Peri-operative immunoadsorption in sensitized renal transplant ...

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Nephrol Dial Transplant (2002) 17: 1503–1508

Original Article

Peri-operative immunoadsorption in sensitized renal transplant recipients Martin Haas1, Georg A. Bo¨hmig1, Zdenka Leko-Mohr1, Markus Exner2, Heinz Regele3, Kurt Derfler1, Walter H. Ho¨rl1 and Wilfred Druml1 1

Division of Nephrology and Dialysis, Department of Internal Medicine III, University of Vienna, Austria, 2Department of Laboratory Medicine, University of Vienna, Austria and 3Division of Ultrastructural Pathology and Cell Biology, Department of Pathology, University of Vienna, Austria

Abstract Background. Re-transplanted kidney allograft recipients with high levels of panel reactive antibodies (PRA) are at increased risk of early immunologic graft loss. In these patients, prophylactic peri-operative antibody depletion by immunoadsorption (IA) could prevent humoral graft injury and thus, in combination with anti-cellular rejection therapy, improve graft survival. Methods. Twenty re-transplanted and broadly immunized cadaver kidney recipients (median PRA reactivity 87%, range 55–100%) were treated with IA (protein A) immediately before transplantation and during the early post-transplantation period (median number of IA sessions 11, range 1–24). Patients received additional prophylactic anti-lymphocyte antibody therapy. Nineteen patients had a negative pre-transplant crossmatch. In one patient, a positive cross-match was rendered negative by the pre-transplant IA session. Results. One-year graft survival was 80% and patient survival 95%. Median (range) serum creatinine in functioning grafts was 1.6 (0.8–2.7) mgudl at discharge and 1.5 (1.0–5.8) mgudl at 1 year. Two grafts were lost due to acute vascular rejection, whereby one rejection occurred after withdrawal of immunosuppression due to septicaemia. One patient had acute cellular rejection, which was reversed by a second course of anti-lymphocyte antibody therapy. Thrombotic microangiopathy and surgical complications were the causes for one graft loss each. Retrospective immunohistochemistry revealed peritubular C4d staining, a presumed marker for humoral alloreactivity, in 12 out of 15 biopsies. Conclusions. These results suggest that prophylactic peri-operative IA and anti-lymphocyte antibody therapy might be an effective therapeutic strategy

Correspondence and offprint requests to: Martin Haas, MD, Division of Nephrology and Dialysis, Department of Internal Medicine III, University of Vienna, Wa¨hringer Guertel 18–20, A-1090 Vienna, Austria. Email: [email protected] #

for the prevention of early graft failure in sensitized re-transplant recipients. Keywords: HLA-antibodies; humoral response; hypersensitized patients; immunoadsorption; kidney re-transplant; kidney transplantation; renal graft rejection

Introduction Broadly immunized re-transplant recipients represent a growing group on the transplant waiting list. Besides the high probability of a positive pre-transplant cytotoxic cross-match, episodes of acute rejections in sensitized patients are frequent and transplant survival is below that observed in non-sensitized recipients [1–3]. Optimal immunosuppressive therapy for highly immunized transplant recipients remains undefined. As a means to improve graft survival, administration of mono- or polyclonal anti-lymphocyte antibodies as part of a quadruple induction therapy has been recommended [4–11]. Still, despite reduction of cellular rejections in these patients, a high incidence of early graft failure was reported [4–8,10–11]. An increasing number of studies emphasize the important pathogenetic role of alloantibodies, which can have a detrimental influence on graft survival [12–18]. Particularly broadly sensitized recipients are susceptible to humoral graft injury [12,13,15,16,18]. On this basis we hypothesized that a more efficient prophylaxis against acute rejection in sensitized patients might be achieved by targeting not only cellular but also humoral immune responses. Extracorporeal immunoadsorption (IA) is considered to be the most efficient way to remove antibodies. A single treatment session is able to decrease IgG levels by 80% [19]. IA has thus been used to effectively

2002 European Renal Association–European Dialysis and Transplant Association


M. Haas et al.

Table 1. Individual parameters and outcome of 20 broadly sensitized transplant recipients treated with quadruple immunosuppresive therapy and peri-operative IA Patient no.

Age (years)


Tx no.

