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Advantages of C2 Monitoring to Avoid Acute Rejection in Pediatric Heart Transplant Recipients
PEDIATRIC TRANSPLANTATION
Advantages of C2 Monitoring to Avoid Acute Rejection in Pediatric Heart Transplant Recipients S. Schubert, MD,a H. Abdul-Khaliq, MD, PhD,a H. B. Lehmkuhl, MD,b M. Hübler, MD,c M. Y. Abd El Rahman, MD,a O. Miera, MD,a P. Ewert, MD, PhD,a Y. Weng, MD,c H. Wei, MD,a B. Krüdewagen, MD,b R. Hetzer, MD, PhD,c and F. Berger, MD, PhDa Background: Inadequate cyclosporine (CsA) blood levels are a major risk factor for acute rejection in transplant recipients. The CsA trough level (C0 level) measured just before the next dose is commonly used to adjust the oral dosage. However, the 2-hour post-CsA dose concentration (C2 level) is favored as the best single-point correlate of CsA area-under-the-curve concentration and may better reflect the immunosuppressive effect of CsA. Because an adequate C2 level has not yet been defined, this study was performed to assess the value of C2 monitoring for the prevention of acute rejection and to define target levels in pediatric heart transplant recipients. Methods: C2 levels were assessed in 50 pediatric heart transplant patients with oral CsA therapy and compared with trough C0 levels using full blood sampling, mass spectrometry and a blinded analysis. Acute graft rejection was detected using intramyocardial electrocardiogram (IMEG) and serial conventional and tissue Doppler echocardiography (TDE). Rejection was confirmed or excluded by endomyocardial biopsy. Results: C2 and not C0 levels were significantly reduced in patients with acute graft rejection (ISHLT Grade ⱖ2). Patients with a C2 level ⬍600 ng/ml had a significantly higher risk of developing acute rejection (100% sensitivity and 82% specificity). Patients with impaired CsA absorption were identified with C2 monitoring and switched to another calcineurin inhibitor. Conclusions: Monitoring of the C2 rather than the C0 level better reflects immunosuppressive efficiency and identifies patients at increased risk of acute rejection. A C2 level of ⬎600 ng/ml should be the target to prevent acute rejection. J Heart Lung Transplant 2006;25:619 –25. Copyright © 2006 by the International Society for Heart and Lung Transplantation.
Much variability in oral cyclosporine (CsA) therapy is known to occur, especially in pediatric patients. This is related to differences in the volume of distribution in gastrointestinal absorption and in the rate of systemic clearance by either hepatic metabolism or renal elimination.1– 4 Inadequate systemic blood concentration of CsA may lead to an increased incidence of acute or chronic graft rejection in de novo transplant patients.5,6 Therefore, trough level (C0 level) monitoring has been established in recent years as the standard procedure for pediatric transplant patients.7,8 The greatest calcineurin inhibition occurs within the first hours after
From the Departments of aCongenital Heart Defects/Pediatric Cardiology, bInternal Medicine/Cardiology and cCardiothoracic and Vascular Surgery, Deutsches Herzzentrum Berlin, Berlin, Germany. Submitted October 31, 2005; revised January 23, 2006; accepted February 11, 2006. Reprint requests: Stephan Schubert, MD, Department of Congenital Heart Defects/Pediatric Cardiology, Deutsches Herzzentrum Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Telephone: ⫹49-304593-2800. Fax: ⫹49-30-4593-2900. E-mail: [email protected] Copyright © 2006 by the International Society for Heart and Lung Transplantation. 1053-2498/06/$–see front matter. doi:10.1016/ j.healun.2006.02.002
oral administration, and monitoring of maximum concentration (Cmax) and the “area under the curve” (AUC) has been suggested to be the best indicator of individual systemic CsA exposure.9 However, AUC monitoring is limited in pediatric patients as it requires multiple blood sampling. Thus, C2 level has been described as the optimal single-timepoint marker for AUC0 – 4hours.10 –13 Target levels for CsA (C2 and C0 level) monitoring have been defined for pediatric liver and kidney transplant patients, although highly variable CsA levels have been described among several studies.3,4,12,14 Nevertheless, greater immunosuppression may be needed in heart or lung transplant patients.4,7,15,16 Although Cantarovich et al stated that “lower” CsA levels (C2 level 300 to 600 ng/ml or C0 level 100 to 200 ng/ml) may be effective to prevent acute rejection in pediatric liver transplant patients, higher CsA blood levels are needed to prevent rejection in heart or heart–lung transplant patients.6,17 Adequate C2 levels for the prevention of acute graft rejection have not yet been defined in these patients.18 The aim of this study was to assess the relative value of C0- and C2-level measurement in adjusting immunosuppression to pre619
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vent acute rejection in pediatric heart transplant patients.
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plant. Within 2 to 5 days, intravenous CsA therapy was generally switched to oral administration, although some toddlers were given their oral CsA (Sandimmun Optoral, or Neoral, Novartis Pharma) by gastric tube in the first weeks. Dose adjustments in these patients were made according to the trough-level blood concentration with a target level of 200 to 300 ng/ml within the first 4 weeks and a reduction to 150 to 200 ng/ml thereafter. Renal and hepatic function was monitored using standard laboratory methods. All patients were receiving triple therapy with either azathioprine (Imurek, GlaxoSmithKline) or mycophenolate mofetil (CellCept, Roche) together with CsA and oral steroid treatment (0.5 to 1 mg/kg/day). Dosing of all immunosuppressive agents was adapted on the basis of standardized trough blood level measurements and current blood count.
