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Interstitial Leukocytes in Right Ventricular Endomyocardial Biopsies After Heart Transplantation in Patients With Complicated Versus Uneventful Postoperative Course
Interstitial Leukocytes in Right Ventricular Endomyocardial Biopsies After Heart Transplantation in Patients With Complicated Versus Uneventful Postoperative Course A. Koch, S. Feucht, B.M. Helmke, T.J. Dengler, M. Haass, F.-U. Sack, M. Karck, and P.A. Schnabel ABSTRACT Backround. Infections and rejections play key roles in morbidity and mortality in the early postoperative period after orthotopic heart transplantation (HTX). The aim of this study was to evaluate whether qualitative and quantitative analyses of various interstitial leukocytes in endomyocardial biopsies during the first 2 weeks after HTX provided early information on these complications. Patients and Methods. During and after HTX, endomyocardial biopsies were obtained in 51 patients. By immunohistochemistry we determined the CD3-, CD4-, CD8-, CD15-, CD20-, CD57-, and CD68-positive cell numbers projected to planimetrically measured areas. To compare morbidity in the postoperative course, the patients were subdivided into complicated versus uncomplicated after 3 months. Results. In the uncomplicated group, the cell counts of CD3-, CD8-, CD57-, and CD68-positive cells were significantly lower than in the complicated group. CD3-, CD4-, and CD8-positive cell numbers showed a significant decrease in the first week among the uncomplicated group. In the complicated group, the cell counts increased significantly in the second week. The numbers of CD57-positive cells were significantly lower during the first and second weeks among the uncomplicated group. Conclusions. Increased T lymphocytes, natural killer cells, and macrophages observed in the second week after HTX indicated increased morbidity. A reduction in CD3-positive cells in the first week indicated a low morbidity risk; an increase indicated a higher risk.
T
HE EARLY POSTOPERATIVE period after clinical heart transplantation (HTX) is complicated by acute infections, rejections, and early right heart failure. An infiltration of the myocardium with various leukocyte subtypes may be important for these processes.1 Leukocytes are classified by their surface antigens into clusters of differentiation (CD).2 A systematic analysis of various leukocyte subgroups during the early perioperative period after HTX has not been undertaken. The aim of this study was to analyze the quantity and differentiation of immunohistochemically characterized leukocytes per area in right ventricular endomyocardial biopsies (EMB) during the early perioperative period after HTX. Furthermore, we examined whether characterization of leukocyte differentiation in EMB was predictive to classify patients with complicated or clinically uneventful courses. © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 947–950 (2008)
PATIENTS AND METHODS EMB from 51 recipients of mean age 51 years (range, 13– 68 years), including 36 males, were obtained during the posttransplantation course: EMB 0 at implantation and/or during reperfusion, and at 1, 2, 3 weeks thereafter (Table 1). In 23 patients, 3 EMB were available; in 21, an additional fourth EMB. In 2 patients, only 2 EMB were available. Four patients had only EMB 0 and 1 patient only EMB 0 and 1. According to the postoperative course, 2 patient From the Department of Cardiac Surgery (A.K., F.-U.S., M.K.), Institute of Pathology (S.F., B.M.H., P.A.S.), Department of Cardiology (T.J.D.), and Department of Cardiology, TheresienHospital-Mannheim (M.H.), University of Heidelberg, Heidelberg, Germany. Address reprint requests to Achim Koch, MD, Department of Cardiac Surgery, University of Heidelberg, INF 110, D-69120 Heidelberg, Germany. E-mail: [email protected] 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.03.035 947
948
KOCH, FEUCHT, HELMKE ET AL Table 1. Results of the Different EMB for the Uncomplicated and Complicated Groups* P
Uncomplicated Complicated CD3 Uncomplicated Complicated P CD4 Uncomplicated Complicated P CD8 Uncomplicated Complicated P CD15 Uncomplicated Complicated P CD20 Uncomplicated Complicated P CD57 Uncomplicated Complicated P CD68 Uncomplicated Complicated P