CIT (h)



Graft biopsies (days after Tx)


Cr (discharge) (mgudl)

Cr (1 year) (mgudl)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

50 28 56 38 36 46 34 29 65 47 56 45 20 36 55 71 49 50 40 56

m m m m m m m f m m f m m m m m m f m m

2 2 3 3 2 5 3 3 2 5 2 2 2 2 2 2 2 2 3 2

23 22 22 26 16 18 10 26 11 23 11 10 17 27 10 27 14 15 14 17

0-2-1 1-1-1 1-2-0 1-1-0 1-2-1 1-1-1 0-0-0 1-0-0 0-1-2 0-0-1 0-1-1 1-1-1 1-2-0 0-0-1 0-0-1 1-1-1 2-2-0 2-1-1 0-1-1 1-1-0

positive positive nobx positive positive positive positive positive nobx nobx positive positive negative positive nobx positive positive negative negative nobx

BL (11) NH, BL, BII (5, 16, 27) nobx BL, BII, NH, BL (16, 21, 43, 76) ATN, TMA (5, 10) ATN, BII (6, 90) ATN (5) ATN (1) nobx nobx TMA (9) ATN (2) RVT with ATN (1) ATN (5) nobx ATN, NH, NH (7, 21, 40) ATN (8) ATN (9) NH (11) nobx

yes yes no non-function non-function yes yes yes yes no no yes non-function yes no yes yes yes no no

2.0 2.7 0.8 HD HD died 2.5 2.5 1.6 1.1 1.2 1.7 HD 2.0 1.2 1.7 2.6 1.6 1.6 1.2

1.7 1.5 1.3 HD HD 2.1 1.5 1.0 1.6 1.1 1.0 HD 5.8 1.2 3.1 1.4 1.5 1.8 1.2

Tx, number of transplantations; CIT, cold ischaemic time; MM, mismatch; DGF, delayed graft function; Cr, creatinine; nobx, no biopsy; BL, borderline rejection; BII, Banff II rejection; NH, normal histology; TMA, thrombotic microangiopathy; ATN, acute tubular necrosis; RVT, renal vein thrombosis.

reverse acute humoral rejection in refractory patients [20–24], which is why we hypothesized that prophylactic IA therapy might be equally efficient in broadly sensitized patients prone to antibody-mediated graft loss.

the Eurotransplant Organization using the standard microcytotoxicity technique described by Terasaki and McClelland [25]. Cytotoxic PRA reactivity was assessed using a panel of cells from 20 to 50 phenotyped donors. For exclusion of IgM, tested sera were pre-treated with dithiotreitol.


Subjects and methods Patients Between the years 1995 and 2000, 20 broadly sensitized (panel reactive antibodies (PRA) greater than 50%) retransplanted cadaveric renal allograft recipients were treated with IA immediately before and after transplantation. Demographic data are listed in Table 1. The median (range) of previous graft survival was 3 (0.1–10) years, and less than 4 years in 16 out of 20 patients. All transplantations were performed within the Eurotransplant region and both donors and recipients were Caucasians. The median donor age was 36.6 (2–66) years and median recipient age 46.5 (20–71) years. Thirteen recipients underwent a second, five a third, and two a fifth kidney transplant. Included patients had been on the transplant waiting list for a median of 30 (8–131) months. According to the rules of Eurotransplant, all patients had a negative historic cytotoxic cross-match. The current cytotoxic cross-match (prior to initiation of IA) was negative in 19 out of 20 recipients. In one patient, a positive pre-IA cross-match was rendered negative by a 3 h course of IA.

Immunological techniques Cytotoxic cross-match testing was performed with donor peripheral blood mononuclear cells according to the rules of

The procedure of IA is reported elsewhere in detail [26]. Briefly, the patients underwent plasma separation using the autopheresis-CTM therapeutic plasma system (Baxter, IL). During each session, 2.5 plasma volumes were run alternatively over two cartridges containing staphylococcal protein A (Excorim, Lund, Sweden). For removal of bound immunoglobulin, the column was flushed with sodium citrate after each cycle and for storage with phosphate-buffered saline and 0.9% NaCl solution. IA was initiated and performed once immediately before renal transplantation and then every 2–3 days until stabilization of graft function (serum creatinine less than 2 mgudl) or graft loss. The number of sessions ranged between 1 and 24 (median 11), since the time to graft function differed. Total serum IgG levels were effectively reduced by IA in all patients and were below the detectable level after the last IA session (less than 1.95 gul) in most of them (Table 2). As assessed in 16 out of 20 patients, PRA levels, determined simultaneously with the IgG levels, were significantly reduced upon IA treatment (87%, range 55–100% before vs 53%, range 13–100% after IA; P-0.001). The median PRA titre of 14 out of 20 patients decreased from 1 : 32 (1 : 2–1 : 32) to 1 : 3 (1 : 2–1 : 8) (Table 2). The basal immunosuppressive therapy consisted of cyclosporine (adjusted to trough levels between 200 and 250 nguml for the early post-transplant period (8 weeks) and then reduced to 100–150 nguml), steroids (according to a local tapering regimen), and mycophenolate mofetil at 2 guday (dose-adjusted according to white blood cells). Nineteen patients received prophylactic therapy with anti-lymphocyte

Apheresis in sensitized graft recipients


Table 2. PRA concentration and titre, and serum IgG level before the first (1) and after the last (2) IA-session Patient no.

