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C4d Analysis in Endomyocardial Biopsies of Heart Transplant Patients: Is There a Correlation with Hemodynamic Data?
C4d Analysis in Endomyocardial Biopsies of Heart Transplant Patients: Is There a Correlation with Hemodynamic Data? M. Boffini, D. Ricci, R. Bonato, M. Ribezzo, E. Simonato, R. Saviolo, L. Checco, C. Comoglio, and M. Rinaldi ABSTRACT Background. Endomyocardial biopsy (EMB) is the gold standard for immunologic follow-up to detect acute cellular rejection after cardiac transplantation. Conversely, protocols for the diagnosis and treatment of antibody-mediated rejection (AMR) are not well defined. Histologically, AMR is diagnosed by the presence of capillary damage associated with complement activation. The aim of this study was to correlate C4d expression of activated complement in EMB with hemodynamic compromise upon right heart catheterization. Methods. Heart transplant patients underwent hemodynamic and histologic follow-up with EMB and right heart catheterization between January 2008 and December 2009 for a total of 491 procedures. The cardiac biopsy was evaluated for acute cellular and AMR by means of the presence of the C4d complement fraction. The histologic results were compared with hemodynamic data registered during right heart catheterization. Results. Comparison of the hemodynamic data of subjects with versus without C4d positivity showed no significant difference. Furthermore, there was no significant difference comparing patients with versus without C4d positivity in the absence of significant acute cellular rejection episodes. (C4d⫺/ACR⫺ vs C4d⫹/ACR⫺). The variation of each single hemodynamic parameter from its basal value (defined as the mean value in case of C4d⫺/ACR⫺) seemed to not be influenced by the presence of C4d⫹. Conclusions. In our experience, C4d has been routinely evaluated in the majority of EMBs. We could not demonstrate a significant correlation of C4d positivity with hemodynamic compromise. These findings suggest that significant allograft dysfunction is not related to C4d positivity. Therefore, the diagnosis of AMR is difficult to establish, because allograft dysfunction is 1 of the 3 fundamental criteria. n contrast to acute cellular rejection, antibody-mediated rejection (AMR) is not a well defined clinical entity regarding diagnosis and treatment.1 AMR is caused by antibodies directed against donor-specific human leukocyte antigens (HLAs), blood group antigen isoagglutinins, or endothelial cell antigens.2 Classically AMR is diagnosed by histologic features of myocardial capillary injury and complement activation associated with allograft dysfunction and the presence of circulating donor-specific antibodies (DSAs).1 Complement activation is demonstrated in endomyocardial biopsies (EMBs) by immunofluorence positivity or immunoperoxidase staining for C4d in capillaries.3-4 The aim of the present study was to investigate a correlation between C4d positivity on EMBs and hemodynamic data obtained at right heart catheterization.
I
MATERIALS AND METHODS We retrospectively reviewed the data of all 44 heart transplant patients, including 38 men and 6 women of overall mean age 52.1 ⫾ 13.14 years in the period between January 2008 and December 2009. All of the patients received induction therapy with antithymocyte globulin. Subsequent immunosuppression was based on a calcineurin inhibitor (cyclosporine or tacrolimus), an antiproliferaFrom the Cardiac Surgery Department (M.B., D.R., R.B., M.R., E.S., M.R.) and Cardiology Department (R.S., L.C.), San Giovanni Battista University Hospital; and Cardiac Surgery Department, Villa Maria Pia Hospital (C.C.), Turin, Italy. Address reprint requests to Prof. Mauro Rinaldi, Cardiac Surgery Department, San Giovanni Battista University Hospital, Corso Bramante 88, 10126 Turin, Italy. E-mail: mauro.rinaldi@ unito.it
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.03.021
© 2011 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
1168
Transplantation Proceedings, 43, 1168 –1170 (2011)
C4d AND HEMODYNAMIC DATA IN CARDIAC GRAFTS
1169
tive agent (mycophenolate mofetil or everolimus), and steroids. In the first year after transplantation, all of the patients underwent periodic hemodynamic and histologic follow-up with an EMB and a right heart catheterization. All histologic specimens were graded according to the International Society for Heart and Lung Transplantation Working Formulation to evaluate the presence of acute cellular rejection. Capillary involvement and immunochemistry for C4d analysis were also performed routinely to detect the presence of AMR regardless of the presence of hemodynamic compromise and/or circulating antibodies directed against donor HLA specificities. AMR episodes were classified according to the revision of the Working Formulation, and histologic AMR as 0 if there was no histologic or immunopathologic feature or 1 if there was immunofluorescence or immunoperoxidase staining for C4d. The present study received approval by our local Ethics Committee.