EMB 0 vs EMB at 1 Week
EMB at 1 Week vs EMB at 2 Weeks
EMB at 2 Weeks vs EMB at 3 Weeks
EMB0
EMB at 1 Week
EMB at 2 Weeks
EMB at 3 Weeks
n ⫽ 17 n ⫽ 28
n ⫽ 18 n ⫽ 28
n ⫽ 18 n ⫽ 28
n⫽9 n ⫽ 12
5 (0–17) 6 (0–21) NS
1 (0–7) 2 (0–122) NS
2 (0–30) 7 (0–328) .022
9 (1–81) 7 (1–216) NS
.001 NS
NS .004
NS NS
1 (0–7) 2 (0–7) NS
0 (0–5) 0 (0–63) NS
1 (0–14) 2 (0–123) NS
2 (0–21) 5 (0–170) NS
.011 NS
NS .037
NS NS
3 (0–11) 2 (0–11) NS
0 (0–4) 1 (0–66) NS
1 (0–20) 4 (0–127) .046
5 (1–57) 4 (0–109) NS
.001 NS
.031 .003
NS NS
4 (0–10) 7 (0–20) NS
10 (0–18) 6 (0–47) NS
7 (1–16) 6 (1–20) NS
2 (0–5) 3 (0–21) NS
.013 NS
.039 NS
.008 NS
0 (0–0) 0 (0–0) NS
0 (0–2) 0 (0–28) NS
0 (0–1) 2 (0–24) NS
0 (0–10) 0 (0–147) NS
NS .012
NS NS
NS NS
1 (0–3) 1 (0–8) NS
1 (0–7) 1 (0–34) .037
1 (0–6) 2 (0–24) .026
4 (0–20) 3 (0–18) NS
NS NS
NS NS
NS .028
75 (46–145) 106 (37–199) NS
68 (28–138) 125 (9–307) .001
66 (29–184) 120 (47–261) NS
.003 .001
NS .016
NS NS
53 (18–110) 49 (3–135) NS
NS, not significant. *The number of cells is given per mm2.
groups were formed: The 18 patients with uncomplicated clinical courses, including 12 males showed no rejection above grade 1R and no systemic infection. Their mean age was 49 years (range, 14 – 63 years). The 29 patients with complicated clinical courses, namely, rejection above grade 1R and/or acute febrile systemic infection were of mean age 51 years (range, 13– 65 years). The cohort included 13 men. There were 8 patients with infection, 7 with rejection, and 14 with rejection and infection. The 4 patients who died were excluded. The investigation period was 6 months after HTX. As 4 recipients died during the first 2 weeks after HTX, only the EMB that were obtained during HTX were included for the mortality examination. For the mortality study the patients were divided into 2 groups: 9 patients who died within half a year after HTX (these subjects showed a mean age of 56 years [range, 43– 68 years], including 8 males, causes of death were right heart failure, multiorgan failure, and ventricular fibrillation); and 42 patients who survived 6 months after HTX (their mean age 50 years [range, 14 – 64 years], including 28 males). All patients received an immunosuppressive protocol of antithymocyte induction therapy, cyclosporine, azathioprine, and prednisolone. During implantation right ventricular trabecules were obtained by scissors. Routine weekly EMB harvested by biotome were fixed in 4% formalin, dehydrated, hardened in acetone, and embedded into paraplast. For determination of CD57 3 m frozen sections were cut. For
immunohistochemical staining the sections were deparaffinized. For evaluation of CD3, CD8, CD15, CD20, and CD68, the sections were incubated in citrate buffer; for CD4, in EDTA buffer (pH 8); and for CD57, in pepsin. After processing with ␥-globulin (Leukon, Vienna, Austria), the samples were incubated with the primary antibody, blocked with goat serum, incubated in a biotin-marked secondary antibody, and finally in streptavidin (BioGenex, San Ramon, Calif, United States) and stained with Fast-Red (DAKO, Carpinteria, Calif, United States). The following primary antibodies were used: CD3 monoclonal rat antibody (Serotec, United Kingdom); CD4 monoclonal mouse antibody (Loxo/Novocastra, United States); CD8, CD15, CD20, and CD68 monoclonal mouse antibodies (Dako, Denmark); and CD57 monoclonal mouse antibody (Zymed, United States). For the secondary antibody we used: CD3 anti-goat antibody (Zymed, United States) or CD4, CD8, CD15, CD20, CD57, and CD68 anti-goat antibody (BioGenex, United States). The antibody-marked cells were quantitated by light microscopy at ⫻250 magnification. The area estimation was undertaken by a semiautomatic imaging analysis system (Kontron, Germany). The number of antibody-marked cells was calculated per mm2 (number of cells/mm2). The data are shown as median, first, third quartile, minimal, and maximal values. The Wilcoxon matched paired signed rank, the Kruskal-Wallis, and the WilcoxonMann-Whitney U tests were applied as appropriate.
