Saudi Journal of Kidney Diseases and Transplantation

: 2015  |  Volume : 26  |  Issue : 6  |  Page : 1232--1240

Immuno-histological assessment of sub-clinical acute and borderline rejection in renal allograft recipients: Data from a transplant center in India

Sonia Badwal1, Arun Kumar2, AK Hooda3, PP Varma4,  
1 Department of Pathology, Armed Forces Medical College, Pune, Maharashtra, India
2 Department of Internal Medicine, Armed Forces Medical College, Pune, Maharashtra, India
3 Department of Medicine and Nephrology, Army Hospital (Research and Referral), New Delhi, India
4 Department of Medicine and Nephrology, Udhampur, India

Correspondence Address:
Sonia Badwal
Department of Pathology, Armed Forces Medical College, Pune, Maharashtra


This single-center study was carried out on living related and unrelated renal transplant recipients (RTRs) to evaluate the usefulness of surveillance biopsies in monitoring stable renal allografts using immuno-histological markers for immune-activation. This is a prospective, longitudinal study. Protocol biopsies of 60 RTRs with stable graft function were evaluated at three, six and 12 months post-transplant. Immuno-histological evaluation was carried out using immune-activation markers (perforins, granzyme and interleukin-2R), phenotypic markers (CD-3 and CD-20), viral markers and C4d. The demographic and clinical profile was recorded for each patient. All cases of acute sub-clinical rejection (SCR) were treated and borderline SCR cases were followed-up without treatment. SCR at three and six months post-transplant was evident in 16.7% and 3.7% of RTRs, respectively. Positive statistical association of SCR was seen with HLA-DR mismatches, whereas patients receiving induction therapy and tacrolimus-based immunosuppression exhibited a lower incidence of SCR. T cell phenotype with persistent expression of immune-activation markers exhibited positive statistical association with interstitial fibrosis and tubular atrophy at 12-month follow-up biopsy. The mean creatinine levels were significantly lower in the protocol biopsy group than the non-protocol biopsy group. No significant difference was found between the mean creatinine levels of the SCR group after treatment and the non-SCR cases within the protocol biopsy group. Early treatment of sub-clinical acute rejection leads to better functional outcomes. However, persistent immune-activation is associated with chronicity and may have implications on long-term graft survival.

How to cite this article:
Badwal S, Kumar A, Hooda A K, Varma P P. Immuno-histological assessment of sub-clinical acute and borderline rejection in renal allograft recipients: Data from a transplant center in India.Saudi J Kidney Dis Transpl 2015;26:1232-1240

How to cite this URL:
Badwal S, Kumar A, Hooda A K, Varma P P. Immuno-histological assessment of sub-clinical acute and borderline rejection in renal allograft recipients: Data from a transplant center in India. Saudi J Kidney Dis Transpl [serial online] 2015 [cited 2022 Jan 27 ];26:1232-1240
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Sub-clinical rejection (SCR) is characterized by histological evidence of rejection in a renal biopsy but without functional deterioration of the graft. SCR is therefore diagnosed only on biopsies taken at a fixed time as per protocol rather than driven by clinical indication. [1] Detection of class II human leukocyte antigen (HLA) molecules, cytokines, adhesion molecules and gene transcripts of cytotoxic molecules in stable grafts with SCR have demonstrated unequivocal presence of immune activation. [2] However, it is still controversial whether patients with SCR will have an unfavorable long-term clinical outcome represented by worsened graft outcome. It is also unclear whether the identification of SCR through protocol biopsies and its treatment will alter the clinical course of the transplanted kidneys. [3] The present study was undertaken to address these diagnostic and clinical dilemmas by evaluating the usefulness of surveillance biopsies in the monitoring of stable transplanted kidneys.

 Materials and Methods

A single-center, prospective, longitudinal study was conducted at the Departments of Pathology and Nephrology of a tertiary care hospital. Clearance from the hospital ethical committee was obtained. Living related/unrelated HLA-matched renal transplant recipients (RTRs) receiving post-transplantation care were evaluated. The inclusion criteria were variation of serum creatinine below 20% of baseline and unchanged immunosuppression at least 15 days prior to the protocol biopsy. Patients with coagulopathy, uncontrolled blood pressure and urinary tract infections were excluded from the study. Informed consent was obtained from each patient prior to inclusion in the study and the patients were given the option of leaving the study as and when they desired.

Clinical evaluation

A performa containing demographic and clinical details such as age, sex, type of transplant, panel-reactive antibodies (PRAs), HLA mismatches, immunosuppressive therapy and posttransplant serum creatinine levels was filled for each patient.