PATIENTS AND METHODS The study design involved prospective assessment and blinded analysis in pediatric heart transplant recipients undergoing routine monitoring of CsA (C2 and C0) blood levels at the German Heart Institute Berlin from January 2001 to October 2005. Fifty pediatric de novo heart transplant recipients in regular ambulatory care were included. All patients had received oral CsA therapy since heart transplantation. The minimum posttransplant time for inclusion in this study was set at 2 months to establish stable conditions of oral CsA therapy and to prevent conflicting results due to early administration errors, early drug interaction or late effects of the induction therapy. Exclusion criteria were intravenous cyclosporine therapy, any co-morbidity from acute or chronic hepatic or renal failure, oral feeding problems, diarrhea, malignancy or post-transplant lymphoproliferative disease (PTLD), acute or chronic infection, blood transfusion within the previous 3 days, or any other concomitant drug treatment with possible interaction with oral CsA therapy. Repeated monitoring of the CsA blood level was performed at 2 different timepoints in a standardized manner after informed consent of the patients or their parents had been obtained: CsA trough blood level concentration (C0 level) was measured just before the next oral administration and the C2 level was measured 2 hours after oral administration with a maximum tolerance of ⫾10 minutes for blood sampling. There were no technical or logistic problems. One hundred microliters of ethylene-diamine tetraacetic acid (EDTA)treated whole blood was prepared with a precipitation reagent. The sample was separated with a high-performance liquid chromatography (HPLC) C18 column after centrifugation. Specific cyclosporine A (molecular weight: 1,219.85/1,202.88 Da) was detected using tandem mass spectrometry (API 3000, Applied Biosystems, Concord, ON, Canada) with linear regression analysis (range 0 to 5,000 ng/ml) and cyclosporine D (molecular weight: 1,233.8/1,216.73 Da) as internal standard. In selected patients serial measurements of AUC were performed after obtaining informed consent of parents for multiple blood sampling.
Detection of Rejection After heart transplantation a double-chamber pacemaker was implanted to record the intramyocardial potentials of both ventricles.19 All patients were monitored daily to exclude acute rejection by observation of the intramyocardial electrocardiogram (IMEG) based on day-by-day changes in the maximal QRS complex amplitude and heart rate.19 Patients with a substantial voltage drop in the QRS complex amplitude of ⬎10% over 2 days and an increase in heart rate were considered to have acute rejection and were examined by conventional echocardiography and tissue Doppler echocardiography (TDE).19 Early annular systolic and diastolic peak wall motion velocity in a 4-chamber view, relaxation time, ejection fraction, left ventricular dimension and systolic Doppler flow parameter were studied using the System Five echocardiography unit and Echo Pac (GE, version 6.3.6, Vingmed, Norway). Changes in the new TDE parameters have been shown to be highly sensitive for the diagnosis of acute rejection.20 –22 A downtrend in the TDE systolic velocity of left and right annular ventricular wall motions with or without wall thickening were highly suggestive of acute rejection. The combination of downtrends of IMEG and TDE wall velocities was indicative for the performance of endomyocardial biopsy. Acute moderate or severe rejection was defined as International Society for Heart and Lung Transplantation (ISHLT) Grade ⱖ2 for endomyocardial biopsies.23–25
Immunosuppression All pediatric de novo heart transplant patients at the German Heart Institute Berlin underwent induction therapy with 2 cycles of anti-thymoglobin antibodies (ATG; Fresenius, Bad Homburg, Germany, or Thymoglobuline, Sangstat, Lyon, France) in combination with intravenous methylprednisolone (Urbasone, Hoechst, Germany) pulse therapy on Days 1 and 2 post-trans-
Statistical Analysis Descriptive statistics were used (including median, mean ⫾ standard deviation). Comparative statistical analyses were performed in a blinded manner by means of paired and unpaired Student’s t-test for continuous variables and chi-square test for categoric variables using SPSS for Windows (release 9.0) and STATVIEW software. p ⬍ 0.05 was considered statistically signifi-
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Table 1. Demographics of Patients
No rejection Rejection
Number 36 14
Age (years) 8.9 ⫾ 5.6 8.1 ⫾ 6.3
Gender (M:F) 14:22 5:9
Height (cm) 120 ⫾ 35 114 ⫾ 45
Weight (kg) 27 ⫾ 17 23 ⫾ 18
Post-transplant time (years) 2.4 ⫾ 1.8 2.2 ⫾ 1.6
Creatinine (mg/dl) 0.7 ⫾ 0.4 0.89 ⫾ 0.4
Urea (mg/dl) 46 ⫾ 36 56 ⫾ 42
Data presented as mean ⫾ SD. p-value was not significant for any of these parameters.
cant. The area under the curve (AUC) was calculated using the composite trapezoidal rule26 and Spearman’s rho test to assess the correlation coefficient for AUC and C0, C2 and C4 blood level. Sensitivity and specificity of C0 and C2 levels were tested using the receiver characteristic (ROC) curve with SPSS. This curve plots the true-positive rate against the false-positive rate for different possible cut-off points of the C0 and C2 levels. RESULTS Fifty pediatric de novo heart transplant recipients were included in this prospective study. C0 and C2 blood levels were measured in standardized form and at different time intervals. Additional blood sampling in these pediatric patients was performed without difficulties. Acute moderate or severe rejection was diagnosed by right heart biopsy in 14 patients with ISHLT Grade ⱖ2.23,25 The pathologist was blinded to the initial diagnostic parameters indicating myocardial rejection. Thirty-six patients had only mild signs or no signs of rejection (Grade ⬍2) according to endomyocardial biopsy. The 2 groups with and without confirmed rejection by means of biopsy were matched for age, gender, weight and post-transplant time, as summarized in Table 1. Median daily dose of oral CsA was 8.6 (range 4 to 21) mg/kg in patients with rejection vs 7.9 (range
3.7 to 24) mg/kg in those without rejection and showed no statistically significant difference (p ⫽ 0.67). Concomitant immunosuppression (rejection vs non-rejection groups) consisted of mycophenolate mofetil (8 of 14 vs 25 of 36), azathioprine (5 of 14 vs 8 of 36) and oral prednisolone (9 of 14 vs 20 of 35) without any statistically significant differences between the 2 groups (p ⫽ 0.74). Hypertension was detected in 55% of patients in both groups (9 of 14 vs 20 of 36), and some patients received anti-hypertensive treatment (angiotensin-converting enzyme [ACE] inhibitor) without statistically significant differences (p ⫽ 0.49). Renal function was stable in all patients with creatinine values within the normal range and no significant differences between the 2 groups (Table 1). Creatinine clearance was calculated according to the Schwartz formula27: glomerular filtration rate was 66.8 ⫾ 15.8 ml/min vs 68.8 ⫾ 12.5 ml/min in patients with rejection vs without rejection, indicating no significant difference (p ⫽ 0.53). C2 Levels and AUC Measurements C2 levels were significantly lower in patients with biopsy-confirmed rejection than in patients without rejection (345 ⫾ 163 ng/ml vs 952 ⫾ 310 ng/ml, p ⬍ 0.001; Figure 1). Lower C2 levels were measured depending on the post-transplant time, with significant differences between the 2 groups (p ⬍ 0.001; Table 2). C0 levels showed no significant differences in relation to post-transplant time in either group (p ⫽ 0.56). The AUC was also calculated in 10 of the study patients, of whom 5 were with and 5 without biopsy-proven rejection. AUC was calculated from serial measurements at 0, 2, 4, 8 and 12 hours after oral CsA administration (Table 3 and Figure 3). AUC0 –12hours, AUC0 – 4hours and Cmax were significantly lower (p ⬍ 0.001) in patients with rejection than in those without rejection (Table 3). AUC measurement was possible only in a limited number of patients due to the need for multiple blood sampling, which is unfavorable in a pediatric patient population. Table 2. C2 Level Depending on Post-transplant Time
Figure 1. C0 and C2 levels in patients with and without rejection (mean ⫾ SD). C2 level was significantly lower in patients with acute rejection (p ⬍ 0.001), whereas C0 level showed no significant difference.