PRA 1 (%)

PRA 2 (%)

Titre 1

Titre 2

IgG 1 (gul)

IgG 2 (gul)

IA total sessions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

84 63 82 65 94 96 98 100 56 93 100 90 73 78 96 100 57 68 55 96

42 50 n.d. 42 70 n.d. n.d. 84 n.d. 50 70 60 13 53 87 100 23 60 53 50

1 : 16 1 : 32 n.d. n.d. 1 : 32 n.d. n.d. 1:8 n.d. 1 : 32 1 : 32 1:8 1 : 16 1 : 32 n.d. 1 : 32 1 : 32 1 : 32 1:2 1 : 32

1:2 1:8 n.d. n.d. 1:2 n.d. n.d. 1:2 n.d. 1:2 1:4 1:4 1:2 1:8 n.d. 1:8 1:4 1:2 1:2 1 : 16

86 83 51 95 150 172 50 148 40 79 120 120 43 83 127 80 78 50 78 189

3.2 -1.95 -1.95 -1.95 -1.95 8.6 -1.95 -1.95 -1.95 -1.95 -1.95 -1.95 -1.95 -1.95 -1.95 -1.95 -1.95 -1.95 -1.95 -1.95

11 4 4 13 1 7 10 24 13 9 12 12 13 11 6 16 12 22 11 9

n.d., not done.

antibody (rabbit, or horse, anti-lymphocyte serum (ALS); initial dose 5–10 mgukguday; Pasteur Merieux S.V., France) for a treatment course of 10–14 days. The dose was adjusted according to the white blood cell count. To minimize reduction of ALS levels by IA as a result of antibody binding to staphylococcal protein A, anti-lymphocyte antibody was infused shortly after each IA treatment session. One patient (no. 16) with a history of allergic reactions towards mono- (OKT3) and polyclonal anti-lymphocyte antibodies received prophylactic anti-IL-2R antibody (20 mg at transplantation and after 5 days; basiliximab (Simulect1), Novartis, NJ, USA). Ganciclovir (tapered according to renal function) was administered to all patients as prophylaxis against CMV infection. Anti-cellular rejection therapy consisted of high-dose steroids (dexamethasone, 100 mguday for 3 days) with or without an additional course of poly- or monoclonal anti-lymphocyte antibody. Three patients were converted from cyclosporine to tacrolimus, in one case because of severe rejection, in the other two cases because of suspected cyclosporine-induced haemolytic uraemic syndrome (HUS).

Renal allograft biopsy Percutaneous renal biopsy was performed in 15 patients (three with initial non-function and 12 with delayed graft function). For histopathological analysis, paraffin-embedded sections were stained with haematoxylin and eosin, Periodic Acid-Schiff, Methenamin-Silver and with the trichrome stain Acidic Fuchsin-Orange-G. The histopathological classification was done according to the Banff Working Classification of Kidney Transplant Pathology [30]. C4d staining on paraffin-embedded sections was made according to a protocol described previously [18].

Statistical analysis All results are expressed as mean"SD or median and range when appropriate. A two-tailed Student’s t-test for paired

data was used to assess differences between two time points within the group. A P value -0.05 was considered to indicate statistical significance.

Results Clinical outcome Clinical data and biopsy results are given in Table 1. In the 20 highly sensitized re-transplant recipients subjected to prophylactic IA treatment, 1-year graft survival was 80%, and patient survival was 95% (Figure 1). Median (range) serum creatinine in functioning grafts (ns16) was 1.6 (0.8–2.7) mgudl at discharge and 1.5 (1.0–5.8) mgudl after 1 year. Eleven recipients (55%) had delayed graft function (DGF) and needed initial haemodialysis for at least 1 week (mean"SD, number of dialysis sessions 8"3). One of these patients (no. 2) showed acute vascular rejection (Banff IIA) in a biopsy performed 4 weeks after transplantation. In two previous biopsy specimens of the same patient, no signs of acute rejection were found. Graft function improved upon steroid bolus therapy and a second course of anti-lymphocyte antibody. In one recipient with DGF (no. 9) initial non-function of the graft was suspected to be due to allograft rejection, but wound infection prevented kidney biopsy. However, high-dose steroids and tacrolimus rescue therapy were given whereon kidney function recovered. A third patient with DGF (no. 11) showed histologic signs of thrombotic microangiopathy in a biopsy performed 9 days after transplantation. Based on laboratory evaluation (thrombocytopenia, substantial increase of LDH levels and Coombsnegative haemolysis) cyclosporine-induced HUS was suspected. After conversion to tacrolimus and