RESULTS
From January 2008 to December 2009, we performed 491 EMBs on a cohort of 44 heart transplant patients. In every EMB, we evaluated both cellular and antibody-mediated rejection. In 6 cases the specimen was not adequate for evaluation of complement deposition. Among the remaining 485 EMBs, 362 (74.6%) were C4d negative and 123 (25.4%) C4d positive; 37 subjects had (84%) ⱖ1 C4d positive specimen. The first positive staining for C4d was observed at a mean of 61.2 days after transplantation (range, 5-313 days). Acute cellular rejection episodes (⬎1R, ex 2 according to the old classification) occurred in 23 patients (52.3%) at a mean of 100.26 days after transplantation (range, 13–330 days). C4d positivity was associated with a significant acute cellular rejection process (C4d⫹/ ACR⫹) 12 patients (27.3%), whereas 25 patients (56.8%) displayed C4d positivity without concomittant acute cellular rejection processes (C4d⫹/ACR⫺). Comparisons between the hemodynamic data of patients with versus without C4d positivity (C4d⫺ vs C4d⫹) are shown in Table 1. No significant difference was observed between the 2 groups. Table 2 compares C4d⫺ versus C4d⫹ subjects who did not experience significant acute cellular rejection episodes (C4d⫺/ACR⫺ vs C4d⫹/ACR⫺), revealing no significant difference in terms of central venous pressure, mean pulmonary artery pressure, cardiac index, or wedge pressure (P ⫽ ns). Table 1. Comparison Between C4dⴚ and C4dⴙ Patients With and Without Acute Cellular Rejection
PAPs PAPd PAPm WP CO CI CVP
C4d⫺ (n ⫽ 362)
C4d⫹ (n ⫽ 123)
P Value
25.28 ⫾ 5.72 10.89 ⫾ 5.72 17.35 ⫾ 6.12 9.67 ⫾ 5.61 5.83 ⫾ 1.66 3.19 ⫾ 0.89 5.94 ⫾ 4.64
22.26 ⫾ 8.05 11.29 ⫾ 5.82 17.76 ⫾ 5.88 10.08 ⫾ 5.67 5.35 ⫾ 1.47 2.94 ⫾ 0.76 6.15 ⫾ 4.52
ns ns ns ns ns ns ns
Abbreviations: PAPs, systolic pulmonary artery pressure; PAPd, diastolic pulmonary artery pressure; PAPm, mean pulmonary artery pressure; WP, wedge pressure; CO, cardiac output; CI, cardiac index; CVP, central venous pressure.
Table 2. Comparison of Patients With and Without C4d Positivity in the Absence of Significant Acute Cellular Rejection (ACR)
PAPs PAPd PAPm WP CO CI CVP
C4d⫺/ACR⫺ (n ⫽ 317)
C4d⫹/ACR⫺ (n ⫽ 99)
P Value
25.29 ⫾ 7.15 10.96 ⫾ 5.61 17.41 ⫾ 6.02 9.69 ⫾ 5.54 5.88 ⫾ 1.66 3.21 ⫾ 0.89 6.04 ⫾ 4.45
26.48 ⫾ 8.35 11.41 ⫾ 5.88 17.82 ⫾ 6.03 10.34 ⫾ 5.87 5.32 ⫾ 1.48 2.91 ⫾ 0.77 6.39 ⫾ 4.61
ns ns ns ns ns ns ns
Abbreviations as in Table 1.