LEUKOCYTE DIFFERENTIATION IN EMB AFTER HTX
RESULTS
CD3-positive cells significantly decreased (P ⬍ .01) after the first week in the uncomplicated group. Among the complicated group it increased significantly (P ⬍ .01) during the second week. After the second week the values were significantly higher (P ⬍ .01) among the complicated than the uncomplicated group. During the first week CD4 significantly decreased in the uncomplicated group (P ⬎ .01). In the second week the CD4 cell count increased significantly (P ⬍ .05) in the complicated group. During the first postoperative week the time course of CD8-positive cells showed a significant decrease in both groups (P ⬍ .01) followed by a significant increase in both groups (P ⬍ .05) in the second week. The complicated group showed significantly higher values (P ⬍ .05). The values for CD15positive cells were constantly low among the complicated collective. For the uncomplicated group the values increased significantly (P ⬍ .05) in the first week followed by a significant decrease (P ⬍ .05) during the second. Both groups showed low cell counts for the CD20-positive cells; in the complicated group an increase was significant (P ⬍ .05) during the first week. The values for the CD57-positive cells were constantly low in the uncomplicated group with the values for the complicated group being slightly but significantly higher, especially in the second week (P ⬍ .05). In both groups the CD68-positive cells significantly increased in the first week (complicated group, P ⬍ .01; uncomplicated group, P ⬍ .01), and significantly further (P ⬍ .05) among the complicated group during the second week. After 2 weeks the values in the uncomplicated group were significantly lower than those in the complicated cohort (P ⬍ .01). Regarding third week biopsies, results were available from 9 uncomplicated and 12 complicated patients. The values for CD3, CD4, CD8, and CD20 increased nonsignificantly in both groups from the second to the third postoperative weeks. Both groups showed decreasing values for CD15-positive cells, which were significant for the uncomplicated group (P ⬍ .01). CD57 showed a significant increase among the uncomplicated group. CD68 showed no relevant differences among the groups. DISCUSSION
The long-term course after HTX is significantly influenced by rejections and infections. Therefore the opportunity to obtain information about preclinical signs of rejection and infection would be a desirable option. A previous retrospective study in patients with rejection episodes showed that within the first postoperative year there were higher numbers of CD3-positive interstitial T lymphocytes in early postoperative biopsies than among patients with uncomplicated courses. The numbers of CD3-positive T cells and of CD68-positive macrophages were elevated among patients with later infections.3 The aim of the present prospective study was to investigate whether integration of an immunohistochemical examination for different leukocyte popula-
949
tions in EMB of transplant recipients provides additional information to the routine diagnostics. The following leukocyte markers were examined for information about the further postoperative course. CD3 (T lymphocytes) as effector cells of cellular rejections are expected to increase during acute rejection episodes. Furthermore, they seem to be elevated during infections in the myocardium.3 The significant difference in their content in the initial EMB showed that high initial values represented a risk factor for early and midterm mortality. On examination of the morbidity a dip in the T-lymphocyte course was remarkable in the first week biopsy. This may be caused by T-lymphocyte suppression by immunosuppression, especially induction therapy with antithymocyte globulin (ATG).4 Interestingly, the values for CD3-positive cells were generally elevated in all EMB in recipients with complicated postoperative courses. In the uncomplicated group values decreased in the first week and significantly increased in the second week. Thus, the values after the second week were significantly higher among