Immunosuppression protocol

Baseline immunosuppression consisted of tacrolimus/mycophenolate mofetil/prednisolone (tacro/mmf/pred) or cylosporine/mycophenolate mofetil/prednisolone (cyclo/mmf/pred). Induction with anti-thymocyte globulin (ATG)/baciliximab was carried out in high-risk cases, e.g high PRA, re-transplants and cadaveric transplants.


Renal biopsy was carried out at three, six and 12 months post-transplantation. A minimum of two cores of renal biopsies were obtained under ultrasound guidance with a springloaded 16-gauge biopsy gun. Formalin-fixed and paraffin-embedded sections were stained with hemotoxylin and eosin (H&E), periodic acid Schiff (PAS) and silver methenamine (PASM). The second renal core was processed for direct immunofluorescence using IgG, IgM, IgA, C3c and C1q-tagged antibodies.

Evaluation of the biopsies was carried out as per the Banff Classification with updates from the ninth Banff conference on allograft pathology, 2008. [4],[5] SCR was defined as absence of clinical signs of acute rejection (AR), serum creatinine variation below 20% for at least two weeks prior to biopsy and allograft histology showing AR (Banff grade 1A or more) or borderline changes.


The immune-histochemical staining (IHC) for immune activation, phenotypic and viral markers was carried out with monoclonal mouse anti-human granzyme antibody (Clone GrB-7, DAKO, Glostrup, Denmark; dilution 1:50), monoclonal mouse anti-human perforin antibody (NCL-Perforin, Clone 5B10, Novocastra Lab. Ltd., UK, dilution 1:20), monoclonal mouse anti-human CD3 T cell antibody (DAKO, Glostrup, Denmark, Clone PC3/188A, pre-diluted) and monoclonal mouse antihuman CD20 B cell antibody (DAKO, Denmark, Clone C8/144B, pre-diluted). Cytoplasmic positivity for more than 5% lymphoid cells was taken as positive for immune activation markers. C4d staining was performed with C4d antibody (pre-diluted protein A purified polyclonal rabbit antibody Biogenex No 572 R dilution 1:50).

Biopsies exhibiting prominent interstitial inflammation were evaluated using IHC markers for Cytomegalovirus (CMV-ab1 mouse monoclonal antibody Labvision dilution 1:80), Epstein-Barr virus (Labvision EBV/LMP ab-1 Clone CS1+CS2+ CS3+CS4 pre-diluted) and Burkitts polyoma virus (BKV, Simian Virus - 40 Large T cell antigen BD Bio-sciences Catalogue No 511454 dilution 1:50). Cells were scored positive when they displayed brown membranous or cytoplasmic patterns. Nuclear staining was considered positive in CMV and BKV viral immune-staining.

Based on histological evaluation, the patients were divided into SCR group and negative for SCR group. The allograft recipients in the SCR group were further divided into AR group (Banff Grade 1A or more) and borderline rejection (BLR) group. The AR group was treated with methylprednisolone as determined by the standard of care and intent to treat. The BLR group was not treated and continued with baseline immunosuppression. In cases of cyclosporine toxicity, the dosage was adjusted and patients with graft pyelonephritis (GPN) were administered six weeks of antibiotic therapy.

 Statistical Analysis

Clinical and histological features at the three-, sixand 12-month biopsies were studied in groups with no SCR and SCR. Results were analyzed using SPSS version 13.


A total of 108 renal transplants were performed during the study period and 60 patients formed part of the study group. The remaining 48 patients were either not meeting the inclusion criteria (n = 17) or refused biopsy (n = 31). Of these 48 patients, 38 were retained in the control group and the others were not included for miscellaneous causes (death, graft nephrectomy, sepsis, etc.).

The mean age of the allograft recipients was 33.4 ± 11.2 years (14-60 years), with males constituting 71.3% and females constituting 28% of the recipients. The mean age of the donors was 43.8 ± 11.8 years (21-69 years), with males constituting 40% and females constituting 60% of the donors. A total of 38/60 patients (63.3%) were recipients of living related donors, 13 (21.7%) were spousal, five (8%) were unrelated and four (6.7%) were cadaveric. Five patients were re-transplants. The mean age of the control group was 36.07 ± 11.2 years, with 26 males and 12 females (n = 38); four of them (10.5%) were re-transplants.

Eight of the 60 patients (13.3%) revealed high PRA or a positive cross-match and were desensitized as per the center protocol. Sixteen of them (26%) were unmatched for HLA-A and B, 35 (58%) were haplo-matches and nine (15%) were haplo-identical. Twenty patients (33.3%) were unmatched for HLA-DR, 30 (50%) were haplo-matches and ten (16.6) were haplo-identical. Forty-nine patients (81.7%) received a combination of tacro/mmf/pred while 11 patients (18.3%) received cyclo/mmf/pred. Forty-one patients (68.3%) received induction either in the form of ATG or basiliximab while the remaining 31.7% did not receive any form of induction therapy.