No rejection (n ⫽ 36) Rejection (n ⫽ 14)
0–1 year (ng/ml) 1,134 ⫾ 356 456 ⫾ 164a
Data presented as mean ⫾ SD. a No rejection vs rejection, p ⬍ 0.001.
1–2 years (ng/ml) 996 ⫾ 266 428 ⫾ 196a
2–5 years (ng/ml) 859 ⫾ 274 244 ⫾ 147a
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Table 3. Area Under the Curve (AUC) Measurement in Patients With and Without Rejection
No rejection (n ⫽ 5) Rejection (n ⫽ 5)
AUC0–12hours (ng · h/ml) 5,530 ⫾ 889 3,615 ⫾ 508a
AUC0–4hours (ng · h/ml) 2,713 ⫾ 536 1,498 ⫾ 132a
Cmax (ng/ml) 966 ⫾ 231 467 ⫾ 38a
Cmin (ng/ml) 219 ⫾ 32 213 ⫾ 29
Data presented as mean ⫾ SD. a Rejection vs no rejection, p ⬍ 0.001.
There was a significant correlation between C2 level, AUC0 – 4hours and AUC0 –12hours (r ⫽ 0.994). C0 level showed no significant correlation (r ⫽ ⫺0.25). Sensitivity and specificity of C0 and C2 levels were tested using the ROC curve (Figure 4). Cut-off Values In another step we calculated the cut-off points at 500, 600 and 700 ng/ml to assess a realistic C2 level for the identification of patients with increased risk of acute rejection. The AUC of the ROC curve for the C2 level (0.982) was significantly higher than that of C0 (0.532; Figure 4). According to the ROC curve we found that 100% of our patients with acute rejection had a C2 level of ⬍600 ng/ml, indicating 100% sensitivity. Specificity was 83% at a C2 level of ⬍600 ng/ml and still 68% at a C2 level of ⬍700 ng/ml, because some patients without rejection also had a C2 level of ⬍700 ng/ml (Figure 2). According to these data a C2 level of ⬍600 ng/ml seems to be the best cut-off point for achieving high sensitivity and good specificity (see Figure 4).
ylprednisolone (Urbason Soluble, Hoechst) and the other 50% received an additional 1 to 3 cycles of anti-thymocyte globulin (ATG, Fresenius HemoCare or Thymoglobuline, Sangstat, Lyon, France). All patients recovered completely and were discharged within 7 to 9 days on improved immunosuppression and low-dose steroid treatment. CsA doses were increased in all patients with biopsy-proven rejection. In 5 patients it was necessary to change immunosuppression from CsA to FK 506 due to reduced resorption of CsA and relapsing episodes of acute rejection. No serious adverse events occurred under increased CsA doses or rejection treatment.
Rejection Treatment Fifty percent of the patients with rejection received intravenous high-dose pulse steroid therapy with meth-
DISCUSSION The results of this prospective observational study confirm the superior value of C2 rather than C0 monitoring of immunosuppressive effectiveness of oral CsA therapy in pediatric heart transplant recipients. To our knowledge this is the first study to demonstrate that the determination of C2 level has significant predictive value, with high sensitivity and specificity, for the early detection of inadequate immunosuppression in pediatric heart transplant patients receiving oral CsA therapy. According to our results, reduced C2 level may result in
Figure 2. C2 blood level in patients with and without rejection. Patients with CyA blood level ⬍600 ng/ml had a higher risk of acute graft rejection. The horizontal line indicates a cut-off value of approximately 600 ng/ml for the C2 level of cyclosporine.
Figure 3. Serial measurements 0 to 12 hours after CyA administration. CyA dosing is marked with an arrow. CyA blood levels are registered at 0, 2, 4, 6, 8 and 12 hours after oral administration. Five of these patients had no signs of rejection. Five patients had biopsy-confirmed acute rejection, a significantly lower Cmax approximately 2 hours after oral administration, and a generally reduced absorption profile.
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Figure 4. Receiver operating characteristic (ROC) curve for C0 and C2 levels. C2 blood level at 600 ng/ml seems to be the best cut-off point, with 100% sensitivity and 80% specificity.