the complicated versus the uncomplicated group. Elevated initial values for CD3-positive cells can be interpreted as the result of processes that attracted T cells during brain death or damage that occurred before transplantation related to ischemia, implantation, and reperfusion.5 In summary, CD3 seems to be an interesting marker indicating the possibility of early complications like death, rejection, and infection and therefore allowing early diagnosis and intervention. The CD4 (T-helper cells) and CD8 (cytotoxic T cells) subpopulations showed the same tendency as CD3. There was a significant decrease after the first week in the uncomplicated group and a significant increase in the complicated group after the second week. Additionally, CD8 showed a significant increase in the uncomplicated group after the second week. However, the level of significance was lower than that of CD3. Thus, CD4 and CD8 did not have the same statistical significance and only confirmed the CD3 results. CD20-positive cells were found rarely and mainly among recipients with complicated courses. They participate in humoral rejections.7,8 The predictive value of this marker seems to be limited. The values of CD57 were not significantly different, remaining constantly low among the uncomplicated group and increasing slightly in the complicated group which showed significantly higher absolute values. Thus, CD57 may provide an early discrimination between recipients with an uncomplicated versus complicated postoperative course. The CD15 (neutrophilic granulocytes) marker showed a significant increase among the uncomplicated group in the first week and a significant decrease during the second week. The later course during the second week differentiated CD68 into 2 directions: a decrease in the uncomplicated group and a further significant increase in the complicated group. Values in the complicated group were significantly higher than those in the uncomplicated cohort. Macro-
950
phages are rarely found in normal myocardium.9 The increase in CD15 and CD68 in the first week can be interpreted as a sign of ongoing repair processes after ischemia- reperfusion injuries.5,6 Furthermore, the suppression of the T-cell response by immunosuppression can cause a compensatory proliferation of non-T cells, eg, neutrophils and macrophages. Higher values of macrophages after the second week can be seen as a marker for increased morbidity, whereas decreasing values seem to point to a benign subsequent course. Each of the T- and B-lymphocyte markers displayed a nonsignificant increase in both groups during the third week. The decrease in CD15 in both groups in the third week was significant among the uncomplicated group, which may represent the end of the repair processes during the third week after an initial ischemia-reperfusion injury. In conclusion, a remarkable decrease among infiltrating T lymphocytes during the first postoperative week seemed to indicate a less complicated course, whereas an increase in the second week indicated a more complicated course. High initial values of CD3-positive cells may indicate an increased risk of mortality in the first year after HTX. A lower count of CD15-positive cells in the second week pointed to an uncomplicated course. Greater numbers of CD57- and CD68-positive cells were observed in increasing numbers among patients with complicated courses. Thus quantitative examination of myocardial infiltrates of CD3, CD15, CD57,
KOCH, FEUCHT, HELMKE ET AL
and CD68 may be predictive of a complicated postoperative course. REFERENCES 1. Winters GL: The pathology of heart allograft rejection. Arch Pathol Lab Med 115:266, 1991 2. Bernard A, Boumsell L: Human leucocyte differentiation antigens. Press Med 13:2311, 1984 3. Schnabel PA, Sykora J, Becker K, et al: Predictive value of infiltrating cells in endomyocardial biopsies during the first two weeks after heart transplantation. 