Sixty biopsies were performed at three months, of which 19 biopsies (31.7%) exhibited rejection; nine cases (15%) showed BLR and 10 cases (16.7%) showed AR. The Banff grades of rejection of these 19 cases are shown in [Table 1]. The other biopsies revealed features of mild patchy acute tubular necrosis (ATN) and calcineurin inhibitor (CNI) toxicity in two cases each and GPN and recurrence of primary glomerular disease (focal and segmental glomerulosclerosis) in one case each, while 35/60 biopsies were histologically normal. {Table 1}

Three cases of SCR showed, in addition to tubulitis, dense interstitial inflammation rich in polymorphonuclear cells with granular and neutrophil casts in the tubular lumina. One biopsy sample revealed dense plasma cell infiltrate along with peri-tubular capillary neutrophil infiltration. A total of 15/35 biopsies were histologically normal, but revealed scanty to mild infiltration of lymphocytes, singly scattered or as small focal clusters [Figure 1], [Figure 2] and [Figure 3].{Figure 1}{Figure 2}{Figure 3}

Immuno-histochemistry revealed T lymphocytes along with positive immune-activation markers in all 19 biopsies with rejection-related diagnosis. Admixture of B and T lymphocytes was seen in biopsies with concomitant diagnosis of GPN, SCR and AMR (four of 19 cases). One case was positive for C4d in glomerular basement membrane and peri-tubular capillaries. Overall, 15/35 cases opined as normal showed CD 3-positive T cells with negative immune activation markers while three of 35 cases were positive for perforins and granzymes along with T cells. All 60 biopsies were negative for viral markers.

The mean age of the patients in the rejection group at three months was 33.9 ± 11.2 years (15-60 years) and those without rejection was 32.17 ± 11.2 years (11-52 years). One patient (1/19) had high PRA and four cases had received induction. One case of grade 1A rejection and two patients with BLR had subtherapeutic tacrolimus levels for which the baseline immunosuppression was optimized. Eighty-one percent (49/60) received tacrolimusbased immunosuppression and reported SCR (acute + borderline) in 28% of the patients (14/49), whereas 45% of the patients (5/11) on a cyclosporine-based regimen exhibited SCR (acute + borderline). Patients receiving induction therapy and on tacrolimus-based immunosuppression had a lower rate of SCR, which was statistically significant [Table 2].{Table 2}

Fifty-eight biopsies were performed at six months. One case of BLR diagnosed at three months was unwilling for second biopsy and a second patient succumbed to systemic infection. Eleven biopsies exhibited abnormal features. All cases of rejection-related diagnosis revealed positivity for immune activation markers and T cells. Four cases revealed CD20positive B lymphocytes and three revealed a weak positivity for immune activation markers. None were positive for viral markers [Table 1].

Forty-six biopsies were carried out at 12 months. Fourteen patients could not be biopsied due to various reasons, including transfer of patients, unwillingness for biopsy, not meeting the criteria for protocol biopsy and death (three cases). There were nine biopsies with abnormal features [Table 1]. Positivity of immune-activation markers persisted in four cases.

Correlation of graft histology at three months was carried out with graft histology at 12 months. Cases diagnosed as BLR recovered spontaneously without treatment, except one who showed persistence of inflammation after six months of follow-up. Four of ten cases with AR showed persistence of inflammation and positivity for immune activation markers; the remaining six recovered completely. There was no difference in the 12-month histology of the graft (persistence of inflammation and immune-activation markers) between the nonrejection group, the BLR group and the AR group after treatment, with a x 2 value of 0.618 (2), P = 0.73 (n = 46). Interstitial fibrosis and tubular atrophy was seen in seven of 46 biopsies at 12 months. Presence of SCR, enhanced expression for immune-activation markers and T cell phenotype were significantly associated with higher predisposition for developing chronicity even in a short-term follow-up of 12 months [Table 3].{Table 3}

The mean creatinine levels of the control group after the follow-up period of 12 months was 1.7 mg/dL (SD ±0.81) and that of the protocol biopsy group was 1.35 mg/dL (SD ±0.46). The difference was statistically significant (unpaired Student's t test, t = 0.031). The mean creatinine level of the BLR group and the AR group (after anti-rejection treatment) after 12 months of follow-up was 1.47 mg% (SD ±0.79) and 1.37 mg% (SD ±0.49), respectively, with no significant difference (P = 0.60).


SCR is AR identified on allograft biopsies without concurrent functional renal impairment. Hence, allograft biopsies are carried out as per the protocol of the institution at a definitive time and are not related to the clinical indication. Evidence was first generated by the clinical trials of Rush et al that treatment of SCR has beneficial effects on allograft histology and renal function. [6] This study was conducted with an intention to treat acute SCR on protocol biopsy as per the standard of care.

