients showed significant association between development of anti-HLA antibodies and that of chronic allograft rejection and survival.3 In a study involving the role of anti-HLA antibodies in 107 cardiac transplant patients, the long-term cardiac transplant survivors showed a significantly lower frequency of anti-HLA antibodies after transplantation.4 PRA . 25% was shown to be a risk factor for long-term survival in a group of cardiac transplant recipients, although increased PRA did not correlate with acute rejection.5 In a review of 463 heart transplants where the relationship of CDC-PRA level and the donor-specific lymphocytotoxic crossmatch was examined, CDC-PRA .10% was shown to be a risk factor for rejection-related events.6 A negative crossmatch in patients with an elevated CDC-PRA did not reduce the risk of death from acute or chronic rejection. This study concluded that the effect of elevated PRA on survival from rejection appeared to override the effect of a negative or positive crossmatch. Heart transplant recipients with CDC-PRA levels .10% compared to ,10% during the first 6 months after transplantation showed decreased long-term survival and increased graft atherosclerosis but no correlation to acute cellular rejection episodes.7 A previous study involving posttransplant monitoring by PRA-STAT has associated high PRA-STAT with graft survival and acute rejection in kidney transplant recipients.8 Although preliminary reports have described the pretransplant evaluation of PRA-STAT in small groups of cardiac transplant recipients,9 –11 our study is unique in correlating clinical outcome with PRA-STAT elevation after cardiac transplantation. The majority of cardiac transplant patients in previous studies have been treated with cyclosporine-based triple immunotherapy, with one study using CDC-PRA excluding patient’s sera obtained during treatment with OKT3 or ATG.2 PRA-STAT testing should overcome the difficulty in differentiating HLA-specific antibodies from other antibodies and, as shown in our study, could be used for monitoring during antilymphocyte induction therapy. CONCLUSION
Fig 6. Correlation of PRA-STAT results at day 10 and graft loss within 60 days posttransplant.
In this study, PRA-STAT results posttransplant correlated significantly with early and late graft loss. Patients at high risk for increased acute rejection within 1 month posttransplant as well as a shorter duration to first rejection episode were identified by PRA-STAT evaluation. Increasing PRASTAT after transplant correlated with increased acute rejection episodes compared to decreasing PRA-STAT value posttransplant.
ANTI-HLA ANTIBODY ROLE OF ALLOGRAFT REJECTION
The study of anti-HLA antibodies by PRA-STAT in sequential samples of sera after cardiac transplantation appears to be a useful prognostic test to identify high risk recipients, and further testing is warranted in larger number of patients. Currently, prospective studies are ongoing to noninvasively identify by PRA-STAT those patients at high risk for acute rejection and graft loss to guide immunosuppressive therapy and optimal outcome posttransplant. REFERENCES 1. Buelow R, Mercier I, Glanville L, et al: Hum Immunol 44:1, 1995 2. Smith JD, Danskine AJ, Rose ML, et al: Transplantation 53:1358, 1992 3. Barr ML, Cohen DJ, Benvenisty AI, et al: Transplant Proc 25:262, 1993
1157 4. Suciu-Foca N, Reed E, Marboe C, et al: Transplantation 51:716, 1991 5. Loh E, Bergin JD, Couper GS, et al: J Heart Lung Transplant 13:194, 1994 6. Lavee J, Kormos RL, Duquesnoy RJ, et al: J Heart Lung Transplant 10:921, 1991 7. Rose EA, Pepino P, Barr ML, et al: J Heart Lung Transplant 11:5120, 1992 8. Kerman RH, Susskind B, Buelow R, et al: Transplantation 62:201, 1996 9. Kerman R, Susskind B, Katz S, et al: Hum Immunol 49:97, 1996 10. Ting A, Vayntrub T, Mercier I, et al: Hum Immunol 49:93, 1996 11. Sundt TM, Ewald GA, Moorhead SL, et al: J Heart Lung Transplant 16:78, 1997