14th Annual Meeting of the European Association of Cardio-Thoracic Surgeons. Abstracts 2000, 578 4. Koch A, Daniel V, Dengler TJ, et al: Effectivity of a T-cell adapted induction-therapy with antithymocyte globulin (ATG) Sangstat. J Heart Lung Transplant 24:708, 2005 5. Kirklin JK, McGiffin DC: Control of the inflammatory response in extended myocardial preservation of the donor heart. Ann Thorac Surg 68:1978, 1999 6. Billingham ME: Cardiac transplantation. In Sale GE (ed): The Pathology of Organ Transplantation, 1st Ed. Boston: Butterworths; 1990, p 133 7. Aranda JM Jr, Scornik JC, Normann SJ, et al: Anti-CD20 monoclonal antibody (rituximab) therapy for acute cardiac humoral rejection: a case report. Transplantation 73:907, 2002 8. Michels PJ, Fishbein MC, Colvin RB: Humoral rejection of human organ transplants. Springer Semin Immunopathol 25:119, 2003 9. Holzinger C, Zuckermann A, Reinwald C, et al: Are T cells from healthy heart really only passengers? Characterization of cardiac tissue T cells. Immunol Lett 53:63, 1992
T
HE EARLY POSTOPERATIVE period after clinical heart transplantation (HTX) is complicated by acute infections, rejections, and early right heart failure. An infiltration of the myocardium with various leukocyte subtypes may be important for these processes.1 Leukocytes are classified by their surface antigens into clusters of differentiation (CD).2 A systematic analysis of various leukocyte subgroups during the early perioperative period after HTX has not been undertaken. The aim of this study was to analyze the quantity and differentiation of immunohistochemically characterized leukocytes per area in right ventricular endomyocardial biopsies (EMB) during the early perioperative period after HTX. Furthermore, we examined whether characterization of leukocyte differentiation in EMB was predictive to classify patients with complicated or clinically uneventful courses. © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 947–950 (2008)
PATIENTS AND METHODS EMB from 51 recipients of mean age 51 years (range, 13– 68 years), including 36 males, were obtained during the posttransplantation course: EMB 0 at implantation and/or during reperfusion, and at 1, 2, 3 weeks thereafter (Table 1). In 23 patients, 3 EMB were available; in 21, an additional fourth EMB. In 2 patients, only 2 EMB were available. Four patients had only EMB 0 and 1 patient only EMB 0 and 1. According to the postoperative course, 2 patient From the Department of Cardiac Surgery (A.K., F.-U.S., M.K.), Institute of Pathology (S.F., B.M.H., P.A.S.), Department of Cardiology (T.J.D.), and Department of Cardiology, TheresienHospital-Mannheim (M.H.), University of Heidelberg, Heidelberg, Germany. Address reprint requests to Achim Koch, MD, Department of Cardiac Surgery, University of Heidelberg, INF 110, D-69120 Heidelberg, Germany. E-mail: [email protected] 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.03.035 947
948
KOCH, FEUCHT, HELMKE ET AL Table 1. Results of the Different EMB for the Uncomplicated and Complicated Groups* P
Uncomplicated Complicated CD3 Uncomplicated Complicated P CD4 Uncomplicated Complicated P CD8 Uncomplicated Complicated P CD15 Uncomplicated Complicated P CD20 Uncomplicated Complicated P CD57 Uncomplicated Complicated P CD68 Uncomplicated Complicated P
EMB 0 vs EMB at 1 Week
EMB at 1 Week vs EMB at 2 Weeks
EMB at 2 Weeks vs EMB at 3 Weeks
EMB0
EMB at 1 Week
EMB at 2 Weeks
EMB at 3 Weeks
n ⫽ 17 n ⫽ 28
n ⫽ 18 n ⫽ 28
n ⫽ 18 n ⫽ 28
n⫽9 n ⫽ 12
5 (0–17) 6 (0–21) NS
1 (0–7) 2 (0–122) NS
2 (0–30) 7 (0–328) .022
9 (1–81) 7 (1–216) NS
.001 NS
NS .004
NS NS
1 (0–7) 2 (0–7) NS
0 (0–5) 0 (0–63) NS
1 (0–14) 2 (0–123) NS
2 (0–21) 5 (0–170) NS
.011 NS
NS .037
NS NS
3 (0–11) 2 (0–11) NS
0 (0–4) 1 (0–66) NS
1 (0–20) 4 (0–127) .046
5 (1–57) 4 (0–109) NS
.001 NS
.031 .003
NS NS
4 (0–10) 7 (0–20) NS
10 (0–18) 6 (0–47) NS
7 (1–16) 6 (1–20) NS
2 (0–5) 3 (0–21) NS
.013 NS
.039 NS
.008 NS
0 (0–0) 0 (0–0) NS
0 (0–2) 0 (0–28) NS
0 (0–1) 2 (0–24) NS
0 (0–10) 0 (0–147) NS
NS .012
NS NS
NS NS
1 (0–3) 1 (0–8) NS
1 (0–7) 1 (0–34) .037
1 (0–6) 2 (0–24) .026
4 (0–20) 3 (0–18) NS
NS NS
NS NS
NS .028
75 (46–145) 106 (37–199) NS
68 (28–138) 125 (9–307) .001
66 (29–184) 120 (47–261) NS
.003 .001
NS .016
NS NS
53 (18–110) 49 (3–135) NS
NS, not significant. *The number of cells is given per mm2.