The rate of SCR varies in different transplant centers depending on the donor and recipient demographics, immunosuppression protocols, induction therapy and timing of the biopsy. SCR is maximally observed within the first few months after transplantation, and then gradually reduces after one year. [1],[7],[8] At an Indian transplant center, the prevalence of SCR at one, six, and 12 months was 17.5%, 11.2% and 10.3%, respectively, among patients receiving tacrolimus-based immunosuppression following living donor transplantation. [9] We found similar SCR rates at three months, but the rate of SCR was much lower at six and 12 months. This may be attributed to close monitoring and better compliance to immunosuppression as our center is a service transplant center. HLA-A and B mismatches did not contribute significantly to the occurrence of SCR.

However, increased association of HLA-DR mismatches with SCR was demonstrated and is thought to be an early allo-immune response triggered by mismatches to major histocompatibility antigens. [10],[11] Results in our study also re-iterate that the overall occurrence of SCR reduces in patients receiving tacrolimus-based maintenance immunosuppression and induction therapy as reported in studies by Nankivell et al, Gloor et al and Shapiro et al. [8],[12] The presence of activated T lymphocytes is considered an important indicator of AR and is involved in targeted killing by perforin or release of granzyme. Biopsies obtained from well-functioning renal allografts at three and six months have frequently shown lymphocytic infiltrates with heightened expression for IL-2/IL-2R, perforin or granzyme B. [13] The present study revealed immune-positivity for perforin, granzyme B and IL-2R in T lymphocytes infiltrating the renal tissue with SCR (acute and borderline SCR). Cases opined as normal biopsies exhibited scanty to mild lymphocytic inflammation rich in T cell subset with negative immune-activation markers, except in three cases displaying positivity for IL-2R and granzyme, indicating immuneactivation. Similar results have been reported by Kummer and Lipman et al, wherein normal biopsies had the lowest, those with clinical rejection the highest and ones with SCR an intermediate amount of immune-activation. [14],[15]

All cases of GPN showed presence of neutrophils in the interstitium and in the tubular lumina, forming neutrophil casts. Cases with GPN however exhibited an admixture of T cells and B cells with focally enhanced positivity for immune activity, emphasizing the fact that graft infections per se predispose the individual to develop AR by unmasking the cryptic antigens. Individuals with neutrophil casts, T lymphocytic tubulitis and diffuse immune activation beyond and away from the neutrophil casts were managed as GPN with AR.

SCR is usually treated with high-dose pulse corticosteroids, but treatment of borderline SCR is a debatable issue. Although Rush et al treated all borderline SCR cases, in our study, we followed the recommendations of Daniel et al and did not treat cases of borderline SCR. [6],[16] All such cases recovered spontaneously without treatment, except one case who showed persistence of inflammation after six months of follow-up. Ten cases of acute SCR who were treated exhibited histological improvement with residual inflammation in four cases at 12 months and were not treated subsequently. As there was no statistical difference at the 12-month graft histology within the rejection and non-rejection groups after treatment, we are inclined to believe that borderline rejection can be left alone with optimization of maintenance immunosuppression. As mean creatinine levels were significantly lower in the protocol biopsy group vis-a-vis the control group, we are in agreement with Rush et al and Kurkuti et al that SCR should be treated. [17]

Although none of the cases qualified for diagnosis of chronic allograft nephropathy (CAN) at the 12-month follow-up biopsy as per the Banff criteria, histological features of chronicity were seen in 11.7% of the biopsies. The SCR group and biopsies exhibiting enhanced expression of immune-activation markers were significantly associated with higher predisposition for developing chronic features in our study. Rush et al in their randomized study suggested that treatment of early SCR decreases chronic tubulo-interstitial scores and late clinical rejection episodes and improves long-term structural and functional outcomes. [6] Immuno-histochemical analysis of graft-infiltrating cells has shown that SCR and clinical rejection are different only in quantity and not quality of immune-activation markers. Unsuppressed SCR may lead to tubulointerstitial damage and progressive CAN, shorter graft survival even after the acute episode has abated. [18],[19],[20],[21] Although our study had a short follow-up duration, the lower incidence of these features in our set of patients is an encouraging sign and possibly the result of treatment of SCR, which is the single most important factor for CAN. [22],[23],[24]


Early treatment of acute SCR leads to better functional outcomes. However, SCR is associated with heightened expression of immuneactivation markers, which is associated with higher predilection for developing chronicity. Nevertheless, data need to be validated in a larger number of patients with longer followup periods. The relative merit of universal protocol biopsy or individualized biopsy in selected high-risk cases will depend on the profile of patients/donors, resources and management strategies of each transplant unit. The results can then be utilized for custommizing the strategies for protocol biopsies for each transplant unit.

Source(s) of support: This study was funded by the Director General of Armed Forces Medical Services.

Conflicting of interest: None declared.


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