The analysis of variation of each single hemodynamic parameter from its basal value (defined as the mean value in case of C4d⫺/ACR⫺) showed no significant difference in diastolic, mean and systolic pulmonary pressure, wedge pressure, cardiac output and cardiac index, or central venous pressure variation among C4d⫹ cases (Table 3). DISCUSSION
Acute cellular rejection is usually diagnosed according to universally accepted protocols regardless of the presence of hemodynamic compromise.5 The diagnosis is mainly histologic,6 and the treatment is independent of allograft function. Conversely, the diagnosis, therapy, and monitoring of AMR are not standardized.1,7 Activation of the complement cascade resulting in tissue injury and coagulation plays a key role in AMR. As a matter of fact, active complement split products (C3, C4, C5) initiate vasoactive responses and are potent mediators of chemotaxis of neutrophils, monocytes, and macrophages.8 Histologically, the process is characterized by myocardial capillary injury, endothelial-cell swelling, intravascular macrophage accumulation, interstitial edema and hemorrhage, intravascular thrombi, and myocyte necrosis without cellular infiltration.6,9,10 Immunopathologic evidence of AMR is finally based on immunofluorence positivity or immunoperoxidase staining for C4d in capillaries.3,4,6,11,12 For the diagnosis of AMR, acute tissue injury with activation of complement and deposition of C4d and detection of circulating DSAs must be associated with the presence of allograft dysfunction.1 However, this term is general. It is not clear whether it refers to a clinical Table 3. Variation of Each Hemodynamic Parameter from Its Basal Value (Defined as the Mean Value in Case of C4dⴚ/ACRⴚ) in Case of C4d Positivity PAPs PAPd PAPm WP CO CI CVP
⫺2% ⫺20% ⫺7% ⫺26% ⫺2% ⫺4% ⫹13%
Abbreviations as in Tables 1 and 2.
P P P P P P P
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
ns ns ns ns ns ns ns
1170
evaluation (eg, New York Heart Association functional class or oxygen consumption [VO2]), or to an instrumental analysis. As far as the instrumental assessment is concerned, the parameters that must be considered are not clear: hemodynamic values such as filling pressures, cardiac output, pulmonary pressures, and/or echocardiographic parameters such as ejection fraction, global, and segmental contractility.13–15 Moreover, in a recent work, Wu et al16 observed that even asymptomatic AMR had a negative impact on survival and cardiac allograft vasculopathy. For these reasons, similarly to acute cellular rejection episodes, AMR may be considered to be a histopathologic entity with a continuous spectrum of clinical manifestations ranging from an asymptomatic condition to severe heart failure.1 In our study, C4d was routinely evaluated in all EMBs. Our analysis of all C4d⫹ biopsies did not demonstrate a significant impairment by hemodynamic data either by comparison of all C4d⫹ vs C4d⫺ EMBs or by significant modifications of the hemodynamic values in the same patient. These results showed that significant allograft dysfunction was not related to C4d positivity. Therefore the diagnosis of AMR is difficult to establish, because allograft dysfunction is 1 of the 3 fundamental criteria. Because the main limitation of the present study was the small number of subjects, a larger cohort is mandatory to support our conclusion. REFERENCES 1. Kfoury AG, Hammond ME: Controversies in defining cardiac antibody-mediated rejection: need for updated criteria. J Heart Lung Transplant 29:389, 2010 2. Uber WE, Self SE, van Bakel AB, et al: Acute antibodymediated rejection following heart transplantation. Am J Transpl 7:2064, 2007 3. Batal I, Girnita A, Zeevi A, et al: Clinical significance of the distribution of C4d deposits in different anatomic compartments of the allograft kidney. Mod Pathol 21:1490, 2008 4. Behr TM, Feucht HE, Richter K, et al: Detection of humoral rejection in human cardiac allografts by assessing the capillary
