were initiated early. From the Departmentof Cardiac Surgery, University ofMunich, Klinikum Grosshadern,. Federal Republic of Germany. Address for reprints: ...
Transplantation
Special Control of Infection and Rejection Episodes After Four Years of Cardiac Transplantation at the University of Munich Hermann Reichenspurner, M.D., Bernhard M. Kemkes, M.D., George Osterholzer, M.D., Bernhard Reble, M.D., Wolfgang Ertel, M.D., Bruno Reichart, M.D., Christian Lersch, M.D., Claus Hammer, M.D., Ralph Haberl, M.D., Gerhard Steinbeck, M.D., and Joachim Gokel, M.D. During the past 4 years, 36 orthotopic heart transplantations and two heart-lung transplantations were performed at Munich University Hospital. Immunosuppressive regimen consisted of cyclosporine A and low dose prednisone. The rejection diagnosis was based on daily cyto-immunological monitoring (CIM) and high frequency electrocardiography. In addition, viral, bacterial, and fungal infections were examined by CIM. The CIM is based on an evaluation of activated lymphocytes, lymphoblasts, and lymphocyte subsets in the mononuclear concentrate isolated from the peripheral blood by Ficoll Hypaque separation. Of the 38 patients, 24 are currently alive from I month to 4 years later (including one heart-lung recipient 1.5 years postoperatively). Altogether, 40 rejection episodes occurred among the patients. The diagnosis was based on CIM (sensitivity = 95%) and on endomyocardial biopsies (sensitivity = 95%). Control of rejection therapy was also done by using these methods. When the biopsies showed ongoing acute rejection, additional antithymocyte globulin or antilymphocyte globulin therapy was administered, relative to the CIM results. When using the endomyocardial biopsies for rejection control only, results showed a very low rate of two to three biopsies per patient in the first 3 months postoperatively. In addition, 16 infection periods were detected: five viral, six bacterial, fourfungal, and one case of toxoplasmosis. The CIM showed typical hints of these inflammations in 12 cases (sensitivity = 75%) before clinical signs were visible. This immediately led tofurther diagnostic examinations and specific anti-infectious therapies, which were initiated early.
From the Department of Cardiac Surgery, University of Munich, Klinikum Grosshadern, Federal Republic of Germany. Address for reprints: Hermann Reichenspurner, M.D., Department of Cardiac Surgery, University of Munich, Klinikum Grosshadern, Marchioninistr. 15, D-8000, Munich 70, Federal Republic of Germany. Texas Heart Institute Journal
5
BETWEEN AUGUST 1981 and August 1985, 130 patients were referred to Munich University Hospital for cardiac transplantation. Ninety-three were accepted based on the usual selection criteria.' During the waiting period, 31 patients died because there were no available donor organs. Of the 63 persons on the waiting list, 38 were transplanted and 25 remained on the current list. The main complications after surgery were acute rejection and infection. Early diagnosis of such inflammatory events was important in regard to successful therapy. After 4 years of cardiac transplantation, this study assesses the special methods of rejection and infection diagnosis and control.
PATIENTS AND METHODS
Organ Procurement The organ procurement program at our
university is attached to the Eurotransplant Organization in Leiden, the Netherlands. That organization procures organs for every patient waiting for heart transplantation in Great Britain, the Netherlands, Belgium, and West Germany. The patients are registered according to blood group and an individual urgency code.
Patients and Immunosuppression The 38 transplantations included 36 heart and two heart-lung transplantations. The immunosuppressive regimen consisted of a
low cyclosporine loading dose (10-14 mg/kg of body weight) and was continued according to the serum level of 80 to 150 ng/ml, and low dose prednisone tapered from 2.0 to 0.15 mg/kg of body weight per day within 6 weeks.2
Cyto-immunological Monitoring (CIM) The CIM method used consists of two parts. Part I is a routine cytological test and requires only 150 ,ul of heparinized blood - for example, taken from the fingertip.3 First, the white blood cell count is determined and a blood smear is stained for white blood cell differentiation. In addition, mononuclear cells, mostly lymphocytes and monocytes, are separated from the peripheral blood on a Ficoll Hypaque gradient in a microcentrifuge. Cytocentrifuge smears of the mononuclear concentrate are visualized by a quick panoptic leucocyte staining. The lymphocytes are differentiated according to their degree of activation as either normal lymphocytes, activated lymphocytes, or lymphoblasts (Fig. 1). The activated lymphocytes are distinguished from normal lymphocytes with regard to their cell size, cytoplasmic basophility and nuclear structure, and by the fact that they have not yet attained the size of fully differentiated lymphoblasts. In addition, large granular lymphocytes are distinguished cytologically from normal lymphocytes. When more than one lymphoblast or more than 50 activated lymphocytes
,sl I..