groups were formed: The 18 patients with uncomplicated clinical courses, including 12 males showed no rejection above grade 1R and no systemic infection. Their mean age was 49 years (range, 14 – 63 years). The 29 patients with complicated clinical courses, namely, rejection above grade 1R and/or acute febrile systemic infection were of mean age 51 years (range, 13– 65 years). The cohort included 13 men. There were 8 patients with infection, 7 with rejection, and 14 with rejection and infection. The 4 patients who died were excluded. The investigation period was 6 months after HTX. As 4 recipients died during the first 2 weeks after HTX, only the EMB that were obtained during HTX were included for the mortality examination. For the mortality study the patients were divided into 2 groups: 9 patients who died within half a year after HTX (these subjects showed a mean age of 56 years [range, 43– 68 years], including 8 males, causes of death were right heart failure, multiorgan failure, and ventricular fibrillation); and 42 patients who survived 6 months after HTX (their mean age 50 years [range, 14 – 64 years], including 28 males). All patients received an immunosuppressive protocol of antithymocyte induction therapy, cyclosporine, azathioprine, and prednisolone. During implantation right ventricular trabecules were obtained by scissors. Routine weekly EMB harvested by biotome were fixed in 4% formalin, dehydrated, hardened in acetone, and embedded into paraplast. For determination of CD57 3 m frozen sections were cut. For
immunohistochemical staining the sections were deparaffinized. For evaluation of CD3, CD8, CD15, CD20, and CD68, the sections were incubated in citrate buffer; for CD4, in EDTA buffer (pH 8); and for CD57, in pepsin. After processing with ␥-globulin (Leukon, Vienna, Austria), the samples were incubated with the primary antibody, blocked with goat serum, incubated in a biotin-marked secondary antibody, and finally in streptavidin (BioGenex, San Ramon, Calif, United States) and stained with Fast-Red (DAKO, Carpinteria, Calif, United States). The following primary antibodies were used: CD3 monoclonal rat antibody (Serotec, United Kingdom); CD4 monoclonal mouse antibody (Loxo/Novocastra, United States); CD8, CD15, CD20, and CD68 monoclonal mouse antibodies (Dako, Denmark); and CD57 monoclonal mouse antibody (Zymed, United States). For the secondary antibody we used: CD3 anti-goat antibody (Zymed, United States) or CD4, CD8, CD15, CD20, CD57, and CD68 anti-goat antibody (BioGenex, United States). The antibody-marked cells were quantitated by light microscopy at ⫻250 magnification. The area estimation was undertaken by a semiautomatic imaging analysis system (Kontron, Germany). The number of antibody-marked cells was calculated per mm2 (number of cells/mm2). The data are shown as median, first, third quartile, minimal, and maximal values. The Wilcoxon matched paired signed rank, the Kruskal-Wallis, and the WilcoxonMann-Whitney U tests were applied as appropriate.
