Iman M. Hamour

C4d Fixing, Luminex Binding Antibodies—A New Tool for Prediction of Graft Failure After Heart Transplantation

The standard method to detect pretransplant antibodies has been the complement dependent cytotoxicity (CDC) test of donor leukocytes. Solid phase assays to detect HLA antibodies in pretransplant serum reveal a greater number of sensitized patients, but their clinical impact is less certain. Here we have developed a method of detecting C4d fixing HLA antibodies on Luminex beads. Pretransplant serum from 565 cardiac transplant patients was retrospectively tested for the presence of HLA antibodies using CDC, HLA coated Luminex beads and C4d deposition on Luminex beads, and the results correlated with graft survival. Whereas 5/565 patients had CDC positive donor specific antibodies (DSA) before their transplant, this number was increased by 19 using Luminex beads. The 1‐year survival of CDC –ve/Luminex +ve patients with DSA (n = 19) was 42% compared with 77% for CDC –ve/Luminex +ve without DSA (n = 39, p = 0.0039). Fixation of C4d (22/67 Luminex positive sera) had a negative effect on graft outcome; 1‐year graft survival was, C4d +ve/DSA +ve (n = 11) 20%, C4d +ve/DSA –ve (n = 11) 91%, C4d –ve DSA +ve (n = 13) 54%, C4d –ve DSA –ve (n = 32) 75%, compared with 75% for antibody‐negative patients (p = 0.0002). In conclusion, detection of Luminex +ve DSA in pretransplant serum provides a powerful negative predictor of graft survival, especially if they bind C4d.

De Novo Donor HLA-Specific Antibodies after Heart Transplantation Are an Independent Predictor of Poor Patient Survival

Preformed donor HLA‐specific antibodies are a known indicator for poor patient survival after cardiac transplantation. The role of de novo donor‐specific antibodies (DSA) formed after cardiac transplantation is less clear. Here we have retrospectively analyzed 243 cardiac transplant recipients, measuring HLA antibody production every year after transplantation up to 13 years post‐transplant. Production of de novo DSA was analyzed in patients who had been negative for DSA prior to their transplant. DSA including transient antibodies were associated with poor patient survival (p = 0.0018, HR = 3.198). However, de novo and persistent DSA was strongly associated with poor patient survival (p = 0.0001 HR = 4.351). Although complement fixing persistent DSA correlated with poor patient survival, this was not increased compared to noncomplement fixing persistent DSA. Multivariable analysis indicated de novo persistent DSA to be an independent predictor of poor patient survival along with HLA‐DR mismatch and donor age. Only increasing donor age was found to be an independent risk factor for earlier development of CAV. In conclusion, patients who are transplanted in the absence of pre‐existing DSA make de novo DSA after transplantation which are associated with poor survival. Early and regular monitoring of post‐transplant DSA is required to identify patients at risk of allograft failure.

Chronic kidney disease after heart transplantation

BACKGROUND Chronic kidney disease (CKD) is a complication of heart transplantation related to calcineurin inhibitor nephrotoxicity. However, it is unclear whether early ciclosporin (CsA) exposure influences CKD in the long term. METHODS We analysed risk factors for CKD in 352 patients who underwent orthotopic heart transplantation (1995-2005). In 2000, we reduced our target CsA levels in the first year after transplantation. RESULTS Actuarial patient survival was 79% at 1 year and 62% at 10 years. Estimated median glomerular filtration rate (eGFR) by the four-variable Modification of Diet in Renal Disease formula was 64 ml/min/1.73 m2 before transplantation, inter-quartile range (IQR) 54-78. After transplantation, the eGFR was 48 (IQR 37-61) at Year 1, and 41(35-57) at Year 10. The cumulative probability of eGFR <45 ml/min/1.73 m2 was 45% at Year 1, 71% at Year 5 and 83% at Year 10. A multivariable logistic regression model was constructed for the development of eGFR <45 ml/min/1.73 m2 by 3 years. The risk factors were post-operative renal replacement therapy for acute renal failure (ARF), P < 0.001; pretransplant diabetes, P = 0.005; increasing recipient age, P < 0.001; female recipient, P = 0.029; female donor, P = 0.04, but not CsA regimen. The cumulative probability of developing stage 5 CKD (eGFR <15) was 3% at Year 5 and 12% at Year 10. Although lower ciclosporin initial levels were associated with less renal dysfunction at Year 1 (P = 0.008), there was no significant effect by Year 3 (P = 0.7). CONCLUSION The incidence of CKD increased with time and was not influenced by the CsA regimen. Some risk factors are not modifiable but measures to reduce the incidence of post-operative ARF may help to reduce CKD.