a
c
FMg. 1 Normal lymphocyte (a), activated lymphocyte (b) and lymphoblast (c) in the mononuclear concentrate, separated from peripheral blood by Ficoll Hypaque gradient. 6
Vol. 13, No. 1, March, 1986
are found (per mm3 peripheral blood), the
cytological part is described as activated, and
a second immunological part has to be added to differentiate rejection from infection. For this part, mononuclear cells are separated from 10 ml of heparinized blood, and aliquots are incubated with the following monoclonal antibodies: OKT 3*, which labels all peripheral T-lymphocytes, OKT 4* against the T-helper and T-inducer cells, and OKT 8* against the T-suppressor and T-cytotoxic cells. The antibody Ly 2t predominantly labels B-lymphocytes.
*Orthopharmaceutical Inc., Raritan, New
_=_ _
Jersey, 08869, USA. Fig. 2 Postoperative chest X ray one year after combined heart-lung transplantation
tO.Y. Medics Inc., APPB 819 SF, 00101 Helsinki 10, Finland.
.~~~~~~~~~
36
38 8
74 !l~~~~~~~
l
',1~ ~ ~ ~ ~ t
_ 9
FIg. 3 Through the Multi-organ Procurement Program between August 1981 and September 1985, there were 131 organs donated from 38 donors. Texas Heart Institute Journal
7
F0
acute rejections
(AR)
non
lethal
I
lethal
37
38 AR diagnosed by CIM { 38 AR diagnosed by EMB
sensitivity=95
%
1.1 AR per patient 8.5 % lethal AR Fig. 4 Rejection episodes in 38 patients after 4 years of cardiac transplantation.
High-flequency Electrocardiography and Neopterine Levels In addition, a daily computerized highfrequency electrocardiogram is performed, analyzing minimal changes in frequency spectrum and in the amplitude of the QRScomplex. After the electrocardiogram, daily urinary levels of neopterine are measured.4
RESULTS Of the 36 heart recipients, 23 are currently alive, surviving from 4 weeks to 4 years posttransplantation. The mean follow-up has been 16.2 + 11.9 months, resulting in an accummulative patient survey of 450.5 months. Death was caused by graft failure in three patients, acute rejection in three (two early and one late), yellow atrophy of the liver in one, aspergillosis infection in two, toxic hepatitis in one, septicemia in one, and cardiac fibrillation of unknown cause in two - one after 60 days and one after 228 days. Ten days after heart-lung transplantation, one patient died of acute yellow dystrophy of 8
the liver. The second heart-lung transplant patient is still alive 1.5 years postoperatively and is functionally in Class I of the New York Heart Association (Fig. 2).
Organ Procurement Of 38 explantations, 25 hearts were retrieved by distant heart procurement in an average distance of 400 miles from Munich. The mean ischemic time was 165 minutes. Altogether, 131 (range of 89 to 227 months) were retrieved from the 38 donors: 36 hearts, 2 heart-lung blocs, 74 kidneys, 9 livers, and 8 pancreases (Fig. 3). No correlation was found between graft ischemic time and postoperative function, as long as an ischemic time of 250 minutes was not extended. In addition, multiorgan procurement had no influence on postoperative graft function in comparison to single organ procurement.
Acute Rejections Altogether, a total of 40 acute rejections occurred. Thirty-eight of these episodes were diagnosed by CIM, and by endomyocardial Vol. 13, No. 1, Marci, 1986
before AR
durng AR
Fig. 5 Changes of leucocytes, T- and B-subsets, activated lymphocytes and lymphoblasts before and during acute cardiac rejection. *p < 0.005; **p < 0.0005
biopsy (EMB). The sensitivity of CIM, as well as EMB, was thus 95% (Fig. 4). Monitoring showed that all rejection crises were characterized by a significant rise in the number of leucocytes and lymphocytes, predominantly T-lymphocytes (p < 0.005) (Fig. 5). This rise was accompanied by the appearance of activated lymphocytes and lymphoblasts in the mononuclear concentrate (p < 0.0005). The six recent rejection episodes were followed by using the high frequency electrocardiograph. This electrocardiograph showed amplitude and frequency changes of the QRS-complex in all of these acute rejection patients. Acute rejection therapy was controlled by EMB and CIM. In three cases of ongoing acute rejection, after three pulses of methylprednisolone combined with the Texas Heart Institute Journal
predominanace of T-lymphoblasts in CIM, antilymphocyte globulin was administered successfully. In one patient, when the acute rejection was accompanied by the presence of B-lymphoblasts and plasmoblasts in CIM, antithymocyte globulin was not capable of resolving the rejection. Because of the presence of activated B-cells in CIM, antilymphocyte globulin was administered successfully in this patient. Sixteen major infections were detected in the transplanted patients: five viral infections (three herpes simplex, one herpes zoster, and one cytomegalovirus); six bacterial infections (one legionnaires' disease, one pulmonary tuberculosis, one pneumotosis cystoides intestinii, one Staphylococcus aureas, and two Pseudomonas aeruginosa); and four fungal 9
3 *
T/B Ly
OKT4/OKT8
2
L 1
-
before VlI
during VI
after VI
Fig. 6 Changes of T/BN lymphocyte ratio and helper/suppressor cell (OKT 4/OKT 8) ratio before, during, and after viral infection. *p < 0.025
cells / 100 mono nuclear cells *
*
30
20
1
10
before Bl or Fl H
during Bl or Fl
Juvenile Granulocytes Ly 2 positive cells
Fig. 7 Changes of juvenile granulocytes and Ly-2 positive B-lymphocytes before and during bacterial or fungal infections. *p < 0.01 10
Vol. 13, No. 1, March, 1986
20 biopsies (n)
without CIM
with CIM
15
10
(ii. i iii Ii :no 2 3 5 6 8 9 10 111416 171821222324252627282930313233343637 = chronic rejection
of
patient 9/85
Mig. 8 Number and reduction of biopsies during the first 90 postoperative days by using cyto-immunological monitoring (CIM) as an indicator for myocardial biopsies.