BOFFINI, RICCI, BONATO ET AL deposition of complement fragment C4d in endomyocardial biopsies. J Heart Lung Transplant 18:904, 1999 5. Kirklin JK, Bourge RC, Naftel DC, et al: Is there a need for surveillance endomyocardial biopsies in heart transplantation? Transplant Proc 34:1857, 2002 6. Stewart S, Winters GL, Fishbein MC, et al: Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant 24:1710, 2005 7. Takemoto SK, Zeevi A, Feng S, et al: National conference to assess antibody-mediated rejection in solid organ transplantation. Am J Transplant 4:1033, 2004 8. Wasowska BA, Lee CY, Halushka MK, et al: New concepts of complement in allorecognition and graft rejection. Cell Immunol 248:18, 2007 9. Hammond EH, Yowell RL, Nunoda S, et al: Vascular (humoral) rejection in heart transplantation: pathologic observations and clinical implications. J Heart Transplant 8:430, 1989 10. Lones MA, Czer LS, Trento A, et al: Clinical-pathologic features of humoral rejection in cardiac allografts: a study in 81 consecutive patients. J Heart Lung Transplant 14:151, 1995 11. Chantranuwat C, Qiao JH, Kobashigawa J, et al: Immunoperoxidase staining for C4d on paraffin-embedded tissue in cardiac allograft endomyocardial biopsies: comparison to frozen tissue immunofluorescence. Immunohistochem Mol Morphol 12:166, 2004 12. Tan CD, Sokos GG, Pidwell DJ, et al: Correlation of donor-specific antibodies, complement and its regulators with graft dysfunction in cardiac antibody-mediated rejection. Am J Transplant 9:2075, 2009 13. Mills RM, Naftel DC, Kirklin JK, et al: Heart transplant rejection with hemodynamic compromise: a multiinstitutional study of the role of endomyocardial cellular infiltrate. Cardiac Transplant Research Database. J Heart Lung Transplant 16:813, 1997 14. Michaels PJ, Espejo ML, Kobashigawa J, et al: Humoral rejection in cardiac transplantation: risk factors, hemodynamic consequences and relationship to transplant coronary artery disease. J Heart Lung Transplant 22:58, 2003 15. Crespo-Leiro MG, Veiga-Barreiro A, Doménech N, et al: Humoral heart rejection (severe allograft dysfunction with no signs of cellular rejection or ischemia): incidence, management, and the value of C4d for diagnosis. Am J Transplant 5:2560, 2005 16. Wu GW, Kobashigawa JA, Fishbein MC, et al: Asymptomatic antibody-mediated rejection after heart transplantation predicts poor outcomes. J Heart Lung Transplant 28:417, 2009
I
MATERIALS AND METHODS We retrospectively reviewed the data of all 44 heart transplant patients, including 38 men and 6 women of overall mean age 52.1 ⫾ 13.14 years in the period between January 2008 and December 2009. All of the patients received induction therapy with antithymocyte globulin. Subsequent immunosuppression was based on a calcineurin inhibitor (cyclosporine or tacrolimus), an antiproliferaFrom the Cardiac Surgery Department (M.B., D.R., R.B., M.R., E.S., M.R.) and Cardiology Department (R.S., L.C.), San Giovanni Battista University Hospital; and Cardiac Surgery Department, Villa Maria Pia Hospital (C.C.), Turin, Italy. Address reprint requests to Prof. Mauro Rinaldi, Cardiac Surgery Department, San Giovanni Battista University Hospital, Corso Bramante 88, 10126 Turin, Italy. E-mail: mauro.rinaldi@ unito.it
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.03.021
© 2011 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
1168
Transplantation Proceedings, 43, 1168 –1170 (2011)
C4d AND HEMODYNAMIC DATA IN CARDIAC GRAFTS
1169
tive agent (mycophenolate mofetil or everolimus), and steroids. In the first year after transplantation, all of the patients underwent periodic hemodynamic and histologic follow-up with an EMB and a right heart catheterization. All histologic specimens were graded according to the International Society for Heart and Lung Transplantation Working Formulation to evaluate the presence of acute cellular rejection. Capillary involvement and immunochemistry for C4d analysis were also performed routinely to detect the presence of AMR regardless of the presence of hemodynamic compromise and/or circulating antibodies directed against donor HLA specificities. AMR episodes were classified according to the revision of the Working Formulation, and histologic AMR as 0 if there was no histologic or immunopathologic feature or 1 if there was immunofluorescence or immunoperoxidase staining for C4d. The present study received approval by our local Ethics Committee.