an increased rate of moderate or severe rejection and may also contribute to increased long-term morbidity with coronary artery vasculopathy (CAV) and graft loss. The main variability in the enteric absorption of CsA occurs during the first hours after oral administration, as described by other studies in liver and kidney transplant patients.28,29 Measurement of C2 level in pediatric and adult liver, kidney and lung transplant patients has been proposed as the best single-point marker of the AUC concentration.30 –32 The highest immunosuppressive effect reflected by maximum calcineurin and interleukin-2 inhibition occurs during the first hours after oral CsA administration.17,29 Therefore, C2 monitoring has greater potential for the accurate surveillance of immunosuppressive effects than trough-level monitoring (C0 level),33–35 and mass spectrometry has become the laboratory standard with the greatest reliability.10,14,36,37 Recent clinical studies in pediatric liver and kidney transplant patients identified a reduced number of rejection episodes if CsA doses were adjusted on the basis of C2 level rather than C0 level monitoring,l with blood level ⬍500 ng/ml leading to an increased number of acute rejections in one study.39 On the other hand, monitoring of the C2 level may help to avoid higher serum levels and thereby prevent additional toxic effects such as neuro- and nephrotoxicity.36 Nevertheless, in some studies—mostly of liver transplant patients—no statistical differences were found with additional C2 monitoring.40,41 This may be related to the differences in the CsA blood levels required in the various types of organ transplantation, with these normally being lower in liver transplant patients.42,43 On the basis of these findings we intend to implement a target C2 level of ⬎600 ng/ml or, with greater safety, ⬎700 ng/ml in patients 0 to 5 years after heart transplantation. A C2 level of ⬍600 ng/ml seems to represent inadequate immunosuppression and may subject the patients to an increased risk of acute or chronic rejection, especially if the C0 level is within the normal
range.44 Nevertheless, investigation of larger numbers of pediatric patients is needed to identify reliable target levels, which can only be achieved by a multicenter study. Poor absorption or fast elimination may occur more frequently in younger children or toddlers, where C2 blood level monitoring helps us to identify patients in whom immunosuppression needs to be changed to tacrolimus (Prograf, Fujisawa) to prevent ongoing acute rejection episodes or transplant coronary artery disease.45 The relatively small number of patients with rejection limits this prospective, observational trial. Clearly, larger numbers of pediatric heart recipients are necessary to evaluate a reliable cut-off point for C2 level in transplant recipients.44 In addition, prospective studies including patients in the early period after heart transplantation are necessary to evaluate the initial pharmacokinetics of pediatric patients. We cannot define different C2 target levels in relation to adjunctive medication (azathioprine or mycophenolate mofetil), because there were no significant differences in adjunctive medication between the 2 groups. Again, inclusion of a larger number of patients is needed to answer this particular question adequately. According to these results we cannot assume there will be a reduction in the incidence of transplant coronary artery disease with this regimen. Follow-up studies are necessary to evaluate C2 monitoring with regard to the development of coronary artery disease and long-term survival of the graft.46 In conclusion, C2 level monitoring is uncomplicated to perform in pediatric patients and is superior to C0 level measurement for adapting CsA dosage and for early identification of pediatric patients with inadequate immunosuppression, which may be explained by impaired enteric resorption and reduced bioavailability. A C2 level of ⬍600 ng/ml seems to predict acute graft rejection in pediatric heart transplant patients with high sensitivity and good specificity, which is in accordance with the data in adult heart transplant patients previ-
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ously reported by Chou et al.44 Thus, serial C2 rather than C0 monitoring identifies those patients at increased risk of inadequate immunosuppression. It may therefore help to reduce acute rejection and prolong graft survival in children after heart transplantation. The authors thank Anne Gale, ELS (Deutsches Herzzentrum Berlin), for editorial support and Julia Stein, MSc (Deutsches Herzzentrum Berlin), for help with statistical analysis of the data.
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29. Oellerich M, Armstrong VW. Two-hour cyclosporine concentration determination: an appropriate tool to monitor neoral therapy? Ther Drug Monit 2002;24:40 – 6. 30. Trompeter R, Fitzpatrick M, Hutchinson C, Johnston A. Longitudinal evaluation of the pharmacokinetics of cyclosporin microemulsion (Neoral) in pediatric renal transplant recipients and assessment of C2 level as a marker for absorption. Pediatr Transplant 2003;7:282– 8. 31. Hu RH, Tsai MK, Lee PH. Evaluation of cyclosporine C2 levels in long-term stable renal allograft recipients. Transplant Proc 2004;36:2105–7. 32. Oellerich M, Armstrong VW, Streit F, Weber L, Tonshoff B. Immunosuppressive drug monitoring of sirolimus and cyclosporine in pediatric patients. Clin Biochem 2004;37: 424 – 8. 33. Barakat O, Peaston R, Rai R, Talbot D, Manas D. Clinical benefit of monitoring cyclosporine C2 and C4 in longterm liver transplant recipients. Transplant Proc 2002;34: 1535–7. 34. Cantarovich M, Besner JG, Barkun JS, Elstein E, Loertscher R. Two-hour cyclosporine level determination is the appropriate tool to monitor Neoral therapy. Clin Transplant 1998;12:243–9. 35. Midtvedt K. Is C0 better than C2 as a determinant of rejection in renal transplant recipients? Kidney Int 2004; 66:869. 36. Morton JM, Aboyoun CL, Malouf MA, Plit ML, Glanville AR. Enhanced clinical utility of de novo cyclosporine C2 monitoring after lung transplantation. J Heart Lung Transplant 2004;23:1035–9. 37. Holt DW, Armstrong VW, Griesmacher A, et al. International Federation of Clinical Chemistry/International Association of Therapeutic Drug Monitoring and Clinical Toxicology working group on immunosuppressive drug monitoring. Ther Drug Monit 2002;24:59 – 67.
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38. Weber LT, Armstrong VW, Shipkova M, et al. Cyclosporin A absorption profiles in pediatric renal transplant recipients predict the risk of acute rejection. Ther Drug Monit 2004;26:415–24. 39. Pape L, Lehnhardt A, Latta K, Ehrich JH, Offner G. Cyclosporin A monitoring by 2-h levels: preliminary target levels in stable pediatric kidney transplant recipients. Clin Transplant 2003;17:546 – 8. 40. Teisseyre J, Markiewicz M, Drewniak T, et al. Switching cyclosporine blood concentration monitoring from C0 to C2 in children late after liver transplantation. Transplant Proc 2003;35:2287– 8. 41. Ganschow R, Richter A, Grabhorn E, et al. C2 blood concentrations of orally administered cyclosporine in pediatric liver graft recipients with a body weight below 10 kg. Pediatr Transplant 2004;8:185– 8. 42. Cantarovich M, Barkun J, Giannetti N, et al. History of C2 monitoring in heart and liver transplant patients treated with cyclosporine microemulsion. Transplant Proc 2004; 36(suppl):442S–7S. 43. Masini JP, Poirier JM, Weissenburger J, et al. Study of cyclosporin A blood level during the early post-livertransplantation period. Therapie 1993;48:163– 6. 44. Chou NK, Chen RJ, Ko WJ, et al. Cyclosporine C2 monitoring is superior to C0 in predicting acute cellular rejection in heart transplant recipients in Taiwan. Transplant Proc 2004;36:2393–5. 45. Barama A, Sepandj F, Gough J, McKenna R. Correlation between Neoral 2 hours postdose levels and histologic findings on surveillance biopsies. Transplant Proc 2004; 36(suppl):465S–7S. 46. Webber SA, Naftel DC, Parker J, et al. Late rejection episodes more than 1 year after pediatric heart transplantation: risk factors and outcomes. J Heart Lung Transplant 2003;22:869 –75.