LEUKOCYTE DIFFERENTIATION IN EMB AFTER HTX
RESULTS
CD3-positive cells significantly decreased (P ⬍ .01) after the first week in the uncomplicated group. Among the complicated group it increased significantly (P ⬍ .01) during the second week. After the second week the values were significantly higher (P ⬍ .01) among the complicated than the uncomplicated group. During the first week CD4 significantly decreased in the uncomplicated group (P ⬎ .01). In the second week the CD4 cell count increased significantly (P ⬍ .05) in the complicated group. During the first postoperative week the time course of CD8-positive cells showed a significant decrease in both groups (P ⬍ .01) followed by a significant increase in both groups (P ⬍ .05) in the second week. The complicated group showed significantly higher values (P ⬍ .05). The values for CD15positive cells were constantly low among the complicated collective. For the uncomplicated group the values increased significantly (P ⬍ .05) in the first week followed by a significant decrease (P ⬍ .05) during the second. Both groups showed low cell counts for the CD20-positive cells; in the complicated group an increase was significant (P ⬍ .05) during the first week. The values for the CD57-positive cells were constantly low in the uncomplicated group with the values for the complicated group being slightly but significantly higher, especially in the second week (P ⬍ .05). In both groups the CD68-positive cells significantly increased in the first week (complicated group, P ⬍ .01; uncomplicated group, P ⬍ .01), and significantly further (P ⬍ .05) among the complicated group during the second week. After 2 weeks the values in the uncomplicated group were significantly lower than those in the complicated cohort (P ⬍ .01). Regarding third week biopsies, results were available from 9 uncomplicated and 12 complicated patients. The values for CD3, CD4, CD8, and CD20 increased nonsignificantly in both groups from the second to the third postoperative weeks. Both groups showed decreasing values for CD15-positive cells, which were significant for the uncomplicated group (P ⬍ .01). CD57 showed a significant increase among the uncomplicated group. CD68 showed no relevant differences among the groups. DISCUSSION
The long-term course after HTX is significantly influenced by rejections and infections. Therefore the opportunity to obtain information about preclinical signs of rejection and infection would be a desirable option. A previous retrospective study in patients with rejection episodes showed that within the first postoperative year there were higher numbers of CD3-positive interstitial T lymphocytes in early postoperative biopsies than among patients with uncomplicated courses. The numbers of CD3-positive T cells and of CD68-positive macrophages were elevated among patients with later infections.3 The aim of the present prospective study was to investigate whether integration of an immunohistochemical examination for different leukocyte popula-
949
tions in EMB of transplant recipients provides additional information to the routine diagnostics. The following leukocyte markers were examined for information about the further postoperative course. CD3 (T lymphocytes) as effector cells of cellular rejections are expected to increase during acute rejection episodes. Furthermore, they seem to be elevated during infections in the myocardium.3 The significant difference in their content in the initial EMB showed that high initial values represented a risk factor for early and midterm mortality. On examination of the morbidity a dip in the T-lymphocyte course was remarkable in the first week biopsy. This may be caused by T-lymphocyte suppression by immunosuppression, especially induction therapy with antithymocyte globulin (ATG).4 Interestingly, the values for CD3-positive cells were generally elevated in all EMB in recipients with complicated postoperative courses. In the uncomplicated group values decreased in the first week and significantly increased in the second week. Thus, the values after the second week were significantly higher among the complicated versus the uncomplicated group. Elevated initial values for CD3-positive cells can be interpreted as the result of processes that attracted T cells during brain death or damage that occurred before transplantation related to ischemia, implantation, and reperfusion.5 In summary, CD3 seems to be an interesting marker indicating the possibility of early complications like death, rejection, and infection