Limited utility of endomyocardial biopsy in the first year after heart transplantation.

Background. Surveillance endomyocardial biopsies (EMBs) are used for the early diagnosis of acute cardiac allograft rejection. Protocols became standardized in an earlier era and their utility with contemporary immunosuppression has not been investigated. Methods. We studied 258 patients after orthotopic heart transplantation comparing 135 patients immunosuppressed by mycophenolate mofetil (MMF) with 123 patients treated by azathioprine (AZA); both with cyclosporine and corticosteroids after induction therapy with rabbit antithymocyte globulin. Fifteen EMBs were scheduled in the first year. Additional EMBs were performed for suspected rejection, after treatment, or for inadequate samples. The MMF group had 1875 EMBs vs. 1854 in the AZA group. Results. The yield of International Society for Heart and Lung Transplantation (ISHLT) grade≥3A biopsy-proven acute rejection (BPAR) was 1.87% per biopsy (35 of 1875) with MMF vs. 3.13% (58 of 1854) with AZA P=0.024. The number of clinically silent BPAR ISHLT grade ≥3A (the true yield of surveillance EMBs) was 1.39% (26 of 1875) of biopsies MMF vs. 2.1% (39 of 1854) AZA, P=0.48. There were five serious complications requiring intervention or causing long-term sequelae; 0.13% (5 of 3729) per biopsy and 1.94% (5 of 258) per patient. The incidence of all definite and potential complications was 1.42% (53 of 3729) per biopsy and 20.5% (53 of 258) per patient. There was no biopsy-related mortality. Conclusion. The yield of BPAR was low in the AZA group and very low in the MMF group. The incidence of complications was also low, but repeated biopsies led to a higher rate per patient. Routine surveillance EMBs and the frequency of such biopsies should be reevaluated in the light of their low yield with current immunosuppression.

Lack of Effect of MICA Antibodies on Graft Survival Following Heart Transplantation

Little is known about the effect of MICA antibodies (Abs) on cardiac allograft function and survival. Pretransplant and posttransplant serum from 491 and 196 adult cardiac allograft recipients, respectively, has been investigated for MICA Abs, donor specificity and the effect of MICA Abs on graft survival, acute rejection episodes (AR) and cardiac allograft vasculopathy (CAV). Patients with HLA Abs (11.6%) were excluded from the analysis. A total of 11.8% of patients had MICA Abs, without HLA Abs, before their transplant. Actuarial graft survival demonstrated slightly better survival of patients with donor‐specific MICA Abs at 1 and 5 years (88.9% and 83.3%) than patients negative for MICA Abs (72% and 63.7%, p = 0.051). After transplantation, 15.8% of patients produced MICA Abs, and in 17 patients these were produced de novo. There was no effect of pretransplant or posttransplant production of MICA Abs on numbers of AR episodes in year 1, or CAV assessed at years 3 and 5. Immunocytochemistry of cardiac biopsies from 11 patients did not demonstrate a presence of MICA. Sera from only 4/69 patients with MICA Abs fixed complement prior to transplantation and from 7/38 patients following transplantation. In conclusion, this study suggests that MICA Abs do not adversely affect the outcome of cardiac transplantation.