RESULTS
From January 2008 to December 2009, we performed 491 EMBs on a cohort of 44 heart transplant patients. In every EMB, we evaluated both cellular and antibody-mediated rejection. In 6 cases the specimen was not adequate for evaluation of complement deposition. Among the remaining 485 EMBs, 362 (74.6%) were C4d negative and 123 (25.4%) C4d positive; 37 subjects had (84%) ⱖ1 C4d positive specimen. The first positive staining for C4d was observed at a mean of 61.2 days after transplantation (range, 5-313 days). Acute cellular rejection episodes (⬎1R, ex 2 according to the old classification) occurred in 23 patients (52.3%) at a mean of 100.26 days after transplantation (range, 13–330 days). C4d positivity was associated with a significant acute cellular rejection process (C4d⫹/ ACR⫹) 12 patients (27.3%), whereas 25 patients (56.8%) displayed C4d positivity without concomittant acute cellular rejection processes (C4d⫹/ACR⫺). Comparisons between the hemodynamic data of patients with versus without C4d positivity (C4d⫺ vs C4d⫹) are shown in Table 1. No significant difference was observed between the 2 groups. Table 2 compares C4d⫺ versus C4d⫹ subjects who did not experience significant acute cellular rejection episodes (C4d⫺/ACR⫺ vs C4d⫹/ACR⫺), revealing no significant difference in terms of central venous pressure, mean pulmonary artery pressure, cardiac index, or wedge pressure (P ⫽ ns). Table 1. Comparison Between C4dⴚ and C4dⴙ Patients With and Without Acute Cellular Rejection
PAPs PAPd PAPm WP CO CI CVP
C4d⫺ (n ⫽ 362)
C4d⫹ (n ⫽ 123)
P Value
25.28 ⫾ 5.72 10.89 ⫾ 5.72 17.35 ⫾ 6.12 9.67 ⫾ 5.61 5.83 ⫾ 1.66 3.19 ⫾ 0.89 5.94 ⫾ 4.64
22.26 ⫾ 8.05 11.29 ⫾ 5.82 17.76 ⫾ 5.88 10.08 ⫾ 5.67 5.35 ⫾ 1.47 2.94 ⫾ 0.76 6.15 ⫾ 4.52
ns ns ns ns ns ns ns
Abbreviations: PAPs, systolic pulmonary artery pressure; PAPd, diastolic pulmonary artery pressure; PAPm, mean pulmonary artery pressure; WP, wedge pressure; CO, cardiac output; CI, cardiac index; CVP, central venous pressure.
Table 2. Comparison of Patients With and Without C4d Positivity in the Absence of Significant Acute Cellular Rejection (ACR)
PAPs PAPd PAPm WP CO CI CVP
C4d⫺/ACR⫺ (n ⫽ 317)
C4d⫹/ACR⫺ (n ⫽ 99)
P Value
25.29 ⫾ 7.15 10.96 ⫾ 5.61 17.41 ⫾ 6.02 9.69 ⫾ 5.54 5.88 ⫾ 1.66 3.21 ⫾ 0.89 6.04 ⫾ 4.45
26.48 ⫾ 8.35 11.41 ⫾ 5.88 17.82 ⫾ 6.03 10.34 ⫾ 5.87 5.32 ⫾ 1.48 2.91 ⫾ 0.77 6.39 ⫾ 4.61
ns ns ns ns ns ns ns
Abbreviations as in Table 1.
The analysis of variation of each single hemodynamic parameter from its basal value (defined as the mean value in case of C4d⫺/ACR⫺) showed no significant difference in diastolic, mean and systolic pulmonary pressure, wedge pressure, cardiac output and cardiac index, or central venous pressure variation among C4d⫹ cases (Table 3). DISCUSSION
Acute cellular rejection is usually diagnosed according to universally accepted protocols regardless of the presence of hemodynamic compromise.5 The diagnosis is mainly histologic,6 and the treatment is independent of allograft function. Conversely, the diagnosis, therapy, and monitoring of AMR are not standardized.1,7 Activation of the complement cascade resulting in tissue injury and coagulation plays a key role in AMR. As a matter of fact, active complement split products (C3, C4, C5) initiate vasoactive responses and are potent mediators of chemotaxis of neutrophils, monocytes, and macrophages.8 Histologically, the process is characterized by myocardial capillary injury, endothelial-cell swelling, intravascular macrophage accumulation, interstitial edema and hemorrhage, intravascular thrombi, and myocyte necrosis without cellular infiltration.6,9,10 Immunopathologic evidence of AMR is finally based on immunofluorence positivity or immunoperoxidase staining for C4d in capillaries.3,4,6,11,12 For the diagnosis of AMR, acute tissue injury with activation of complement and deposition of C4d and detection of circulating DSAs must be associated with the presence of allograft dysfunction.1 However, this term is general. It is not clear whether it refers to a clinical Table 3. Variation of Each Hemodynamic Parameter from Its Basal Value (Defined as the Mean Value in Case of C4dⴚ/ACRⴚ) in Case of C4d Positivity PAPs PAPd PAPm WP CO CI CVP
⫺2% ⫺20% ⫺7% ⫺26% ⫺2% ⫺4% ⫹13%
Abbreviations as in Tables 1 and 2.