Advantages of C2 Monitoring to Avoid Acute Rejection in Pediatric Heart Transplant Recipients S. Schubert, MD,a H. Abdul-Khaliq, MD, PhD,a H. B. Lehmkuhl, MD,b M. Hübler, MD,c M. Y. Abd El Rahman, MD,a O. Miera, MD,a P. Ewert, MD, PhD,a Y. Weng, MD,c H. Wei, MD,a B. Krüdewagen, MD,b R. Hetzer, MD, PhD,c and F. Berger, MD, PhDa Background: Inadequate cyclosporine (CsA) blood levels are a major risk factor for acute rejection in transplant recipients. The CsA trough level (C0 level) measured just before the next dose is commonly used to adjust the oral dosage. However, the 2-hour post-CsA dose concentration (C2 level) is favored as the best single-point correlate of CsA area-under-the-curve concentration and may better reflect the immunosuppressive effect of CsA. Because an adequate C2 level has not yet been defined, this study was performed to assess the value of C2 monitoring for the prevention of acute rejection and to define target levels in pediatric heart transplant recipients. Methods: C2 levels were assessed in 50 pediatric heart transplant patients with oral CsA therapy and compared with trough C0 levels using full blood sampling, mass spectrometry and a blinded analysis. Acute graft rejection was detected using intramyocardial electrocardiogram (IMEG) and serial conventional and tissue Doppler echocardiography (TDE). Rejection was confirmed or excluded by endomyocardial biopsy. Results: C2 and not C0 levels were significantly reduced in patients with acute graft rejection (ISHLT Grade ⱖ2). Patients with a C2 level ⬍600 ng/ml had a significantly higher risk of developing acute rejection (100% sensitivity and 82% specificity). Patients with impaired CsA absorption were identified with C2 monitoring and switched to another calcineurin inhibitor. Conclusions: Monitoring of the C2 rather than the C0 level better reflects immunosuppressive efficiency and identifies patients at increased risk of acute rejection. A C2 level of ⬎600 ng/ml should be the target to prevent acute rejection. J Heart Lung Transplant 2006;25:619 –25. Copyright © 2006 by the International Society for Heart and Lung Transplantation.
Much variability in oral cyclosporine (CsA) therapy is known to occur, especially in pediatric patients. This is related to differences in the volume of distribution in gastrointestinal absorption and in the rate of systemic clearance by either hepatic metabolism or renal elimination.1– 4 Inadequate systemic blood concentration of CsA may lead to an increased incidence of acute or chronic graft rejection in de novo transplant patients.5,6 Therefore, trough level (C0 level) monitoring has been established in recent years as the standard procedure for pediatric transplant patients.7,8 The greatest calcineurin inhibition occurs within the first hours after
From the Departments of aCongenital Heart Defects/Pediatric Cardiology, bInternal Medicine/Cardiology and cCardiothoracic and Vascular Surgery, Deutsches Herzzentrum Berlin, Berlin, Germany. Submitted October 31, 2005; revised January 23, 2006; accepted February 11, 2006. Reprint requests: Stephan Schubert, MD, Department of Congenital Heart Defects/Pediatric Cardiology, Deutsches Herzzentrum Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Telephone: ⫹49-304593-2800. Fax: ⫹49-30-4593-2900. E-mail: [email protected] Copyright © 2006 by the International Society for Heart and Lung Transplantation. 1053-2498/06/$–see front matter. doi:10.1016/ j.healun.2006.02.002
oral administration, and monitoring of maximum concentration (Cmax) and the “area under the curve” (AUC) has been suggested to be the best indicator of individual systemic CsA exposure.9 However, AUC monitoring is limited in pediatric patients as it requires multiple blood sampling. Thus, C2 level has been described as the optimal single-timepoint marker for AUC0 – 4hours.10 –13 Target levels for CsA (C2 and C0 level) monitoring have been defined for pediatric liver and kidney transplant patients, although highly variable CsA levels have been described among several studies.3,4,12,14 Nevertheless, greater immunosuppression may be needed in heart or lung transplant patients.4,7,15,16 Although Cantarovich et al stated that “lower” CsA levels (C2 level 300 to 600 ng/ml or C0 level 100 to 200 ng/ml) may be effective to prevent acute rejection in pediatric liver transplant patients, higher CsA blood levels are needed to prevent rejection in heart or heart–lung transplant patients.6,17 Adequate C2 levels for the prevention of acute graft rejection have not yet been defined in these patients.18 The aim of this study was to assess the relative value of C0- and C2-level measurement in adjusting immunosuppression to pre619
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vent acute rejection in pediatric heart transplant patients.
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plant. Within 2 to 5 days, intravenous CsA therapy was generally switched to oral administration, although some toddlers were given their oral CsA (Sandimmun Optoral, or Neoral, Novartis Pharma) by gastric tube in the first weeks. Dose adjustments in these patients were made according to the trough-level blood concentration with a target level of 200 to 300 ng/ml within the first 4 weeks and a reduction to 150 to 200 ng/ml thereafter. Renal and hepatic function was monitored using standard laboratory methods. All patients were receiving triple therapy with either azathioprine (Imurek, GlaxoSmithKline) or mycophenolate mofetil (CellCept, Roche) together with CsA and oral steroid treatment (0.5 to 1 mg/kg/day). Dosing of all immunosuppressive agents was adapted on the basis of standardized trough blood level measurements and current blood count.