Mycophenolate mofetil may allow cyclosporine and steroid sparing in de novo heart transplant patients

Background. Mycophenolate mofetil (MMF) provides superior prophylaxis against acute rejection when compared with azathioprine (AZA) in heart and renal transplantation. However, it remains unclear whether this results in improved survival or reduced morbidity after heart transplantation. Method. In a sequential study, 240 cardiac transplant patients were treated with either MMF (n=119) or AZA (n=121) both in combination with cyclosporine and corticosteroids after rabbit antithymocyte globulin induction. Results. By protocol lower cyclosporine levels were targeted in the MMF group during the first year (e.g. 203±52 ng/mL MMF vs. 236±59 ng/mL AZA, P=0.0006 at 6 months). Patient survival at 1 year (82% MMF vs. 79% AZA, P=0.55) and at 3 years was similar in both groups. The cumulative probability of receiving antirejection treatment within 1 year was lower in the MMF group, as was biopsy-proven acute rejection with International Society of Heart and Lung Transplantation grade ≥3A (24% vs. 35%, P=0.03). The MMF group also had fewer episodes requiring cytolytic therapy (6% vs. 13%, P=0.04) and more patients had steroids withdrawn by 1 year (66% vs. 32%, P<0.001). Renal function was better in the MMF group with lower creatinine levels at 1 year (133±45 vs. 155±46 &mgr;mol/L, P=0.0004). Calculated creatinine clearance (Cockcroft and Gault formula) at 1 year was also better (MMF 74±32 mL/min vs. AZA 62±24 mL/min, P=0.004). Conclusion. Our results suggest that immunosuppression with MMF rather than AZA may allow lower cyclosporine levels, better renal function, and increased steroid weaning at 1 year while also achieving better control of acute rejection.

A reevaluation of the role of IgM non-HLA antibodies in cardiac transplantation

Background. Preexisting IgG antibodies to donor human leukocyte antigens (HLA) are a risk factor for rapid allograft rejection. However, non-HLA antibodies, of the IgM class, also called autoreactive antibodies, are not believed to affect graft survival. The aim of this study was to determine the incidence and clinical relevance of pretransplant lymphocytotoxic non-HLA IgM antibodies on long-term cardiac allograft survival. Methods. A retrospective study of 616 adult recipients of cardiac allografts, transplanted at this center between 1991 and 2003, has been performed. Antibodies in pretransplant sera were initially defined using complement-dependent cytotoxicity assays, and subsequently analyzed for HLA specificities using solid phase assays. Results. HLA antibodies were present in 69 of 616 heart recipients (58 IgG, 11 IgM); in 22 of these, the antibodies were donor-specific. Non-HLA IgM antibodies were detected in 59 of 616 recipients who did not have HLA-specific antibodies; these patients had a 1, 2, 5, and 10 year survival of 55.9%, 54.2%, 49.9%, and 43.3% compared with 75.8%, 73.7%, 66.6%, and 52.8% for those without antibodies (P=0.0085 log-rank test). Multivariate analysis demonstrated pretransplant non-HLA IgM antibodies to be an independent risk factor for mortality (P=0.0001). Myocardial histology of postmortem heart and cardiac biopsies suggested an association with ischemic damage and “primary” allograft failure. Conclusions. We propose the hypothesis that the presence of cytotoxic IgM antibodies to non-HLAs before heart transplantation maybe a risk factor for early allograft failure.

Permanent pacemaker implantation early and late after heart transplantation: Clinical indication, risk factors and prognostic implications