infections (two aspergillosis, two candidiasis, and one toxoplasmosis). In CIM during all viral infections, the TH-TS cell ratio became less than 1 (0.71 0.05) in contrast to the normal ratio (1.55 0.24, p < 0.025) (Fig. 6). More than 20% of large granular lymphocytes were found in the mononuclear concentrate (Fig. 7). During four of the bacterial and three of the fungal infections, the CIM showed an increase of Ly-2 positive B-cells in the mononuclear concentrate (p < 0.01) (Fig. 8). In addition, juvenile polymorphs appeared in the mononuclear concentrate (p < 0.01) (Fig. 8). These hints were given by CIM before clinical signs of infections were visible. DISCUSSION The two major complications after cardiac transplantation are acute rejection and infection. Early noninvasive diagnosis of these inflammations seems to be extremely necessary for optional therapy. In 95% of all acute rejection patients, lymphoblasts and more than 50 activated lymphocytes appeared in the mononuclear concentrate of the peripheral blood.3 In order to increase the specificity of Texas Heart Institute Joumnal
CIM, high frequency electrocardiographs performed daily showed typical alterations of the QRS complex during acute rejection. Thus, rejection diagnosis was based on these two examinations. Endomyocardial biopsies were mainly performed to control rejection therapy with methylprednisolone. Ongoing acute rejection in the biopsies, together with the presence of T-lymphoblasts or B-lymphoblasts in CIM, gave the indication for additional antithymocyte- or antilymphocyte globulin therapy. Using this kind of monitoring, the biopsies were reduced to only two to three per patient in the first 3 months after transplantation, which corresponds to a reduction of 75% in comparison to routinely biopsied patients (Fig. 8). The rate of lethal rejections was only 8.5% among the examined patients. During infection episodes, the monitoring showed early and typical changes. During viral infections, the appearance of activated lymphocytes and lymphoblasts was accompanied by more than 20% of large granular lymphocytes in the mononuclear concentrate and an inversion of the helper! suppressor cell ratio. During bacterial or fungal infections, more than 20% of the 11
cells in the mononuclear concentrate were juvenile polymorphs, and the percentage of Ly-2 positive B-cells significantly increased. These early hints in CIM were used to indicate microbiological or virological examinations very early. Thus, when clinical symptoms appeared, a specific anti-infectious therapy was started at a very early stage of the infection, and unnecessary antibiotics were avoided.
REFERENCES 1. Baumgartner WA, Borkon SM, Achuff SC, Baughman KL, Traill TA, Reitz BA. Heart and heart-lung transplantation: Program, development, organization, and initiation. Heart Transplantation 1985; 4:197-202.
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2. Reichart B, Uberfuhr P, Welz A, Kemkes BM, Funccius W, Reble B, Klinner W, Reichenspumer H, Ertel W, Hammer C, Gokel JM. Heart Transplantation at the University of Munich; The first one and one-half years. Heart Transplantation 1983; 4:266-269. 3. Reichenspumer H, Ertel W, Hammer C. Lersch C, Reichart B, Uberfuhr P, Welz A, Reble B, Kemkes BM, Gokel JM. Immunologic monitoring of heart transplant patients under cyclosporine immunosuppression. Transplantation Proceedings 1984; 16(5):1251-1254. 4. Margreiter R, Fuchs D, Hausen A, Huber C, Reibnegger G, Spielberger M, Wachter H. Neopterin as a new biochemical marker for diagnosis of allograft rejection. Transplantation 1983; 36(6): 650-653.
Vol. 13, No. 1, March, 1986