P P P P P P P
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
ns ns ns ns ns ns ns
1170
evaluation (eg, New York Heart Association functional class or oxygen consumption [VO2]), or to an instrumental analysis. As far as the instrumental assessment is concerned, the parameters that must be considered are not clear: hemodynamic values such as filling pressures, cardiac output, pulmonary pressures, and/or echocardiographic parameters such as ejection fraction, global, and segmental contractility.13–15 Moreover, in a recent work, Wu et al16 observed that even asymptomatic AMR had a negative impact on survival and cardiac allograft vasculopathy. For these reasons, similarly to acute cellular rejection episodes, AMR may be considered to be a histopathologic entity with a continuous spectrum of clinical manifestations ranging from an asymptomatic condition to severe heart failure.1 In our study, C4d was routinely evaluated in all EMBs. Our analysis of all C4d⫹ biopsies did not demonstrate a significant impairment by hemodynamic data either by comparison of all C4d⫹ vs C4d⫺ EMBs or by significant modifications of the hemodynamic values in the same patient. These results showed that significant allograft dysfunction was not related to C4d positivity. Therefore the diagnosis of AMR is difficult to establish, because allograft dysfunction is 1 of the 3 fundamental criteria. Because the main limitation of the present study was the small number of subjects, a larger cohort is mandatory to support our conclusion. REFERENCES 1. Kfoury AG, Hammond ME: Controversies in defining cardiac antibody-mediated rejection: need for updated criteria. J Heart Lung Transplant 29:389, 2010 2. Uber WE, Self SE, van Bakel AB, et al: Acute antibodymediated rejection following heart transplantation. Am J Transpl 7:2064, 2007 3. Batal I, Girnita A, Zeevi A, et al: Clinical significance of the distribution of C4d deposits in different anatomic compartments of the allograft kidney. Mod Pathol 21:1490, 2008 4. Behr TM, Feucht HE, Richter K, et al: Detection of humoral rejection in human cardiac allografts by assessing the capillary
BOFFINI, RICCI, BONATO ET AL deposition of complement fragment C4d in endomyocardial biopsies. J Heart Lung Transplant 18:904, 1999 5. Kirklin JK, Bourge RC, Naftel DC, et al: Is there a need for surveillance endomyocardial biopsies in heart transplantation? Transplant Proc 34:1857, 2002 6. Stewart S, Winters GL, Fishbein MC, et al: Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant 24:1710, 2005 7. Takemoto SK, Zeevi A, Feng S, et al: National conference to assess antibody-mediated rejection in solid organ transplantation. Am J Transplant 4:1033, 2004 8. Wasowska BA, Lee CY, Halushka MK, et al: New concepts of complement in allorecognition and graft rejection. Cell Immunol 248:18, 2007 9. Hammond EH, Yowell RL, Nunoda S, et al: Vascular (humoral) rejection in heart transplantation: pathologic observations and clinical implications. J Heart Transplant 8:430, 1989 10. Lones MA, Czer LS, Trento A, et al: Clinical-pathologic features of humoral rejection in cardiac allografts: a study in 81 consecutive patients. J Heart Lung Transplant 14:151, 1995 11. Chantranuwat C, Qiao JH, Kobashigawa J, et al: Immunoperoxidase staining for C4d on paraffin-embedded tissue in cardiac allograft endomyocardial biopsies: comparison to frozen tissue immunofluorescence. Immunohistochem Mol Morphol 12:166, 2004 12. Tan CD, Sokos GG, Pidwell DJ, et al: Correlation of donor-specific antibodies, complement and its regulators with graft dysfunction in cardiac antibody-mediated rejection. Am J Transplant 9:2075, 2009 13. Mills RM, Naftel DC, Kirklin JK, et al: Heart transplant rejection with hemodynamic compromise: a multiinstitutional study of the role of endomyocardial cellular infiltrate. Cardiac Transplant Research Database. J Heart Lung Transplant 16:813, 1997 14. Michaels PJ, Espejo ML, Kobashigawa J, et al: Humoral rejection in cardiac transplantation: risk factors, hemodynamic consequences and relationship to transplant coronary artery disease. J Heart Lung Transplant 22:58, 2003 15. Crespo-Leiro MG, Veiga-Barreiro A, Doménech N, et al: Humoral heart rejection (severe allograft dysfunction with no signs of cellular rejection or ischemia): incidence, management, and the value of C4d for diagnosis. Am J Transplant 5:2560, 2005 16. Wu GW, Kobashigawa JA, Fishbein MC, et al: Asymptomatic antibody-mediated rejection after heart transplantation predicts poor outcomes. J Heart Lung Transplant 28:417, 2009