M. Haas et al.

Fig. 1. One-year graft and patient survival in 20 broadly sensitized recipients treated with prophylactic IA and quadruple therapy.

initiation of plasma exchange in addition to IA, renal function improved and the patient was discharged with excellent graft function. In eight recipients, early renal biopsy revealed acute tubular necrosis (ATN) as morphologic correlate of DGF. Graft function recovered in all patients without additional immunosuppression. One of these patients (no. 6), however, developed severe methicillin-resistant Staphylococcus aureus septicaemia 6 weeks after transplantation. Following stepwise withdrawal of basal immunosuppression, severe acute rejection (Banff IIA) occurred and made transplant nephrectomy necessary. About 1 week later, the patient died from sepsis and multisystem organ failure. Another three recipients lost their graft during the early post-transplantation period. One allograft (no. 13) was lost because of surgical complication (renal vein thrombosis) two days after transplantation. In another patient (no. 4) with graft loss, a first biopsy (day 16) performed because of initial nonfunction of the transplanted kidney revealed Banff borderline lesion. The presence of intracapillary granulocytes in this specimen pointed to humoral graft injury. In a subsequent biopsy (day 21), borderline changes had progressed to acute vascular rejection (Banff IIA), whereon monoclonal antibody therapy with OKT3 (5 mguday for 10 days) was initiated. Two subsequent biopsies revealed a complete remission of cellular rejection and disappearance of intracapillary granulocytes. Nevertheless, graft function did not recover and the patient remained on dialysis. The fourth graft loss occurred in a patient (no. 5) with an initially well functioning transplant. At day 5, severe graft dysfunction occurred together with thrombocytopenia and laboratory signs of haemolysis. This and the histologic finding of thrombotic microangiopathy (TMA) led to the diagnosis of

cyclosporine-induced HUS. IA and cyclosporine were converted to plasma exchange and tacrolimus, however, graft function did not recover and the patient remained on haemodialysis. C4d staining C4d staining was performed in all 15 histologically examined patients (in 10 of them immunohistochemistry was done retrospectively). In 12 recipients (80%) at least one biopsy stained positive for C4d in PTC. In nine C4d-positive patients, biopsy revealed no unequivocal signs of cellular rejection (ATN, ns6; TMA, ns2; Banff borderline, ns1). The remaining three C4d-positive patients showed features of vascular rejection. Three recipients were C4d negative. One of them had renal vein thrombosis. In the other two patients biopsy revealed normal histology or ATN, respectively. All grafts lost, except the one with renal vein thrombosis, were C4d positive. Infectious complications With the exception of the patient dying from severe septicaemia, no severe infection episodes occurred. Two patients developed mild CMV infection. Both patients were successfully treated with i.v. ganciclovir. Urinary tract infection in five and bacteraemia in two patients responded well to antibiotic therapy.

Discussion In our study, we report the first prophylactic immunosuppressive regimen in broadly sensitized re-transplanted kidney graft recipients targeted specifically at both cellular and humoral alloreactivity.