PATIENTS AND METHODS The study design involved prospective assessment and blinded analysis in pediatric heart transplant recipients undergoing routine monitoring of CsA (C2 and C0) blood levels at the German Heart Institute Berlin from January 2001 to October 2005. Fifty pediatric de novo heart transplant recipients in regular ambulatory care were included. All patients had received oral CsA therapy since heart transplantation. The minimum posttransplant time for inclusion in this study was set at 2 months to establish stable conditions of oral CsA therapy and to prevent conflicting results due to early administration errors, early drug interaction or late effects of the induction therapy. Exclusion criteria were intravenous cyclosporine therapy, any co-morbidity from acute or chronic hepatic or renal failure, oral feeding problems, diarrhea, malignancy or post-transplant lymphoproliferative disease (PTLD), acute or chronic infection, blood transfusion within the previous 3 days, or any other concomitant drug treatment with possible interaction with oral CsA therapy. Repeated monitoring of the CsA blood level was performed at 2 different timepoints in a standardized manner after informed consent of the patients or their parents had been obtained: CsA trough blood level concentration (C0 level) was measured just before the next oral administration and the C2 level was measured 2 hours after oral administration with a maximum tolerance of ⫾10 minutes for blood sampling. There were no technical or logistic problems. One hundred microliters of ethylene-diamine tetraacetic acid (EDTA)treated whole blood was prepared with a precipitation reagent. The sample was separated with a high-performance liquid chromatography (HPLC) C18 column after centrifugation. Specific cyclosporine A (molecular weight: 1,219.85/1,202.88 Da) was detected using tandem mass spectrometry (API 3000, Applied Biosystems, Concord, ON, Canada) with linear regression analysis (range 0 to 5,000 ng/ml) and cyclosporine D (molecular weight: 1,233.8/1,216.73 Da) as internal standard. In selected patients serial measurements of AUC were performed after obtaining informed consent of parents for multiple blood sampling.
Detection of Rejection After heart transplantation a double-chamber pacemaker was implanted to record the intramyocardial potentials of both ventricles.19 All patients were monitored daily to exclude acute rejection by observation of the intramyocardial electrocardiogram (IMEG) based on day-by-day changes in the maximal QRS complex amplitude and heart rate.19 Patients with a substantial voltage drop in the QRS complex amplitude of ⬎10% over 2 days and an increase in heart rate were considered to have acute rejection and were examined by conventional echocardiography and tissue Doppler echocardiography (TDE).19 Early annular systolic and diastolic peak wall motion velocity in a 4-chamber view, relaxation time, ejection fraction, left ventricular dimension and systolic Doppler flow parameter were studied using the System Five echocardiography unit and Echo Pac (GE, version 6.3.6, Vingmed, Norway). Changes in the new TDE parameters have been shown to be highly sensitive for the diagnosis of acute rejection.20 –22 A downtrend in the TDE systolic velocity of left and right annular ventricular wall motions with or without wall thickening were highly suggestive of acute rejection. The combination of downtrends of IMEG and TDE wall velocities was indicative for the performance of endomyocardial biopsy. Acute moderate or severe rejection was defined as International Society for Heart and Lung Transplantation (ISHLT) Grade ⱖ2 for endomyocardial biopsies.23–25
Immunosuppression All pediatric de novo heart transplant patients at the German Heart Institute Berlin underwent induction therapy with 2 cycles of anti-thymoglobin antibodies (ATG; Fresenius, Bad Homburg, Germany, or Thymoglobuline, Sangstat, Lyon, France) in combination with intravenous methylprednisolone (Urbasone, Hoechst, Germany) pulse therapy on Days 1 and 2 post-trans-
Statistical Analysis Descriptive statistics were used (including median, mean ⫾ standard deviation). Comparative statistical analyses were performed in a blinded manner by means of paired and unpaired Student’s t-test for continuous variables and chi-square test for categoric variables using SPSS for Windows (release 9.0) and STATVIEW software. p ⬍ 0.05 was considered statistically signifi-
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Table 1. Demographics of Patients
No rejection Rejection
Number 36 14
Age (years) 8.9 ⫾ 5.6 8.1 ⫾ 6.3
Gender (M:F) 14:22 5:9
Height (cm) 120 ⫾ 35 114 ⫾ 45
Weight (kg) 27 ⫾ 17 23 ⫾ 18
Post-transplant time (years) 2.4 ⫾ 1.8 2.2 ⫾ 1.6
Creatinine (mg/dl) 0.7 ⫾ 0.4 0.89 ⫾ 0.4
Urea (mg/dl) 46 ⫾ 36 56 ⫾ 42
Data presented as mean ⫾ SD. p-value was not significant for any of these parameters.
cant. The area under the curve (AUC) was calculated using the composite trapezoidal rule26 and Spearman’s rho test to assess the correlation coefficient for AUC and C0, C2 and C4 blood level. Sensitivity and specificity of C0 and C2 levels were tested using the receiver characteristic (ROC) curve with SPSS. This curve plots the true-positive rate against the false-positive rate for different possible cut-off points of the C0 and C2 levels. RESULTS Fifty pediatric de novo heart transplant recipients were included in this prospective study. C0 and C2 blood levels were measured in standardized form and at different time intervals. Additional blood sampling in these pediatric patients was performed without difficulties. Acute moderate or severe rejection was diagnosed by right heart biopsy in 14 patients with ISHLT Grade ⱖ2.23,25 The pathologist was blinded to the initial diagnostic parameters indicating myocardial rejection. Thirty-six patients had only mild signs or no signs of rejection (Grade ⬍2) according to endomyocardial biopsy. The 2 groups with and without confirmed rejection by means of biopsy were matched for age, gender, weight and post-transplant time, as summarized in Table 1. Median daily dose of oral CsA was 8.6 (range 4 to 21) mg/kg in patients with rejection vs 7.9 (range
3.7 to 24) mg/kg in those without rejection and showed no statistically significant difference (p ⫽ 0.67). Concomitant immunosuppression (rejection vs non-rejection groups) consisted of mycophenolate mofetil (8 of 14 vs 25 of 36), azathioprine (5 of 14 vs 8 of 36) and oral prednisolone (9 of 14 vs 20 of 35) without any statistically significant differences between the 2 groups (p ⫽ 0.74). Hypertension was detected in 55% of patients in both groups (9 of 14 vs 20 of 36), and some patients received anti-hypertensive treatment (angiotensin-converting enzyme [ACE] inhibitor) without statistically significant differences (p ⫽ 0.49). Renal function was stable in all patients with creatinine values within the normal range and no significant differences between the 2 groups (Table 1). Creatinine clearance was calculated according to the Schwartz formula27: glomerular filtration rate was 66.8 ⫾ 15.8 ml/min vs 68.8 ⫾ 12.5 ml/min in patients with rejection vs without rejection, indicating no significant difference (p ⫽ 0.53). C2 Levels and AUC Measurements C2 levels were significantly lower in patients with biopsy-confirmed rejection than in patients without rejection (345 ⫾ 163 ng/ml vs 952 ⫾ 310 ng/ml, p ⬍ 0.001; Figure 1). Lower C2 levels were measured depending on the post-transplant time, with significant differences between the 2 groups (p ⬍ 0.001; Table 2). C0 levels showed no significant differences in relation to post-transplant time in either group (p ⫽ 0.56). The AUC was also calculated in 10 of the study patients, of whom 5 were with and 5 without biopsy-proven rejection. AUC was calculated from serial measurements at 0, 2, 4, 8 and 12 hours after oral CsA administration (Table 3 and Figure 3). AUC0 –12hours, AUC0 – 4hours and Cmax were significantly lower (p ⬍ 0.001) in patients with rejection than in those without rejection (Table 3). AUC measurement was possible only in a limited number of patients due to the need for multiple blood sampling, which is unfavorable in a pediatric patient population. Table 2. C2 Level Depending on Post-transplant Time
Figure 1. C0 and C2 levels in patients with and without rejection (mean ⫾ SD). C2 level was significantly lower in patients with acute rejection (p ⬍ 0.001), whereas C0 level showed no significant difference.