and therefore allowing early diagnosis and intervention. The CD4 (T-helper cells) and CD8 (cytotoxic T cells) subpopulations showed the same tendency as CD3. There was a significant decrease after the first week in the uncomplicated group and a significant increase in the complicated group after the second week. Additionally, CD8 showed a significant increase in the uncomplicated group after the second week. However, the level of significance was lower than that of CD3. Thus, CD4 and CD8 did not have the same statistical significance and only confirmed the CD3 results. CD20-positive cells were found rarely and mainly among recipients with complicated courses. They participate in humoral rejections.7,8 The predictive value of this marker seems to be limited. The values of CD57 were not significantly different, remaining constantly low among the uncomplicated group and increasing slightly in the complicated group which showed significantly higher absolute values. Thus, CD57 may provide an early discrimination between recipients with an uncomplicated versus complicated postoperative course. The CD15 (neutrophilic granulocytes) marker showed a significant increase among the uncomplicated group in the first week and a significant decrease during the second week. The later course during the second week differentiated CD68 into 2 directions: a decrease in the uncomplicated group and a further significant increase in the complicated group. Values in the complicated group were significantly higher than those in the uncomplicated cohort. Macro-
950
phages are rarely found in normal myocardium.9 The increase in CD15 and CD68 in the first week can be interpreted as a sign of ongoing repair processes after ischemia- reperfusion injuries.5,6 Furthermore, the suppression of the T-cell response by immunosuppression can cause a compensatory proliferation of non-T cells, eg, neutrophils and macrophages. Higher values of macrophages after the second week can be seen as a marker for increased morbidity, whereas decreasing values seem to point to a benign subsequent course. Each of the T- and B-lymphocyte markers displayed a nonsignificant increase in both groups during the third week. The decrease in CD15 in both groups in the third week was significant among the uncomplicated group, which may represent the end of the repair processes during the third week after an initial ischemia-reperfusion injury. In conclusion, a remarkable decrease among infiltrating T lymphocytes during the first postoperative week seemed to indicate a less complicated course, whereas an increase in the second week indicated a more complicated course. High initial values of CD3-positive cells may indicate an increased risk of mortality in the first year after HTX. A lower count of CD15-positive cells in the second week pointed to an uncomplicated course. Greater numbers of CD57- and CD68-positive cells were observed in increasing numbers among patients with complicated courses. Thus quantitative examination of myocardial infiltrates of CD3, CD15, CD57,
KOCH, FEUCHT, HELMKE ET AL
and CD68 may be predictive of a complicated postoperative course. REFERENCES 1. Winters GL: The pathology of heart allograft rejection. Arch Pathol Lab Med 115:266, 1991 2. Bernard A, Boumsell L: Human leucocyte differentiation antigens. Press Med 13:2311, 1984 3. Schnabel PA, Sykora J, Becker K, et al: Predictive value of infiltrating cells in endomyocardial biopsies during the first two weeks after heart transplantation. 14th Annual Meeting of the European Association of Cardio-Thoracic Surgeons. Abstracts 2000, 578 4. Koch A, Daniel V, Dengler TJ, et al: Effectivity of a T-cell adapted induction-therapy with antithymocyte globulin (ATG) Sangstat. J Heart Lung Transplant 24:708, 2005 5. Kirklin JK, McGiffin DC: Control of the inflammatory response in extended myocardial preservation of the donor heart. Ann Thorac Surg 68:1978, 1999 6. Billingham ME: Cardiac transplantation. In Sale GE (ed): The Pathology of Organ Transplantation, 1st Ed. Boston: Butterworths; 1990, p 133 7. Aranda JM Jr, Scornik JC, Normann SJ, et al: Anti-CD20 monoclonal antibody (rituximab) therapy for acute cardiac humoral rejection: a case report. Transplantation 73:907, 2002 8. Michels PJ, Fishbein MC, Colvin RB: Humoral rejection of human organ transplants. Springer Semin Immunopathol 25:119, 2003 9. Holzinger C, Zuckermann A, Reinwald C, et al: Are T cells from healthy heart really only passengers? Characterization of cardiac tissue T cells. Immunol Lett 53:63, 1992