Apheresis in sensitized graft recipients

Our reported 1-year graft survival of 80% compares favourably to the 60–70% graft survival of sensitized recipients shown by previous studies aimed mainly at inhibiting cellular rejection [4–8,10,11]. Schroeder et al. [10] described an early graft loss due to hyperacute or acute rejection in 22% of the sensitized recipients treated with prophylactic quadruple therapy, and a 1-year graft survival of only 70%. Apparently, different results were presented by Thibaudin et al. [9], who reported a high graft survival in patients with more than 5% PRA treated with prophylactic quadruple therapy, but the data cannot be extrapolated to broadly sensitized recipients. Despite the classification of their patients in different groups of sensitization, the authors did not specify the graft survival for each group. Thus, it remains unclear how many of the approximately 50% acute rejections in the broadly sensitized recipients led to graft failure [9]. Furthermore the majority of the patients were not retransplanted and thus probably less prone to graft failure than our population. In those reports describing the outcome in sensitized recipients receiving a second or subsequent graft, 1-year survival was reported to be even worse than in sensitized first transplants. In the study of Gaston et al. [4], 40% of the patients receiving a re-transplant, in the presence of a negative pre-transplant CDC-cross-match, lost the graft within 1 year. As quadruple therapy did not improve the outcome, it was concluded that induction therapy with ALS is without benefit in patients undergoing repetitive transplantation. The data are in accordance with our own experiences. Between the years 1993 and 1995 highly sensitized and re-transplanted patients at our transplant centre were regularly treated with prophylactic quadruple immunosuppressive therapy. The 1-year graft survival during this period was similarly only 60%. It could be argued that transplant outcomes have been improving as immunosuppressive strategies have been changing in the latter years. In fact, according to USRDS, 1-year graft survival for patients with a PRA greater than 30% was 84% in 1997 and that of repeat transplants 82%. However, transplant survival of patients with both risk factors, i.e. high PRA concentrations and re-transplantation, remains clearly below non sensitized recipients. The Eurotransplant Foundation reports a 1-year graft survival of 70% (95% CI 67–73) in 1045 sensitized (PRA greater than 50%) recipients receiving a second or subsequent graft during the years 1995–1999 (Drs J. Smits and G. Persijn, Eurotransplant Foundation, personal communication). This outcome underlines the importance of alternative immunosuppressive strategies in sensitized re-transplant recipients. The remarkable low incidence of immunologic graft losses seems particularly unusual given that 80% of the patients had a positive C4d staining. The high sensitivity of C4d in the diagnosis of acute humoral rejection and the close correlation to donor-specific antibodies has only recently been confirmed [28,29]. Thus, graft survival in C4d-positive patients is


markedly decreased and has been reported to be as low as 60% [30]. Even if an aggressive post-transplant anti-rejection therapy is applied, including mycophenolate mofetil, tacrolimus and plasmapheresis, graft survival has been increased to at most 70% [28]. Our results stand in clear contrast to the reported low transplant survival in patients with anti-donor antibodies. Although the number of C4d-positive biopsies was almost three times as high as usual [28,29], 83% of the C4d-positive grafts survived beyond 1 year. In only two patients immune-mediated graft loss occurred. In both of them graft loss was attributed to vascular rejection, occurring after the end of induction therapy. Our finding of positive C4d staining in these recipients, in one of them associated with an accumulation of granulocytes in peritubulary capillaries, points at a contribution of humoral immune mechanisms to graft failure. In a third recipient, the finding of thrombotic microangiopathy together with distinct laboratory features led to the diagnosis of cyclosporine-induced HUS. Retrospective analysis, however, revealed endothelial C4d deposits. Capillary microthrombi with or without C4d deposits have earlier been described to be associated with antibody-positive rejection [1,30,31]. Accordingly, thrombotic microangiopathy in this patient could also be interpreted as a sign of refractory humoral injury. The general paucity of additional histological features for acute rejection (i.e. interstitial inflammatory infiltrates, vasculitis, tubulitis, polymorphonuclear cells in the peritubulary capillaries) could be explained by prophylactic anti-cellular and antihumoral therapy, which might have mitigated the histological picture. ATN was a common biopsy finding in our patients and was often associated with capillary C4d deposits. Previously, we were able to demonstrate that C4d deposits in acute humoral rejection persist despite successful anti-rejection therapy with IA [24]. Hence, C4d-positive ATN without other features of humoral rejection might represent a discrete lesion of antibody-mediated graft injury. In these patients, the complete manifestation of humoral rejection might have been prevented by early anti-humoral therapy. Although IA is highly effective in removing immunoglobulins, other methods might prove to be similarly successful. Plasma exchange has been shown to effectively reverse humoral rejection [32] and might be a promising prophylactic regimen when combined with i.v. IgG [33]. In addition, administration of a specific anti-CD20 monoclonal antibody (rituximab) directed against B cells might hinder donor-specific antibody synthesis. So far, however, none of these treatments has been administered to a larger transplant population as prophylactic post-transplant therapy and their efficacy in this setting still needs to be ascertained. In conclusion, our data suggest a beneficial effect of prophylactic peri-operative IA and anti-lymphocyte antibody therapy on graft survival in broadly sensitized renal re-transplant recipients. Inhibition of both


the humoral and cellular arm of the immune system might comprise an increased graft survival in this specific group of high-risk patients, however, a prospective and randomized trial is warranted to definitely answer this question.

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