No rejection (n ⫽ 36) Rejection (n ⫽ 14)
0–1 year (ng/ml) 1,134 ⫾ 356 456 ⫾ 164a
Data presented as mean ⫾ SD. a No rejection vs rejection, p ⬍ 0.001.
1–2 years (ng/ml) 996 ⫾ 266 428 ⫾ 196a
2–5 years (ng/ml) 859 ⫾ 274 244 ⫾ 147a
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Table 3. Area Under the Curve (AUC) Measurement in Patients With and Without Rejection
No rejection (n ⫽ 5) Rejection (n ⫽ 5)
AUC0–12hours (ng · h/ml) 5,530 ⫾ 889 3,615 ⫾ 508a
AUC0–4hours (ng · h/ml) 2,713 ⫾ 536 1,498 ⫾ 132a
Cmax (ng/ml) 966 ⫾ 231 467 ⫾ 38a
Cmin (ng/ml) 219 ⫾ 32 213 ⫾ 29
Data presented as mean ⫾ SD. a Rejection vs no rejection, p ⬍ 0.001.
There was a significant correlation between C2 level, AUC0 – 4hours and AUC0 –12hours (r ⫽ 0.994). C0 level showed no significant correlation (r ⫽ ⫺0.25). Sensitivity and specificity of C0 and C2 levels were tested using the ROC curve (Figure 4). Cut-off Values In another step we calculated the cut-off points at 500, 600 and 700 ng/ml to assess a realistic C2 level for the identification of patients with increased risk of acute rejection. The AUC of the ROC curve for the C2 level (0.982) was significantly higher than that of C0 (0.532; Figure 4). According to the ROC curve we found that 100% of our patients with acute rejection had a C2 level of ⬍600 ng/ml, indicating 100% sensitivity. Specificity was 83% at a C2 level of ⬍600 ng/ml and still 68% at a C2 level of ⬍700 ng/ml, because some patients without rejection also had a C2 level of ⬍700 ng/ml (Figure 2). According to these data a C2 level of ⬍600 ng/ml seems to be the best cut-off point for achieving high sensitivity and good specificity (see Figure 4).
ylprednisolone (Urbason Soluble, Hoechst) and the other 50% received an additional 1 to 3 cycles of anti-thymocyte globulin (ATG, Fresenius HemoCare or Thymoglobuline, Sangstat, Lyon, France). All patients recovered completely and were discharged within 7 to 9 days on improved immunosuppression and low-dose steroid treatment. CsA doses were increased in all patients with biopsy-proven rejection. In 5 patients it was necessary to change immunosuppression from CsA to FK 506 due to reduced resorption of CsA and relapsing episodes of acute rejection. No serious adverse events occurred under increased CsA doses or rejection treatment.
Rejection Treatment Fifty percent of the patients with rejection received intravenous high-dose pulse steroid therapy with meth-
DISCUSSION The results of this prospective observational study confirm the superior value of C2 rather than C0 monitoring of immunosuppressive effectiveness of oral CsA therapy in pediatric heart transplant recipients. To our knowledge this is the first study to demonstrate that the determination of C2 level has significant predictive value, with high sensitivity and specificity, for the early detection of inadequate immunosuppression in pediatric heart transplant patients receiving oral CsA therapy. According to our results, reduced C2 level may result in
Figure 2. C2 blood level in patients with and without rejection. Patients with CyA blood level ⬍600 ng/ml had a higher risk of acute graft rejection. The horizontal line indicates a cut-off value of approximately 600 ng/ml for the C2 level of cyclosporine.
Figure 3. Serial measurements 0 to 12 hours after CyA administration. CyA dosing is marked with an arrow. CyA blood levels are registered at 0, 2, 4, 6, 8 and 12 hours after oral administration. Five of these patients had no signs of rejection. Five patients had biopsy-confirmed acute rejection, a significantly lower Cmax approximately 2 hours after oral administration, and a generally reduced absorption profile.
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Figure 4. Receiver operating characteristic (ROC) curve for C0 and C2 levels. C2 blood level at 600 ng/ml seems to be the best cut-off point, with 100% sensitivity and 80% specificity.