BACKGROUND Permanent pacemaker implantation (PPM) early after cardiac transplantation has been shown not to predict a worse outcome. However, the requirement for pacing late after transplantation and its prognostic implications are not fully known. We describe the clinical indications, risk factors and long-term outcome in patients who required pacing early and late after transplantation. METHODS The transplant database, medical records and pacing database/records were reviewed for all patients undergoing de novo orthotopic cardiac transplantation (n = 389) at our institution between January 1995 and May 2006. RESULTS A total of 48 patients (12.3%) received a pacemaker after transplantation. Of these patients, 30 were paced early, pre-hospital discharge (25 ± 19 days post-transplantation), and 18 patients had late pacing (3.0 ± 3.3 years post-transplantation). There were no differences in clinical characteristics, use of anti-arrhythmic drugs or length-of-stay post-transplantation between early and late groups. Early indications for pacing were more often sino-atrial (SA) disease (24 of 30, 80%), whereas atrio-ventricular (AV) disease was more likely to occur later (p = 0.03). Risk factors for PPM included use of biatrial anastomosis (p = 0.001) and donor age (p = 0.002). Prior rejection was a univariate but not multivariate (p = 0.09) predictor of the need for PPM. Development of cardiac allograft vasculopathy was not predictive. There was no significant difference in mortality between late and early PPM patients or between late PPM patients and the non-paced patients who survived transplantation and initial stay. CONCLUSIONS Patients who required PPM late after orthotopic cardiac transplantation had a prognosis comparable to those paced early and those who did not require PPM. The independent risk factors for PPM were biatrial anastomosis and increasing donor age. SA-nodal dysfunction as an indication for PPM was more prevalent early after transplantation, whereas atrioventricular (AV) disease more commonly presented late. The requirement for pacing late after transplantation was not associated with rejection or cardiac allograft vasculopathy.

Impaired left ventricular systolic function early after heart transplantation is associated with cardiac allograft vasculopathy.

Cardiac allograft vasculopathy (CAV) is a major cause of death more than 1 year after heart transplantation. We evaluated the role and possible predictive value of different etiological factors on development of CAV as diagnosed by quantitative coronary angiography (QCA). A total of 121 patients were studied with baseline QCA and 117 had a follow‐up study at 1 year to assess the relationship of mean lumen diameter loss (MLDL) in main coronary arteries to immunological and non‐immunological factors potentially affecting long‐term survival. Out of them, 103 patients were males (85%), 114 (94%) patients were Caucasians and mean age was 48.5 ± 10 years. Univariate analysis showed that MLDL at 1 year was inversely related to echocardiographic fractional shortening (FS) measured within the first week after transplantation (p = 0.0098) and to intracranial hemorrhage as cause of donor death (p = 0.04) and was directly related to male donors (p = 0.0008), domino transplants (p = 0.037) and donor negative cytomegalovirus (CMV) status (p = 0.022). Multivariate analysis showed that initial FS (p = 0.006) and donor intracranial hemorrhage as a cause of death (p = 0.042) were inversely related to MLDL whereas donor male sex (p = 0.003) and prednisolone treatment throughout the first year (p = 0.012) were directly related. Thus, left ventricular systolic dysfunction early after heart transplantation was associated with subsequent development of CAV.

Transfer from ciclosporin to mycophenolate-sirolimus immunosuppression for chronic renal disease after heart transplantation: safety and efficacy of two regimens

BACKGROUND Chronic kidney disease is common after heart transplantation, and is related to ciclosporin (CsA) therapy. We compared the safety and efficacy of two ciclosporin withdrawal regimens. METHODS CsA was stopped and sirolimus (SRL) commenced immediately and the transfer was covered with prednisolone. Those on azathioprine (AZA) were transferred to MMF. In protocol A, the SRL target concentration was 16 (12-20) ng/ml; in protocol B, the target concentration was 7(5-10) ng/ml, but mycophenolate (MMF) and steroids were commenced prior to the transfer. RESULTS Baseline characteristics were similar in both groups except that group B were switched later after transplantation. Renal function improved significantly in both groups; this was maintained up to 1 year. Two patients in group A experienced acute rejection (ISHLT grade 3A or 2R); none was seen in group B. Six patients (46%) remained on protocol A and 22 (85%) remained on protocol B at 1 year. CONCLUSIONS MMF-SRL substitution resulted in a rapid but partial improvement in renal function; the lower dose SRL regimen was better tolerated.