an increased rate of moderate or severe rejection and may also contribute to increased long-term morbidity with coronary artery vasculopathy (CAV) and graft loss. The main variability in the enteric absorption of CsA occurs during the first hours after oral administration, as described by other studies in liver and kidney transplant patients.28,29 Measurement of C2 level in pediatric and adult liver, kidney and lung transplant patients has been proposed as the best single-point marker of the AUC concentration.30 –32 The highest immunosuppressive effect reflected by maximum calcineurin and interleukin-2 inhibition occurs during the first hours after oral CsA administration.17,29 Therefore, C2 monitoring has greater potential for the accurate surveillance of immunosuppressive effects than trough-level monitoring (C0 level),33–35 and mass spectrometry has become the laboratory standard with the greatest reliability.10,14,36,37 Recent clinical studies in pediatric liver and kidney transplant patients identified a reduced number of rejection episodes if CsA doses were adjusted on the basis of C2 level rather than C0 level monitoring,l with blood level ⬍500 ng/ml leading to an increased number of acute rejections in one study.39 On the other hand, monitoring of the C2 level may help to avoid higher serum levels and thereby prevent additional toxic effects such as neuro- and nephrotoxicity.36 Nevertheless, in some studies—mostly of liver transplant patients—no statistical differences were found with additional C2 monitoring.40,41 This may be related to the differences in the CsA blood levels required in the various types of organ transplantation, with these normally being lower in liver transplant patients.42,43 On the basis of these findings we intend to implement a target C2 level of ⬎600 ng/ml or, with greater safety, ⬎700 ng/ml in patients 0 to 5 years after heart transplantation. A C2 level of ⬍600 ng/ml seems to represent inadequate immunosuppression and may subject the patients to an increased risk of acute or chronic rejection, especially if the C0 level is within the normal
range.44 Nevertheless, investigation of larger numbers of pediatric patients is needed to identify reliable target levels, which can only be achieved by a multicenter study. Poor absorption or fast elimination may occur more frequently in younger children or toddlers, where C2 blood level monitoring helps us to identify patients in whom immunosuppression needs to be changed to tacrolimus (Prograf, Fujisawa) to prevent ongoing acute rejection episodes or transplant coronary artery disease.45 The relatively small number of patients with rejection limits this prospective, observational trial. Clearly, larger numbers of pediatric heart recipients are necessary to evaluate a reliable cut-off point for C2 level in transplant recipients.44 In addition, prospective studies including patients in the early period after heart transplantation are necessary to evaluate the initial pharmacokinetics of pediatric patients. We cannot define different C2 target levels in relation to adjunctive medication (azathioprine or mycophenolate mofetil), because there were no significant differences in adjunctive medication between the 2 groups. Again, inclusion of a larger number of patients is needed to answer this particular question adequately. According to these results we cannot assume there will be a reduction in the incidence of transplant coronary artery disease with this regimen. Follow-up studies are necessary to evaluate C2 monitoring with regard to the development of coronary artery disease and long-term survival of the graft.46 In conclusion, C2 level monitoring is uncomplicated to perform in pediatric patients and is superior to C0 level measurement for adapting CsA dosage and for early identification of pediatric patients with inadequate immunosuppression, which may be explained by impaired enteric resorption and reduced bioavailability. A C2 level of ⬍600 ng/ml seems to predict acute graft rejection in pediatric heart transplant patients with high sensitivity and good specificity, which is in accordance with the data in adult heart transplant patients previ-
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ously reported by Chou et al.44 Thus, serial C2 rather than C0 monitoring identifies those patients at increased risk of inadequate immunosuppression. It may therefore help to reduce acute rejection and prolong graft survival in children after heart transplantation. The authors thank Anne Gale, ELS (Deutsches Herzzentrum Berlin), for editorial support and Julia Stein, MSc (Deutsches Herzzentrum Berlin), for help with statistical analysis of the data.
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29. Oellerich M, Armstrong VW. Two-hour cyclosporine concentration determination: an appropriate tool to monitor neoral therapy? Ther Drug Monit 2002;24:40 – 6. 30. Trompeter R, Fitzpatrick M, Hutchinson C, Johnston A. Longitudinal evaluation of the pharmacokinetics of cyclosporin microemulsion (Neoral) in pediatric renal transplant recipients and assessment of C2 level as a marker for absorption. Pediatr Transplant 2003;7:282– 8. 31. Hu RH, Tsai MK, Lee PH. Evaluation of cyclosporine C2 levels in long-term stable renal allograft recipients. Transplant Proc 2004;36:2105–7. 32. Oellerich M, Armstrong VW, Streit F, Weber L, Tonshoff B. Immunosuppressive drug monitoring of sirolimus and cyclosporine in pediatric patients. Clin Biochem 2004;37: 424 – 8. 33. Barakat O, Peaston R, Rai R, Talbot D, Manas D. Clinical benefit of monitoring cyclosporine C2 and C4 in longterm liver transplant recipients. Transplant Proc 2002;34: 1535–7. 34. Cantarovich M, Besner JG, Barkun JS, Elstein E, Loertscher R. Two-hour cyclosporine level determination is the appropriate tool to monitor Neoral therapy. Clin Transplant 1998;12:243–9. 35. Midtvedt K. Is C0 better than C2 as a determinant of rejection in renal transplant recipients? Kidney Int 2004; 66:869. 36. Morton JM, Aboyoun CL, Malouf MA, Plit ML, Glanville AR. Enhanced clinical utility of de novo cyclosporine C2 monitoring after lung transplantation. J Heart Lung Transplant 2004;23:1035–9. 37. Holt DW, Armstrong VW, Griesmacher A, et al. International Federation of Clinical Chemistry/International Association of Therapeutic Drug Monitoring and Clinical Toxicology working group on immunosuppressive drug monitoring. Ther Drug Monit 2002;24:59 – 67.
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38. Weber LT, Armstrong VW, Shipkova M, et al. Cyclosporin A absorption profiles in pediatric renal transplant recipients predict the risk of acute rejection. Ther Drug Monit 2004;26:415–24. 39. Pape L, Lehnhardt A, Latta K, Ehrich JH, Offner G. Cyclosporin A monitoring by 2-h levels: preliminary target levels in stable pediatric kidney transplant recipients. Clin Transplant 2003;17:546 – 8. 40. Teisseyre J, Markiewicz M, Drewniak T, et al. Switching cyclosporine blood concentration monitoring from C0 to C2 in children late after liver transplantation. Transplant Proc 2003;35:2287– 8. 41. Ganschow R, Richter A, Grabhorn E, et al. C2 blood concentrations of orally administered cyclosporine in pediatric liver graft recipients with a body weight below 10 kg. Pediatr Transplant 2004;8:185– 8. 42. Cantarovich M, Barkun J, Giannetti N, et al. History of C2 monitoring in heart and liver transplant patients treated with cyclosporine microemulsion. Transplant Proc 2004; 36(suppl):442S–7S. 43. Masini JP, Poirier JM, Weissenburger J, et al. Study of cyclosporin A blood level during the early post-livertransplantation period. Therapie 1993;48:163– 6. 44. Chou NK, Chen RJ, Ko WJ, et al. Cyclosporine C2 monitoring is superior to C0 in predicting acute cellular rejection in heart transplant recipients in Taiwan. Transplant Proc 2004;36:2393–5. 45. Barama A, Sepandj F, Gough J, McKenna R. Correlation between Neoral 2 hours postdose levels and histologic findings on surveillance biopsies. Transplant Proc 2004; 36(suppl):465S–7S. 46. Webber SA, Naftel DC, Parker J, et al. Late rejection episodes more than 1 year after pediatric heart transplantation: risk factors and outcomes. J Heart Lung Transplant 2003;22:869 –75.